WorldWideScience

Sample records for brain microvascular endothelial

  1. Pathways for insulin access to the brain: the role of the microvascular endothelial cell.

    Science.gov (United States)

    Meijer, Rick I; Gray, Sarah M; Aylor, Kevin W; Barrett, Eugene J

    2016-11-01

    Insulin affects multiple important central nervous system (CNS) functions including memory and appetite, yet the pathway(s) by which insulin reaches brain interstitial fluid (bISF) has not been clarified. Recent studies demonstrate that to reach bISF, subarachnoid cerebrospinal fluid (CSF) courses through the Virchow-Robin space (VRS) which sheaths penetrating pial vessels down to the capillary level. Whether insulin predominantly enters the VRS and bISF by local transport through the blood-brain barrier, or by being secreted into the CSF by the choroid plexus, is unknown. We injected 125 I-TyrA14-insulin or regular insulin intravenously and compared the rates of insulin reaching subarachnoid CSF with its plasma clearance by brain tissue samples (an index of microvascular endothelial cell binding/uptake/transport). The latter process was more than 40-fold more rapid. We then showed that selective insulin receptor blockade or 4 wk of high-fat feeding each inhibited microvascular brain 125 I-TyrA14-insulin clearance. We further confirmed that 125 I-TyrA14-insulin was internalized by brain microvascular endothelial cells, indicating that the in vivo tissue association reflected cellular transport, not simply microvascular tracer binding. Copyright © 2016 the American Physiological Society.

  2. Vascular Endothelial Growth Factor Receptor 1 Contributes to Escherichia coli K1 Invasion of Human Brain Microvascular Endothelial Cells through the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway▿ †

    OpenAIRE

    Zhao, Wei-Dong; Liu, Wei; Fang, Wen-Gang; Kim, Kwang Sik; Chen, Yu-Hua

    2010-01-01

    Escherichia coli is the most common Gram-negative organism causing neonatal meningitis. Previous studies demonstrated that E. coli K1 invasion of brain microvascular endothelial cells (BMEC) is required for penetration into the central nervous system, but the microbe-host interactions that are involved in this process remain incompletely understood. Here we report the involvement of vascular endothelial growth factor receptor 1 (VEGFR1) expressed on human brain microvascular endothelial cells...

  3. Effect of shear stress on iPSC-derived human brain microvascular endothelial cells (dhBMECs).

    Science.gov (United States)

    DeStefano, Jackson G; Xu, Zinnia S; Williams, Ashley J; Yimam, Nahom; Searson, Peter C

    2017-08-04

    The endothelial cells that form the lumen of capillaries and microvessels are an important component of the blood-brain barrier. Cell phenotype is regulated by transducing a range of biomechanical and biochemical signals in the local microenvironment. Here we report on the role of shear stress in modulating the morphology, motility, proliferation, apoptosis, and protein and gene expression, of confluent monolayers of human brain microvascular endothelial cells derived from induced pluripotent stem cells. To assess the response of derived human brain microvascular endothelial cells (dhBMECs) to shear stress, confluent monolayers were formed in a microfluidic device. Monolayers were subjected to a shear stress of 4 or 12 dyne cm -2 for 40 h. Static conditions were used as the control. Live cell imaging was used to assess cell morphology, cell speed, persistence, and the rates of proliferation and apoptosis as a function of time. In addition, immunofluorescence imaging and protein and gene expression analysis of key markers of the blood-brain barrier were performed. Human brain microvascular endothelial cells exhibit a unique phenotype in response to shear stress compared to static conditions: (1) they do not elongate and align, (2) the rates of proliferation and apoptosis decrease significantly, (3) the mean displacement of individual cells within the monolayer over time is significantly decreased, (4) there is no cytoskeletal reorganization or formation of stress fibers within the cell, and (5) there is no change in expression levels of key blood-brain barrier markers. The characteristic response of dhBMECs to shear stress is significantly different from human and animal-derived endothelial cells from other tissues, suggesting that this unique phenotype that may be important in maintenance of the blood-brain barrier. The implications of this work are that: (1) in confluent monolayers of dhBMECs, tight junctions are formed under static conditions, (2) the formation

  4. Effect of Antimicrobial Compounds on Balamuthia mandrillaris Encystment and Human Brain Microvascular Endothelial Cell Cytopathogenicity▿

    Science.gov (United States)

    Siddiqui, Ruqaiyyah; Matin, Abdul; Warhurst, David; Stins, Monique; Khan, Naveed Ahmed

    2007-01-01

    Cycloheximide, ketoconazole, or preexposure of organisms to cytochalasin D prevented Balamuthia mandrillaris-associated cytopathogenicity in human brain microvascular endothelial cells, which constitute the blood-brain barrier. In an assay for inhibition of cyst production, these three agents prevented the production of cysts, suggesting that the biosynthesis of proteins and ergosterol and the polymerization of actin are important in cytopathogenicity and encystment. PMID:17875991

  5. Effect of Antimicrobial Compounds on Balamuthia mandrillaris Encystment and Human Brain Microvascular Endothelial Cell Cytopathogenicity▿

    OpenAIRE

    Siddiqui, Ruqaiyyah; Matin, Abdul; Warhurst, David; Stins, Monique; Khan, Naveed Ahmed

    2007-01-01

    Cycloheximide, ketoconazole, or preexposure of organisms to cytochalasin D prevented Balamuthia mandrillaris-associated cytopathogenicity in human brain microvascular endothelial cells, which constitute the blood-brain barrier. In an assay for inhibition of cyst production, these three agents prevented the production of cysts, suggesting that the biosynthesis of proteins and ergosterol and the polymerization of actin are important in cytopathogenicity and encystment.

  6. Quercetin protects human brain microvascular endothelial cells from fibrillar β-amyloid1–40-induced toxicity

    Directory of Open Access Journals (Sweden)

    Yongjie Li

    2015-01-01

    Full Text Available Amyloid beta-peptides (Aβ are known to undergo active transport across the blood-brain barrier, and cerebral amyloid angiopathy has been shown to be a prominent feature in the majority of Alzheimer׳s disease. Quercetin is a natural flavonoid molecule and has been demonstrated to have potent neuroprotective effects, but its protective effect on endothelial cells under Aβ-damaged condition is unclear. In the present study, the protective effects of quercetin on brain microvascular endothelial cells injured by fibrillar Aβ1–40 (fAβ1–40 were observed. The results show that fAβ1–40-induced cytotoxicity in human brain microvascular endothelial cells (hBMECs can be relieved by quercetin treatment. Quercetin increases cell viability, reduces the release of lactate dehydrogenase, and relieves nuclear condensation. Quercetin also alleviates intracellular reactive oxygen species generation and increases superoxide dismutase activity. Moreover, it strengthens the barrier integrity through the preservation of the transendothelial electrical resistance value, the relief of aggravated permeability, and the increase of characteristic enzyme levels after being exposed to fAβ1–40. In conclusion, quercetin protects hBMECs from fAβ1–40-induced toxicity.

  7. Human Brain Microvascular Endothelial Cells and Umbilical Vein Endothelial Cells Differentially Facilitate Leukocyte Recruitment and Utilize Chemokines for T Cell Migration

    Directory of Open Access Journals (Sweden)

    Shumei Man

    2008-01-01

    Full Text Available Endothelial cells that functionally express blood brain barrier (BBB properties are useful surrogates for studying leukocyte-endothelial cell interactions at the BBB. In this study, we compared two different endothelial cellular models: transfected human brain microvascular endothelial cells (THBMECs and human umbilical vein endothelial cells (HUVECs. With each grow under optimal conditions, confluent THBMEC cultures showed continuous occludin and ZO-1 immunoreactivity, while HUVEC cultures exhibited punctate ZO-1 expression at sites of cell-cell contact only. Confluent THBMEC cultures on 24-well collagen-coated transwell inserts had significantly higher transendothelial electrical resistance (TEER and lower solute permeability than HUVECs. Confluent THBMECs were more restrictive for mononuclear cell migration than HUVECs. Only THBMECs utilized abluminal CCL5 to facilitate T-lymphocyte migration in vitro although both THBMECs and HUVECs employed CCL3 to facilitate T cell migration. These data establish baseline conditions for using THBMECs to develop in vitro BBB models for studying leukocyte-endothelial interactions during neuroinflammation.

  8. Zika Virus Infects, Activates, and Crosses Brain Microvascular Endothelial Cells, without Barrier Disruption

    Science.gov (United States)

    Papa, Michelle P.; Meuren, Lana M.; Coelho, Sharton V. A.; Lucas, Carolina G. de Oliveira; Mustafá, Yasmin M.; Lemos Matassoli, Flavio; Silveira, Paola P.; Frost, Paula S.; Pezzuto, Paula; Ribeiro, Milene R.; Tanuri, Amilcar; Nogueira, Mauricio L.; Campanati, Loraine; Bozza, Marcelo T.; Paula Neto, Heitor A.; Pimentel-Coelho, Pedro M.; Figueiredo, Claudia P.; de Aguiar, Renato S.; de Arruda, Luciana B.

    2017-01-01

    Zika virus (ZIKV) has been associated to central nervous system (CNS) harm, and virus was detected in the brain and cerebrospinal fluids of microcephaly and meningoencephalitis cases. However, the mechanism by which the virus reaches the CNS is unclear. Here, we addressed the effects of ZIKV replication in human brain microvascular endothelial cells (HBMECs), as an in vitro model of blood brain barrier (BBB), and evaluated virus extravasation and BBB integrity in an in vivo mouse experimental model. HBMECs were productively infected by African and Brazilian ZIKV strains (ZIKVMR766 and ZIKVPE243), which induce increased production of type I and type III IFN, inflammatory cytokines and chemokines. Infection with ZIKVMR766 promoted earlier cellular death, in comparison to ZIKVPE243, but infection with either strain did not result in enhanced endothelial permeability. Despite the maintenance of endothelial integrity, infectious virus particles crossed the monolayer by endocytosis/exocytosis-dependent replication pathway or by transcytosis. Remarkably, both viruses' strains infected IFNAR deficient mice, with high viral load being detected in the brains, without BBB disruption, which was only detected at later time points after infection. These data suggest that ZIKV infects and activates endothelial cells, and might reach the CNS through basolateral release, transcytosis or transinfection processes. These findings further improve the current knowledge regarding ZIKV dissemination pathways. PMID:29312238

  9. Differentiation state determines neural effects on microvascular endothelial cells

    International Nuclear Information System (INIS)

    Muffley, Lara A.; Pan, Shin-Chen; Smith, Andria N.; Ga, Maricar; Hocking, Anne M.; Gibran, Nicole S.

    2012-01-01

    Growing evidence indicates that nerves and capillaries interact paracrinely in uninjured skin and cutaneous wounds. Although mature neurons are the predominant neural cell in the skin, neural progenitor cells have also been detected in uninjured adult skin. The aim of this study was to characterize differential paracrine effects of neural progenitor cells and mature sensory neurons on dermal microvascular endothelial cells. Our results suggest that neural progenitor cells and mature sensory neurons have unique secretory profiles and distinct effects on dermal microvascular endothelial cell proliferation, migration, and nitric oxide production. Neural progenitor cells and dorsal root ganglion neurons secrete different proteins related to angiogenesis. Specific to neural progenitor cells were dipeptidyl peptidase-4, IGFBP-2, pentraxin-3, serpin f1, TIMP-1, TIMP-4 and VEGF. In contrast, endostatin, FGF-1, MCP-1 and thrombospondin-2 were specific to dorsal root ganglion neurons. Microvascular endothelial cell proliferation was inhibited by dorsal root ganglion neurons but unaffected by neural progenitor cells. In contrast, microvascular endothelial cell migration in a scratch wound assay was inhibited by neural progenitor cells and unaffected by dorsal root ganglion neurons. In addition, nitric oxide production by microvascular endothelial cells was increased by dorsal root ganglion neurons but unaffected by neural progenitor cells. -- Highlights: ► Dorsal root ganglion neurons, not neural progenitor cells, regulate microvascular endothelial cell proliferation. ► Neural progenitor cells, not dorsal root ganglion neurons, regulate microvascular endothelial cell migration. ► Neural progenitor cells and dorsal root ganglion neurons do not effect microvascular endothelial tube formation. ► Dorsal root ganglion neurons, not neural progenitor cells, regulate microvascular endothelial cell production of nitric oxide. ► Neural progenitor cells and dorsal root

  10. Brain endothelial dysfunction in cerebral adrenoleukodystrophy.

    Science.gov (United States)

    Musolino, Patricia L; Gong, Yi; Snyder, Juliet M T; Jimenez, Sandra; Lok, Josephine; Lo, Eng H; Moser, Ann B; Grabowski, Eric F; Frosch, Matthew P; Eichler, Florian S

    2015-11-01

    See Aubourg (doi:10.1093/awv271) for a scientific commentary on this article.X-linked adrenoleukodystrophy is caused by mutations in the ABCD1 gene leading to accumulation of very long chain fatty acids. Its most severe neurological manifestation is cerebral adrenoleukodystrophy. Here we demonstrate that progressive inflammatory demyelination in cerebral adrenoleukodystrophy coincides with blood-brain barrier dysfunction, increased MMP9 expression, and changes in endothelial tight junction proteins as well as adhesion molecules. ABCD1, but not its closest homologue ABCD2, is highly expressed in human brain microvascular endothelial cells, far exceeding its expression in the systemic vasculature. Silencing of ABCD1 in human brain microvascular endothelial cells causes accumulation of very long chain fatty acids, but much later than the immediate upregulation of adhesion molecules and decrease in tight junction proteins. This results in greater adhesion and transmigration of monocytes across the endothelium. PCR-array screening of human brain microvascular endothelial cells after ABCD1 silencing revealed downregulation of both mRNA and protein levels of the transcription factor c-MYC (encoded by MYC). Interestingly, MYC silencing mimicked the effects of ABCD1 silencing on CLDN5 and ICAM1 without decreasing the levels of ABCD1 protein itself. Together, these data demonstrate that ABCD1 deficiency induces significant alterations in brain endothelium via c-MYC and may thereby contribute to the increased trafficking of leucocytes across the blood-brain barrier as seen in cerebral adrenouleukodystrophy. © The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  11. Verocytotoxin-induced apoptosis of human microvascular endothelial cells.

    Science.gov (United States)

    Pijpers, A H; van Setten, P A; van den Heuvel, L P; Assmann, K J; Dijkman, H B; Pennings, A H; Monnens, L A; van Hinsbergh, V W

    2001-04-01

    The pathogenesis of the epidemic form of hemolytic uremic syndrome is characterized by endothelial cell damage. In this study, the role of apoptosis in verocytotoxin (VT)-mediated endothelial cell death in human glomerular microvascular endothelial cells (GMVEC), human umbilical vein endothelial cells, and foreskin microvascular endothelial cells (FMVEC) was investigated. VT induced apoptosis in GMVEC and human umbilical vein endothelial cells when the cells were prestimulated with the inflammatory mediator tumor necrosis factor-alpha (TNF-alpha). FMVEC displayed strong binding of VT and high susceptibility to VT under basal conditions, which made them suitable for the study of VT-induced apoptosis without TNF-alpha interference. On the basis of functional (flow cytometry and immunofluorescence microscopy using FITC-conjugated annexin V and propidium iodide), morphologic (transmission electron microscopy), and molecular (agarose gel electrophoresis of cellular DNA fragments) criteria, it was documented that VT induced programmed cell death in microvascular endothelial cells in a dose- and time-dependent manner. Furthermore, whereas partial inhibition of protein synthesis by VT was associated with a considerable number of apoptotic cells, comparable inhibition of protein synthesis by cycloheximide was not. This suggests that additional pathways, independent of protein synthesis inhibition, may be involved in VT-mediated apoptosis in microvascular endothelial cells. Specific inhibition of caspases by Ac-Asp-Glu-Val-Asp-CHO, but not by Ac-Tyr-Val-Ala-Asp-CHO, was accompanied by inhibition of VT-induced apoptosis in FMVEC and TNF-alpha-treated GMVEC. These data indicate that VT can induce apoptosis in human microvascular endothelial cells.

  12. 2-Chlorohexadecanoic acid induces ER stress and mitochondrial dysfunction in brain microvascular endothelial cells

    Directory of Open Access Journals (Sweden)

    Eva Bernhart

    2018-05-01

    Full Text Available Peripheral leukocytes induce blood-brain barrier (BBB dysfunction through the release of cytotoxic mediators. These include hypochlorous acid (HOCl that is formed via the myeloperoxidase-H2O2-chloride system of activated phagocytes. HOCl targets the endogenous pool of ether phospholipids (plasmalogens generating chlorinated inflammatory mediators like e.g. 2-chlorohexadecanal and its conversion product 2-chlorohexadecanoic acid (2-ClHA. In the cerebrovasculature these compounds inflict damage to brain microvascular endothelial cells (BMVEC that form the morphological basis of the BBB. To follow subcellular trafficking of 2-ClHA we synthesized a ‘clickable’ alkyne derivative (2-ClHyA that phenocopied the biological activity of the parent compound. Confocal and superresolution structured illumination microscopy revealed accumulation of 2-ClHyA in the endoplasmic reticulum (ER and mitochondria of human BMVEC (hCMEC/D3 cell line. 2-ClHA and its alkyne analogue interfered with protein palmitoylation, induced ER-stress markers, reduced the ER ATP content, and activated transcription and secretion of interleukin (IL−6 as well as IL-8. 2-ClHA disrupted the mitochondrial membrane potential and induced procaspase-3 and PARP cleavage. The protein kinase R-like ER kinase (PERK inhibitor GSK2606414 suppressed 2-ClHA-mediated activating transcription factor 4 synthesis and IL-6/8 secretion, but showed no effect on endothelial barrier dysfunction and cleavage of procaspase-3. Our data indicate that 2-ClHA induces potent lipotoxic responses in brain endothelial cells and could have implications in inflammation-induced BBB dysfunction.

  13. The anti-apoptotic effect of fluid mechanics preconditioning by cells membrane and mitochondria in rats brain microvascular endothelial cells.

    Science.gov (United States)

    Tian, Shan; Zhu, Fengping; Hu, Ruiping; Tian, Song; Chen, Xingxing; Lou, Dan; Cao, Bing; Chen, Qiulei; Li, Bai; Li, Fang; Bai, Yulong; Wu, Yi; Zhu, Yulian

    2018-01-01

    Exercise preconditioning is a simple and effective way to prevent ischemia. This paper further provided the mechanism in hemodynamic aspects at the cellular level. To study the anti-apoptotic effects of fluid mechanics preconditioning, Cultured rats brain microvascular endothelial cells were given fluid intervention in a parallel plate flow chamber before oxygen glucose deprivation. It showed that fluid mechanics preconditioning could inhibit the apoptosis of endothelial cells, and this process might be mediated by the shear stress activation of Tie-2 on cells membrane surface and Bcl-2 on the mitochondria surface. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. High glucose, glucose fluctuation and carbonyl stress enhance brain microvascular endothelial barrier dysfunction: Implications for diabetic cerebral microvasculature.

    Science.gov (United States)

    Li, Wei; Maloney, Ronald E; Aw, Tak Yee

    2015-08-01

    We previously demonstrated that in normal glucose (5mM), methylglyoxal (MG, a model of carbonyl stress) induced brain microvascular endothelial cell (IHEC) dysfunction that was associated with occludin glycation and prevented by N-acetylcysteine (NAC). Herein, we investigated the impact of high glucose and low GSH, conditions that mimicked the diabetic state, on MG-induced IHEC dysfunction. MG-induced loss of transendothelial electrical resistance (TEER) was potentiated in IHECs cultured for 7 or 12 days in 25 mM glucose (hyperglycemia); moreover, barrier function remained disrupted 6h after cell transfer to normal glucose media (acute glycemic fluctuation). Notably, basal occludin glycation was elevated under these glycemic states. TEER loss was exaggerated by inhibition of glutathione (GSH) synthesis and abrogated by NAC, which corresponded to GSH decreases and increases, respectively. Significantly, glyoxalase II activity was attenuated in hyperglycemic cells. Moreover, hyperglycemia and GSH inhibition increased MG accumulation, consistent with a compromised capacity for MG elimination. α-Oxoaldehydes (MG plus glyoxal) levels were elevated in streptozotocin-induced diabetic rat plasma. Immunohistochemistry revealed a prevalence of MG-positive, but fewer occludin-positive microvessels in the diabetic brain in vivo, and Western analysis confirmed an increase in MG-occludin adducts. These results provide the first evidence that hyperglycemia and acute glucose fluctuation promote MG-occludin formation and exacerbate brain microvascular endothelial dysfunction. Low occludin expression and high glycated-occludin contents in diabetic brain in vivo are factors that would contribute to the dysfunction of the cerebral microvasculature during diabetes. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  15. High glucose, glucose fluctuation and carbonyl stress enhance brain microvascular endothelial barrier dysfunction: Implications for diabetic cerebral microvasculature

    Directory of Open Access Journals (Sweden)

    Wei Li

    2015-08-01

    Full Text Available We previously demonstrated that in normal glucose (5 mM, methylglyoxal (MG, a model of carbonyl stress induced brain microvascular endothelial cell (IHEC dysfunction that was associated with occludin glycation and prevented by N-acetylcysteine (NAC. Herein, we investigated the impact of high glucose and low GSH, conditions that mimicked the diabetic state, on MG-induced IHEC dysfunction. MG-induced loss of transendothelial electrical resistance (TEER was potentiated in IHECs cultured for 7 or 12 days in 25 mM glucose (hyperglycemia; moreover, barrier function remained disrupted 6 h after cell transfer to normal glucose media (acute glycemic fluctuation. Notably, basal occludin glycation was elevated under these glycemic states. TEER loss was exaggerated by inhibition of glutathione (GSH synthesis and abrogated by NAC, which corresponded to GSH decreases and increases, respectively. Significantly, glyoxalase II activity was attenuated in hyperglycemic cells. Moreover, hyperglycemia and GSH inhibition increased MG accumulation, consistent with a compromised capacity for MG elimination. α-Oxoaldehydes (MG plus glyoxal levels were elevated in streptozotocin-induced diabetic rat plasma. Immunohistochemistry revealed a prevalence of MG-positive, but fewer occludin-positive microvessels in the diabetic brain in vivo, and Western analysis confirmed an increase in MG–occludin adducts. These results provide the first evidence that hyperglycemia and acute glucose fluctuation promote MG–occludin formation and exacerbate brain microvascular endothelial dysfunction. Low occludin expression and high glycated-occludin contents in diabetic brain in vivo are factors that would contribute to the dysfunction of the cerebral microvasculature during diabetes.

  16. Endothelial progenitor cells physiology and metabolic plasticity in brain angiogenesis and blood-brain barrier modeling

    Directory of Open Access Journals (Sweden)

    Natalia Malinovskaya

    2016-12-01

    Full Text Available Currently, there is a considerable interest to the assessment of blood-brain barrier (BBB development as a part of cerebral angiogenesis developmental program. Embryonic and adult angiogenesis in the brain is governed by the coordinated activity of endothelial progenitor cells, brain microvascular endothelial cells, and non-endothelial cells contributing to the establishment of the BBB (pericytes, astrocytes, neurons. Metabolic and functional plasticity of endothelial progenitor cells controls their timely recruitment, precise homing to the brain microvessels, and efficient support of brain angiogenesis. Deciphering endothelial progenitor cells physiology would provide novel engineering approaches to establish adequate microfluidically-supported BBB models and brain microphysiological systems for translational studies.

  17. Glial cell ceruloplasmin and hepcidin differentially regulate iron efflux from brain microvascular endothelial cells.

    Science.gov (United States)

    McCarthy, Ryan C; Kosman, Daniel J

    2014-01-01

    We have used an in vitro model system to probe the iron transport pathway across the brain microvascular endothelial cells (BMVEC) of the blood-brain barrier (BBB). This model consists of human BMVEC (hBMVEC) and C6 glioma cells (as an astrocytic cell line) grown in a transwell, a cell culture system commonly used to quantify metabolite flux across a cell-derived barrier. We found that iron efflux from hBMVEC through the ferrous iron permease ferroportin (Fpn) was stimulated by secretion of the soluble form of the multi-copper ferroxidase, ceruloplasmin (sCp) from the co-cultured C6 cells. Reciprocally, expression of sCp mRNA in the C6 cells was increased by neighboring hBMVEC. In addition, data indicate that C6 cell-secreted hepcidin stimulates internalization of hBMVEC Fpn but only when the end-feet projections characteristic of this glia-derived cell line are proximal to the endothelial cells. This hepcidin-dependent loss of Fpn correlated with knock-down of iron efflux from the hBMVEC; this result was consistent with the mechanism by which hepcidin regulates iron efflux in mammalian cells. In summary, the data support a model of iron trafficking across the BBB in which the capillary endothelium induce the underlying astrocytes to produce the ferroxidase activity needed to support Fpn-mediated iron efflux. Reciprocally, astrocyte proximity modulates the effective concentration of hepcidin at the endothelial cell membrane and thus the surface expression of hBMVEC Fpn. These results are independent of the source of hBMVEC iron (transferrin or non-transferrin bound) indicating that the model developed here is broadly applicable to brain iron homeostasis.

  18. Interleukin 6-Mediated Endothelial Barrier Disturbances Can Be Attenuated by Blockade of the IL6 Receptor Expressed in Brain Microvascular Endothelial Cells.

    Science.gov (United States)

    Blecharz-Lang, Kinga G; Wagner, Josephin; Fries, Alexa; Nieminen-Kelhä, Melina; Rösner, Jörg; Schneider, Ulf C; Vajkoczy, Peter

    2018-02-10

    Compromised blood-brain barrier (BBB) by dysregulation of cellular junctions is a hallmark of many cerebrovascular disorders due to the pro-inflammatory cytokines action. Interleukin 6 (IL6) is implicated in inflammatory processes and in secondary brain injury after subarachnoid hemorrhage (SAH) but its role in the maintenance of cerebral endothelium still requires a precise elucidation. Although IL6 has been shown to exert pro-inflammatory action on brain microvascular endothelial cells (ECs), the expression of one of the IL6 receptors, the IL6R is controversially discussed. In attempt to reach more clarity in this issue, we present here an evident baseline expression of the IL6R in BBB endothelium in vivo and in an in vitro model of the BBB, the cEND cell line. A significantly increased expression of IL6R and its ligand was observed in BBB capillaries 2 days after experimental SAH in mice. In vitro, we saw IL6 administration resulting in an intracellular and extracellular elevation of IL6 protein, which was accompanied by a reduced expression of tight and adherens junctions, claudin-5, occludin, and vascular-endothelial (VE-) cadherin. By functional assays, we could demonstrate IL6-incubated brain ECs to lose their endothelial integrity that can be attenuated by inhibiting the IL6R. Blockade of the IL6R by a neutralizing antibody has reconstituted the intercellular junction expression to the control level and caused a restoration of the transendothelial electrical resistance of the cEND cell monolayer. Our findings add depth to the current understanding of the involvement of the endothelial IL6R in the loss of EC integrity implicating potential therapy options.

  19. Endothelial glycocalyx dysfunction in disease: albuminuria and increased microvascular permeability.

    Science.gov (United States)

    Salmon, Andrew H J; Satchell, Simon C

    2012-03-01

    Appreciation of the glomerular microcirculation as a specialized microcirculatory bed, rather than as an entirely separate entity, affords important insights into both glomerular and systemic microvascular pathophysiology. In this review we compare regulation of permeability in systemic and glomerular microcirculations, focusing particularly on the role of the endothelial glycocalyx, and consider the implications for disease processes. The luminal surface of vascular endothelium throughout the body is covered with endothelial glycocalyx, comprising surface-anchored proteoglycans, supplemented with adsorbed soluble proteoglycans, glycosaminoglycans and plasma constituents. In both continuous and fenestrated microvessels, this endothelial glycocalyx provides resistance to the transcapillary escape of water and macromolecules, acting as an integral component of the multilayered barrier provided by the walls of these microvessels (ie acting in concert with clefts or fenestrae across endothelial cell layers, basement membranes and pericytes). Dysfunction of any of these capillary wall components, including the endothelial glycocalyx, can disrupt normal microvascular permeability. Because of its ubiquitous nature, damage to the endothelial glycocalyx alters the permeability of multiple capillary beds: in the glomerulus this is clinically apparent as albuminuria. Generalized damage to the endothelial glycocalyx can therefore manifest as both albuminuria and increased systemic microvascular permeability. This triad of altered endothelial glycocalyx, albuminuria and increased systemic microvascular permeability occurs in a number of important diseases, such as diabetes, with accumulating evidence for a similar phenomenon in ischaemia-reperfusion injury and infectious disease. The detection of albuminuria therefore has implications for the function of the microcirculation as a whole. The importance of the endothelial glycocalyx for other aspects of vascular function

  20. Microvascular endothelial function and cognitive performance: The ELSA-Brasil cohort study.

    Science.gov (United States)

    Brant, Luisa; Bos, Daniel; Araujo, Larissa Fortunato; Ikram, M Arfan; Ribeiro, Antonio Lp; Barreto, Sandhi M

    2018-06-01

    Impaired microvascular endothelial function may be implicated in the etiology of cognitive decline. Yet, current data on this association are inconsistent. Our objective is to investigate the relation of microvascular endothelial function to cognitive performance in the ELSA-Brasil cohort study. A total of 1521 participants from ELSA-Brasil free of dementia underwent peripheral arterial tonometry (PAT) to quantify microvascular endothelial function (PAT-ratio and mean baseline pulse amplitude (BPA)) and cognitive tests that covered the domains of memory, verbal fluency, and executive function at baseline. Cognitive tests in participants aged 55 years old and above were repeated during the second examination (mean follow-up: 3.5 (0.3) years). Linear regression and generalized linear models were used to evaluate the association between endothelial function, global cognitive performance, and performance on specific cognitive domains. In unadjusted cross-sectional analyses, we found that BPA and PAT-ratio were associated with worse global cognitive performance (mean difference for BPA: -0.07, 95% CI: -0.11; -0.03, p<0.01; mean difference for PAT-ratio: 0.11, 95% CI: 0.01; 0.20, p=0.02), worse performance on learning, recall, and word recognition tests (BPA: -0.87, 95% CI: -1.21; -0.52, p<0.01; PAT-ratio: 1.58, 95% CI: 0.80; 2.36, p<0.01), and only BPA was associated with worse performance in verbal fluency tests (-0.70, 95% CI: -1.19; -0.21, p<0.01). Adjustments for age, sex, and level of education rendered the associations statistically non-significant. Longitudinally, there was no association between microvascular endothelial and cognitive functions. The associations between microvascular endothelial function and cognition are explained by age, sex, and educational level. Measures of microvascular endothelial function may be of limited value with regard to preclinical cognitive deficits.

  1. Morphine induces expression of platelet-derived growth factor in human brain microvascular endothelial cells: implication for vascular permeability.

    Directory of Open Access Journals (Sweden)

    Hongxiu Wen

    Full Text Available Despite the advent of antiretroviral therapy, complications of HIV-1 infection with concurrent drug abuse are an emerging problem. Morphine, often abused by HIV-infected patients, is known to accelerate neuroinflammation associated with HIV-1 infection. Detailed molecular mechanisms of morphine action however, remain poorly understood. Platelet-derived growth factor (PDGF has been implicated in a number of pathological conditions, primarily due to its potent mitogenic and permeability effects. Whether morphine exposure results in enhanced vascular permeability in brain endothelial cells, likely via induction of PDGF, remains to be established. In the present study, we demonstrated morphine-mediated induction of PDGF-BB in human brain microvascular endothelial cells, an effect that was abrogated by the opioid receptor antagonist-naltrexone. Pharmacological blockade (cell signaling and loss-of-function (Egr-1 approaches demonstrated the role of mitogen-activated protein kinases (MAPKs, PI3K/Akt and the downstream transcription factor Egr-1 respectively, in morphine-mediated induction of PDGF-BB. Functional significance of increased PDGF-BB manifested as increased breach of the endothelial barrier as evidenced by decreased expression of the tight junction protein ZO-1 in an in vitro model system. Understanding the regulation of PDGF expression may provide insights into the development of potential therapeutic targets for intervention of morphine-mediated neuroinflammation.

  2. Exposure to lipopolysaccharide and/or unconjugated bilirubin impair the integrity and function of brain microvascular endothelial cells.

    Directory of Open Access Journals (Sweden)

    Filipa L Cardoso

    Full Text Available BACKGROUND: Sepsis and jaundice are common conditions in newborns that can lead to brain damage. Though lipopolysaccharide (LPS is known to alter the integrity of the blood-brain barrier (BBB, little is known on the effects of unconjugated bilirubin (UCB and even less on the joint effects of UCB and LPS on brain microvascular endothelial cells (BMEC. METHODOLOGY/PRINCIPAL FINDINGS: Monolayers of primary rat BMEC were treated with 1 µg/ml LPS and/or 50 µM UCB, in the presence of 100 µM human serum albumin, for 4 or 24 h. Co-cultures of BMEC with astroglial cells, a more complex BBB model, were used in selected experiments. LPS led to apoptosis and UCB induced both apoptotic and necrotic-like cell death. LPS and UCB led to inhibition of P-glycoprotein and activation of matrix metalloproteinases-2 and -9 in mono-cultures. Transmission electron microscopy evidenced apoptotic bodies, as well as damaged mitochondria and rough endoplasmic reticulum in BMEC by either insult. Shorter cell contacts and increased caveolae-like invaginations were noticeable in LPS-treated cells and loss of intercellular junctions was observed upon treatment with UCB. Both compounds triggered impairment of endothelial permeability and transendothelial electrical resistance both in mono- and co-cultures. The functional changes were confirmed by alterations in immunostaining for junctional proteins β-catenin, ZO-1 and claudin-5. Enlargement of intercellular spaces, and redistribution of junctional proteins were found in BMEC after exposure to LPS and UCB. CONCLUSIONS: LPS and/or UCB exert direct toxic effects on BMEC, with distinct temporal profiles and mechanisms of action. Therefore, the impairment of brain endothelial integrity upon exposure to these neurotoxins may favor their access to the brain, thus increasing the risk of injury and requiring adequate clinical management of sepsis and jaundice in the neonatal period.

  3. The protective role of isorhamnetin on human brain microvascular endothelial cells from cytotoxicity induced by methylglyoxal and oxygen-glucose deprivation.

    Science.gov (United States)

    Li, Wenlu; Chen, Zhigang; Yan, Min; He, Ping; Chen, Zhong; Dai, Haibin

    2016-02-01

    As the first target of stroke, cerebral endothelial cells play a key role in brain vascular repair and maintenance, and their function is impeded in diabetes. Methylglyoxal (MGO), a reactive dicarbonyl produced during glucose metabolism, accumulates in diabetic patients. MGO and MGO-induced advanced glycation end-products (AGEs) could ameliorate stroke-induced brain vascular damage, closely related with ECs dysfunction. Using MGO plus oxygen-glucose deprivation (OGD) to mimic diabetic stroke, we reported the protective effect of isorhamnetin on OGD-induced cytotoxicity after MGO treatment on primary human brain microvascular endothelial cells (HBMEC) and explored the underlying mechanisms. Treatment of MGO for 24 h significantly enhanced 3-h OGD-induced HBMEC toxic effect, which was inhibited by pretreatment of isorhamnetin (100 μmol/L). Moreover, the protective effect of isorhamnetin is multiple function dependent, which includes anti-inflammation, anti-oxidative stress and anti-apoptosis effects. Besides its well-known inhibition on the mitochondria-dependent or intrinsic apoptotic pathway, isorhamnetin also reduced activation of the extrinsic apoptotic pathway, as characterized by the decreased expression and activity of caspase 3 and caspase 8. Furthermore, pretreatment with isorhamnetin specifically inhibited FAS/FASL expression and suppressed nuclear factor-kappa B nuclear translocation. Taken together, our results indicated that isorhamnetin protected against OGD-induced cytotoxicity after MGO treatment in cultured HBMEC due to its multiple protective effects and could inhibit Fas-mediated extrinsic apoptosis. Therefore, isorhamnetin is a promising reagent for the treatment of hyperglycemia and ischemia-induced cerebral vascular degeneration. A proposed model of the potential protective mechanism of isorhamnetin, a metabolite of quercetin, on methylglyoxal (MGO) treatment plus oxygen-glucose deprivation (OGD) exposure-induced cytotoxicity in cultured human

  4. Adhesive properties of Enterobacter sakazakii to human epithelial and brain microvascular endothelial cells

    Directory of Open Access Journals (Sweden)

    Pospischil Andreas

    2006-06-01

    Full Text Available Abstract Background Enterobacter sakazakii is an opportunistic pathogen that has been associated with sporadic cases and outbreaks causing meningitis, necrotizing enterocolitis and sepsis especially in neonates. However, up to now little is known about the mechanisms of pathogenicity in E. sakazakii. A necessary state in the successful colonization, establishment and ultimately production of disease by microbial pathogens is the ability to adhere to host surfaces such as mucous membranes, gastric and intestinal epithelial or endothelial tissue. This study examined for the first time the adherence ability of 50 E. sakazakii strains to the two epithelial cell lines HEp-2 and Caco-2, as well as the brain microvascular endothelial cell line HBMEC. Furthermore, the effects of bacterial culture conditions on the adherence behaviour were investigated. An attempt was made to characterize the factors involved in adherence. Results Two distinctive adherence patterns, a diffuse adhesion and the formation of localized clusters of bacteria on the cell surface could be distinguished on all three cell lines. In some strains, a mixture of both patterns was observed. Adherence was maximal during late exponential phase, and increased with higher MOI. The adhesion capacity of E. sakazakii to HBMEC cells was affected by the addition of blood to the bacteria growth medium. Mannose, hemagglutination, trypsin digestion experiments and transmission electron microscopy suggested that the adhesion of E. sakazakii to the epithelial and endothelial cells is mainly non-fimbrial based. Conclusion Adherence experiments show heterogeneity within different E. sakazakii strains. In agreement with studies on E. cloacae, we found no relationship between the adhesive capacities in E. sakazakii and the eventual production of specific fimbriae. Further studies will have to be carried out in order to determine the adhesin(s involved in the interaction of E. sakazakii with cells and to

  5. The effects of anti-obesity intervention with orlistat and sibutramine on microvascular endothelial function.

    Science.gov (United States)

    Al-Tahami, Belqes Abdullah Mohammad; Ismail, Ab Aziz Al-Safi; Bee, Yvonne Tee Get; Awang, Siti Azima; Salha Wan Abdul Rani, Wan Rimei; Sanip, Zulkefli; Rasool, Aida Hanum Ghulam

    2015-01-01

    Obesity is associated with impaired microvascular endothelial function. We aimed to determine the effects of orlistat and sibutramine treatment on microvascular endothelial function, anthropometric and lipid profile, blood pressure (BP), and heart rate (HR). 76 subjects were recruited and randomized to receive orlistat 120 mg three times daily or sibutramine 10 mg daily for 9 months. Baseline weight, BMI, BP, HR and lipid profile were taken. Microvascular endothelial function was assessed using laser Doppler fluximetry and iontophoresis process. Maximum change (max), percent change (% change) and peak flux (peak) in perfusion to acetylcholine (ACh) and sodium nitroprusside (SNP) iontophoresis were used to quantify endothelium dependent and independent vasodilatations. 24 subjects in both groups completed the trial. After treatment, weight and BMI were decreased for both groups. AChmax, ACh % change and ACh peak were increased in orlistat-treated group but no difference was observed for sibutramine-treated group. BP and total cholesterol (TC) were reduced for orlistat-treated group. HR was reduced for orlistat-treated group but was increased in sibutramine-treated group. 9 months treatment with orlistat significantly improved microvascular endothelial function. This was associated with reductions in weight, BMI, BP, HR, TC and low density lipoprotein cholesterol. No effect was seen in microvascular endothelial function with sibutramine.

  6. Assessment of macrovascular endothelial function using pulse wave analysis and its association with microvascular reactivity in healthy subjects.

    Science.gov (United States)

    Ibrahim, N N I N; Rasool, A H G

    2017-08-01

    Pulse wave analysis (PWA) and laser Doppler fluximetry (LDF) are non-invasive methods of assessing macrovascular endothelial function and microvascular reactivity respectively. The aim of this study was to assess the correlation between macrovascular endothelial function assessed by PWA and microvascular reactivity assessed by LDF. 297 healthy and non-smoking subjects (159 females, mean age (±SD) 23.56 ± 4.54 years) underwent microvascular reactivity assessment using LDF followed by macrovascular endothelial function assessments using PWA. Pearson's correlation showed no correlation between macrovascular endothelial function and microvascular reactivity (r = -0.10, P = 0.12). There was no significant correlation between macrovascular endothelial function assessed by PWA and microvascular reactivity assessed by LDF in healthy subjects. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  7. ZO-1 expression is suppressed by GM-CSF via miR-96/ERG in brain microvascular endothelial cells.

    Science.gov (United States)

    Zhang, Hu; Zhang, Shuhong; Zhang, Jilin; Liu, Dongxin; Wei, Jiayi; Fang, Wengang; Zhao, Weidong; Chen, Yuhua; Shang, Deshu

    2018-05-01

    The level of granulocyte-macrophage colony-stimulating factor (GM-CSF) increases in some disorders such as vascular dementia, Alzheimer's disease, and multiple sclerosis. We previously reported that in Alzheimer's disease patients, a high level of GM-CSF in the brain parenchyma downregulated expression of ZO-1, a blood-brain barrier tight junction protein, and facilitated the infiltration of peripheral monocytes across the blood-brain barrier. However, the molecular mechanism underlying regulation of ZO-1 expression by GM-CSF is unclear. Herein, we found that the erythroblast transformation-specific (ETS) transcription factor ERG cooperated with the proto-oncogene protein c-MYC in regulation of ZO-1 transcription in brain microvascular endothelial cells (BMECs). The ERG expression was suppressed by miR-96 which was increased by GM-CSF through the phosphoinositide-3 kinase (PI3K)/Akt pathway. Inhibition of miR-96 prevented ZO-1 down-regulation induced by GM-CSF both in vitro and in vivo. Our results revealed the mechanism of ZO-1 expression reduced by GM-CSF, and provided a potential target, miR-96, which could block ZO-1 down-regulation caused by GM-CSF in BMECs.

  8. Is endothelial microvascular function equally impaired among patients with chronic Chagas and ischemic cardiomyopathy?

    Science.gov (United States)

    Borges, Juliana Pereira; Mendes, Fernanda de Souza Nogueira Sardinha; Lopes, Gabriella de Oliveira; Sousa, Andréa Silvestre de; Mediano, Mauro Felippe Felix; Tibiriçá, Eduardo

    2018-08-15

    Chronic Chagas cardiomyopathy (CCC) and cardiomyopathies due to other etiologies involve differences in pathophysiological pathways that are still unclear. Systemic microvascular abnormalities are associated with the pathogenesis of ischemic heart disease. However, systemic microvascular endothelial function in CCC remains to be elucidated. Thus, we compared the microvascular endothelial function of patients presenting with CCC to those with ischemic cardiomyopathy disease. Microvascular reactivity was assessed in 21 patients with cardiomyopathy secondary to Chagas disease, 21 patients with cardiomyopathy secondary to ischemic disease and 21 healthy controls. Microvascular blood flow was assessed in the skin of the forearm using laser speckle contrast imaging coupled with iontophoresis of acetylcholine (ACh). Peak increase in forearm blood flow with ACh iontophoresis in relation to baseline was greater in healthy controls than in patients with heart disease (controls: 162.7 ± 58.4% vs. ischemic heart disease: 74.1 ± 48.3% and Chagas: 85.1 ± 68.1%; p < 0.0001). Patients with Chagas and ischemic cardiomyopathy presented similar ACh-induced changes from baseline in skin blood flow (p = 0.55). Endothelial microvascular function was equally impaired among patients with CCC and ischemic cardiomyopathy. Copyright © 2018 Elsevier B.V. All rights reserved.

  9. Novel effects of edaravone on human brain microvascular endothelial cells revealed by a proteomic approach.

    Science.gov (United States)

    Onodera, Hidetaka; Arito, Mitsumi; Sato, Toshiyuki; Ito, Hidemichi; Hashimoto, Takuo; Tanaka, Yuichiro; Kurokawa, Manae S; Okamoto, Kazuki; Suematsu, Naoya; Kato, Tomohiro

    2013-10-09

    Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) is a free radical scavenger used for acute ischemic stroke. However, it is not known whether edaravone works only as a free radical scavenger or possess other pharmacological actions. Therefore, we elucidated the effects of edaravone on human brain microvascular endothelial cells (HBMECs) by 2 dimensional fluorescence difference gel electrophoresis (2D-DIGE). We found 38 protein spots the intensity of which was significantly altered 1.3 fold on average (pedaravone treatment and successfully identified 17 proteins of those. Four of those 17 proteins were cytoskeleton proteins or cytoskeleton-regulating proteins. Therefore, we subsequently investigated the change of size and shape of the cells, the actin network, and the tight junction of HBMEC by immunocytochemistry. As a result, most edaravone-treated HBMECs became larger and rounder compared with those that were not treated. Furthermore, edaravone-treated HBMECs formed gathering zona occludens (ZO)-1, a tight junction protein, along the junction of the cells. In addition, we found that edaravone suppressed interleukin (IL)-1β-induced secretion of monocyte chemoattractant protein-1 (MCP-1), which was reported to increase cell permeability. We found a novel function of edaravone is the promotion of tight junction formations of vascular endothelial cells partly via the down-regulation of MCP-1 secretion. These data provide fundamental and useful information in the clinical use of edaravone in patients with cerebral vascular diseases. © 2013 Elsevier B.V. All rights reserved.

  10. Propionyl-L-Carnitine Enhances Wound Healing and Counteracts Microvascular Endothelial Cell Dysfunction.

    Directory of Open Access Journals (Sweden)

    Maria Giovanna Scioli

    Full Text Available Impaired wound healing represents a high cost for health care systems. Endothelial dysfunction characterizes dermal microangiopathy and contributes to delayed wound healing and chronic ulcers. Endothelial dysfunction impairs cutaneous microvascular blood flow by inducing an imbalance between vasorelaxation and vasoconstriction as a consequence of reduced nitric oxide (NO production and the increase of oxidative stress and inflammation. Propionyl-L-carnitine (PLC is a natural derivative of carnitine that has been reported to ameliorate post-ischemic blood flow recovery.We investigated the effects of PLC in rat skin flap and cutaneous wound healing. A daily oral PLC treatment improved skin flap viability and associated with reactive oxygen species (ROS reduction, inducible nitric oxide synthase (iNOS and NO up-regulation, accelerated wound healing and increased capillary density, likely favoring dermal angiogenesis by up-regulation for iNOS, vascular endothelial growth factor (VEGF, placental growth factor (PlGF and reduction of NADPH-oxidase 4 (Nox4 expression. In serum-deprived human dermal microvascular endothelial cell cultures, PLC ameliorated endothelial dysfunction by increasing iNOS, PlGF, VEGF receptors 1 and 2 expression and NO level. In addition, PLC counteracted serum deprivation-induced impairment of mitochondrial β-oxidation, Nox4 and cellular adhesion molecule (CAM expression, ROS generation and leukocyte adhesion. Moreover, dermal microvascular endothelial cell dysfunction was prevented by Nox4 inhibition. Interestingly, inhibition of β-oxidation counteracted the beneficial effects of PLC on oxidative stress and endothelial dysfunction.PLC treatment improved rat skin flap viability, accelerated wound healing and dermal angiogenesis. The beneficial effects of PLC likely derived from improvement of mitochondrial β-oxidation and reduction of Nox4-mediated oxidative stress and endothelial dysfunction. Antioxidant therapy and

  11. Endothelial Progenitor Cells in Diabetic Microvascular Complications: Friends or Foes?

    Science.gov (United States)

    Yu, Cai-Guo; Zhang, Ning; Yuan, Sha-Sha; Ma, Yan; Yang, Long-Yan; Feng, Ying-Mei; Zhao, Dong

    2016-01-01

    Despite being featured as metabolic disorder, diabetic patients are largely affected by hyperglycemia-induced vascular abnormality. Accumulated evidence has confirmed the beneficial effect of endothelial progenitor cells (EPCs) in coronary heart disease. However, antivascular endothelial growth factor (anti-VEGF) treatment is the main therapy for diabetic retinopathy and nephropathy, indicating the uncertain role of EPCs in the pathogenesis of diabetic microvascular disease. In this review, we first illustrate how hyperglycemia induces metabolic and epigenetic changes in EPCs, which exerts deleterious impact on their number and function. We then discuss how abnormal angiogenesis develops in eyes and kidneys under diabetes condition, focusing on "VEGF uncoupling with nitric oxide" and "competitive angiopoietin 1/angiopoietin 2" mechanisms that are shared in both organs. Next, we dissect the nature of EPCs in diabetic microvascular complications. After we overview the current EPCs-related strategies, we point out new EPCs-associated options for future exploration. Ultimately, we hope that this review would uncover the mysterious nature of EPCs in diabetic microvascular disease for therapeutics.

  12. Let-7i attenuates human brain microvascular endothelial cell damage in oxygen glucose deprivation model by decreasing toll-like receptor 4 expression.

    Science.gov (United States)

    Xiang, Wei; Tian, Canhui; Peng, Shunli; Zhou, Liang; Pan, Suyue; Deng, Zhen

    2017-11-04

    The let-7 family of microRNAs (miRNAs) plays an important role on endothelial cell function. However, there have been few studies on their role under ischemic conditions. In this study, we demonstrate that let-7i, belonging to the let-7 family, rescues human brain microvascular endothelial cells (HBMECs) in an oxygen-glucose deprivation (OGD) model. Our data show that the expression of let-7 family miRNAs was downregulated after OGD. Overexpression of let-7i significantly alleviated cell death and improved survival of OGD-treated HBMECs. Let-7i also protected permeability in an in vitro blood brain barrier (BBB) model. Further, let-7i downregulated the expression of toll-like receptor 4 (TLR4), an inflammation trigger. Moreover, overexpression of let-7i decreased matrix metallopeptidase 9 (MMP9) and inducible nitric oxide synthase (iNOS) expression under OGD. Upon silencing TLR4 expression in HBMECs, the anti-inflammatory effect of let-7i was abolished. Our research suggests that let-7i promotes OGD-induced inflammation via downregulating TLR4 expression. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Ghrelin stimulates angiogenesis in human microvascular endothelial cells: Implications beyond GH release

    International Nuclear Information System (INIS)

    Li Aihua; Cheng Guangli; Zhu Genghui; Tarnawski, Andrzej S.

    2007-01-01

    Ghrelin, a peptide hormone isolated from the stomach, releases growth hormone and stimulates appetite. Ghrelin is also expressed in pancreas, kidneys, cardiovascular system and in endothelial cells. The precise role of ghrelin in endothelial cell functions remains unknown. We examined the expression of ghrelin and its receptor (GHSR1) mRNAs and proteins in human microvascular endothelial cells (HMVEC) and determined whether ghrelin affects in these cells proliferation, migration and in vitro angiogenesis; and whether MAPK/ERK2 signaling is important for the latter action. We found that ghrelin and GHSR1 are constitutively expressed in HMVEC. Treatment of HMVEC with exogenous ghrelin significantly increased in these cells proliferation, migration, in vitro angiogenesis and ERK2 phosphorylation. MEK/ERK2 inhibitor, PD 98059 abolished ghrelin-induced in vitro angiogenesis. This is First demonstration that ghrelin and its receptor are expressed in human microvascular endothelial cells and that ghrelin stimulates HMVEC proliferation, migration, and angiogenesis through activation of ERK2 signaling

  14. West Nile virus-induced cell adhesion molecules on human brain microvascular endothelial cells regulate leukocyte adhesion and modulate permeability of the in vitro blood-brain barrier model.

    Directory of Open Access Journals (Sweden)

    Kelsey Roe

    Full Text Available Characterizing the mechanisms by which West Nile virus (WNV causes blood-brain barrier (BBB disruption, leukocyte infiltration into the brain and neuroinflammation is important to understand the pathogenesis of WNV encephalitis. Here, we examined the role of endothelial cell adhesion molecules (CAMs in mediating the adhesion and transendothelial migration of leukocytes across human brain microvascular endothelial cells (HBMVE. Infection with WNV (NY99 strain significantly induced ICAM-1, VCAM-1, and E-selectin in human endothelial cells and infected mice brain, although the levels of their ligands on leukocytes (VLA-4, LFA-1and MAC-1 did not alter. The permeability of the in vitro BBB model increased dramatically following the transmigration of monocytes and lymphocytes across the models infected with WNV, which was reversed in the presence of a cocktail of blocking antibodies against ICAM-1, VCAM-1, and E-selectin. Further, WNV infection of HBMVE significantly increased leukocyte adhesion to the HBMVE monolayer and transmigration across the infected BBB model. The blockade of these CAMs reduced the adhesion and transmigration of leukocytes across the infected BBB model. Further, comparison of infection with highly neuroinvasive NY99 and non-lethal (Eg101 strain of WNV demonstrated similar level of virus replication and fold-increase of CAMs in HBMVE cells suggesting that the non-neuropathogenic response of Eg101 is not because of its inability to infect HBMVE cells. Collectively, these results suggest that increased expression of specific CAMs is a pathological event associated with WNV infection and may contribute to leukocyte infiltration and BBB disruption in vivo. Our data further implicate that strategies to block CAMs to reduce BBB disruption may limit neuroinflammation and virus-CNS entry via 'Trojan horse' route, and improve WNV disease outcome.

  15. Endothelial network formed with human dermal microvascular endothelial cells in autologous multicellular skin substitutes.

    Science.gov (United States)

    Ponec, Maria; El Ghalbzouri, Abdoelwaheb; Dijkman, Remco; Kempenaar, Johanna; van der Pluijm, Gabri; Koolwijk, Pieter

    2004-01-01

    A human skin equivalent from a single skin biopsy harboring keratinocytes and melanocytes in the epidermal compartment, and fibroblasts and microvascular dermal endothelial cells in the dermal compartment was developed. The results of the study revealed that the nature of the extracellular matrix of the dermal compartments plays an important role in establishment of endothelial network in vitro. With rat-tail type I collagen matrices only lateral but not vertical expansion of endothelial networks was observed. In contrast, the presence of extracellular matrix of entirely human origin facilitated proper spatial organization of the endothelial network. Namely, when human dermal fibroblasts and microvascular endothelial cells were seeded on the bottom of an inert filter and subsequently epidermal cells were seeded on top of it, fibroblasts produced extracellular matrix throughout which numerous branched tubes were spreading three-dimensionally. Fibroblasts also facilitated the formation of basement membrane at the epidermal/matrix interface. Under all culture conditions, fully differentiated epidermis was formed with numerous melanocytes present in the basal epidermal cell layer. The results of the competitive RT-PCR revealed that both keratinocytes and fibroblasts expressed VEGF-A, -B, -C, aFGF and bFGF mRNA, whereas fibroblasts also expressed VEGF-D mRNA. At protein level, keratinocytes produced 10 times higher amounts of VEGF-A than fibroblasts did. The generation of multicellular skin equivalent from a single human skin biopsy will stimulate further developments for its application in the treatment of full-thickness skin defects. The potential development of biodegradable, biocompatible material suitable for these purposes is a great challenge for future research.

  16. Tetramethylpyrazine Protects Against Oxygen-Glucose Deprivation-Induced Brain Microvascular Endothelial Cells Injury via Rho/Rho-kinase Signaling Pathway.

    Science.gov (United States)

    Yang, Guang; Qian, Chen; Wang, Ning; Lin, Chenyu; Wang, Yan; Wang, Guangyun; Piao, Xinxin

    2017-05-01

    Tetramethylpyrazine (TMP, also known as Ligustrazine), which is isolated from Chinese Herb Medicine Ligustium wollichii Franchat (Chuan Xiong), has been widely used in China for the treatment of ischemic stroke by Chinese herbalists. Brain microvascular endothelial cells (BMECs) are the integral parts of the blood-brain barrier (BBB), protecting BMECs against oxygen-glucose deprivation (OGD) which is important for the treatment of ischemic stroke. Here, we investigated the protective mechanisms of TMP, focusing on OGD-injured BMECs and the Rho/Rho-kinase (Rho-associated kinases, ROCK) signaling pathway. The model of OGD-injured BMECs was established in this study. BMECs were identified by von Willebrand factor III staining and exposed to fasudil, or TMP at different concentrations (14.3, 28.6, 57.3 µM) for 2 h before 24 h of OGD injury. The effect of each treatment was examined by cell viability assays, measurement of intracellular reactive oxygen species (ROS), and transendothelial electric resistance and western blot analysis (caspase-3, endothelial nitric oxide synthase (eNOS), RhoA, Rac1). Our results show that TMP significantly attenuated apoptosis and the permeability of BMECs induced by OGD. In addition, TMP could notably down-regulate the characteristic proteins in Rho/ROCK signaling pathway such as RhoA and Rac1, which triggered abnormal changes of eNOS and ROS, respectively. Altogether, our results show that TMP has a strong protective effect against OGD-induced BMECs injury and suggest that the mechanism might be related to the inhibition of the Rho/ROCK signaling pathway.

  17. Vascular endothelial growth factor is upregulated by l-dopa in the parkinsonian brain: implications for the development of dyskinesia

    Science.gov (United States)

    Francardo, Veronica; Lindgren, Hanna S.; Sillivan, Stephanie E.; O’Sullivan, Sean S.; Luksik, Andrew S.; Vassoler, Fair M.; Lees, Andrew J.; Konradi, Christine

    2011-01-01

    Angiogenesis and increased permeability of the blood–brain barrier have been reported to occur in animal models of Parkinson’s disease and l-dopa-induced dyskinesia, but the significance of these phenomena has remained unclear. Using a validated rat model of l-dopa-induced dyskinesia, this study demonstrates that chronic treatment with l-dopa dose dependently induces the expression of vascular endothelial growth factor in the basal ganglia nuclei. Vascular endothelial growth factor was abundantly expressed in astrocytes and astrocytic processes in the proximity of blood vessels. When co-administered with l-dopa, a small molecule inhibitor of vascular endothelial growth factor signalling significantly attenuated the development of dyskinesia and completely blocked the angiogenic response and associated increase in blood–brain barrier permeability induced by the treatment. The occurrence of angiogenesis and vascular endothelial growth factor upregulation was verified in post-mortem basal ganglia tissue from patients with Parkinson’s disease with a history of dyskinesia, who exhibited increased microvascular density, microvascular nestin expression and an upregulation of vascular endothelial growth factor messenger ribonucleic acid. These congruent findings in the rat model and human patients indicate that vascular endothelial growth factor is implicated in the pathophysiology of l-dopa-induced dyskinesia and emphasize an involvement of the microvascular compartment in the adverse effects of l-dopa pharmacotherapy in Parkinson’s disease. PMID:21771855

  18. In vitro model of cerebral ischemia by using brain microvascular endothelial cells derived from human induced pluripotent stem cells.

    Science.gov (United States)

    Kokubu, Yasuhiro; Yamaguchi, Tomoko; Kawabata, Kenji

    2017-04-29

    Brain-derived microvascular endothelial cells (BMECs), which play a central role in blood brain barrier (BBB), can be used for the evaluation of drug transport into the brain. Although human BMEC cell lines have already been reported, they lack original properties such as barrier integrity. Pluripotent stem cells (PSCs) can be used for various applications such as regenerative therapy, drug screening, and pathological study. In the recent study, an induction method of BMECs from PSCs has been established, making it possible to more precisely study the in vitro human BBB function. Here, using induced pluripotent stem (iPS) cell-derived BMECs, we examined the effects of oxygen-glucose deprivation (OGD) and OGD/reoxygenation (OGD/R) on BBB permeability. OGD disrupted the barrier function, and the dysfunction was rapidly restored by re-supply of the oxygen and glucose. Interestingly, TNF-α, which is known to be secreted from astrocytes and microglia in the cerebral ischemia, prevented the restoration of OGD-induced barrier dysfunction in an apoptosis-independent manner. Thus, we could establish the in vitro BBB disease model that mimics the cerebral ischemia by using iPS cell-derived BMECs. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Thrombin stimulates albumin transcytosis in lung microvascular endothelial cells via activation of acid sphingomyelinase.

    Science.gov (United States)

    Kuebler, Wolfgang M; Wittenberg, Claudia; Lee, Warren L; Reppien, Eike; Goldenberg, Neil M; Lindner, Karsten; Gao, Yizhuo; Winoto-Morbach, Supandi; Drab, Marek; Mühlfeld, Christian; Dombrowsky, Heike; Ochs, Matthias; Schütze, Stefan; Uhlig, Stefan

    2016-04-15

    Transcellular albumin transport occurs via caveolae that are abundant in lung microvascular endothelial cells. Stimulation of albumin transcytosis by proinflammatory mediators may contribute to alveolar protein leak in lung injury, yet the regulation of albumin transport and its underlying molecular mechanisms are so far incompletely understood. Here we tested the hypothesis that thrombin may stimulate transcellular albumin transport across lung microvascular endothelial cells in an acid-sphingomyelinase dependent manner. Thrombin increased the transport of fluorescently labeled albumin across confluent human lung microvascular endothelial cell (HMVEC-L) monolayers to an extent that markedly exceeds the rate of passive diffusion. Thrombin activated acid sphingomyelinase (ASM) and increased ceramide production in HMVEC-L, but not in bovine pulmonary artery cells, which showed little albumin transport in response to thrombin. Thrombin increased total caveolin-1 (cav-1) content in both whole cell lysates and lipid rafts from HMVEC-L, and this effect was blocked by inhibition of ASM or de novo protein biosynthesis. Thrombin-induced uptake of albumin into lung microvascular endothelial cells was confirmed in isolated-perfused lungs by real-time fluorescence imaging and electron microscopy of gold-labeled albumin. Inhibition of ASM attenuated thrombin-induced albumin transport both in confluent HMVEC-L and in intact lungs, whereas HMVEC-L treatment with exogenous ASM increased albumin transport and enriched lipid rafts in cav-1. Our findings indicate that thrombin stimulates transcellular albumin transport in an acid sphingomyelinase-dependent manner by inducing de novo synthesis of cav-1 and its recruitment to membrane lipid rafts. Copyright © 2016 the American Physiological Society.

  20. Modeling Group B Streptococcus and Blood-Brain Barrier Interaction by Using Induced Pluripotent Stem Cell-Derived Brain Endothelial Cells

    OpenAIRE

    Kim, Brandon J.; Bee, Olivia B.; McDonagh, Maura A.; Stebbins, Matthew J.; Palecek, Sean P.; Doran, Kelly S.; Shusta, Eric V.

    2017-01-01

    ABSTRACT Bacterial meningitis is a serious infection of the central nervous system (CNS) that occurs after bacteria interact with and penetrate the blood-brain barrier (BBB). The BBB is comprised of highly specialized brain microvascular endothelial cells (BMECs) that function to separate the circulation from the CNS and act as a formidable barrier for toxins and pathogens. Certain bacteria, such as Streptococcus agalactiae (group B Streptococcus [GBS]), possess the ability to interact with a...

  1. Blood-brain barrier dysfunction and amyloid precursor protein accumulation in microvascular compartment following ischemia-reperfusion brain injury with 1-year survival.

    Science.gov (United States)

    Pluta, R

    2003-01-01

    This study examined the late microvascular consequences of brain ischemia due to cardiac arrest in rats. In reacted vibratome sections scattered foci of extravasated horseradish peroxidase were noted throughout the brain and did not appear to be restricted to any specific area of brain. Ultrastructural investigation of leaky sites frequently presented platelets adhering to the endothelium of venules and capillaries. Endothelial cells demonstrated pathological changes with evidence of perivascular astrocytic swelling. At the same time, we noted C-terminal of amyloid precursor protein/beta-amyloid peptide (CAPP/betaA) deposits in cerebral blood vessels, with a halo of CAPP/betaA immunoreactivity in the surrounding parenchyma suggested diffusion of CAPP/betaA out of the vascular compartment. Changes predominated in the hippocampus, cerebral and entorhinal cortex, corpus callosum, thalamus, basal ganglia and around the lateral ventricles. These data implicate delayed abnormal endothelial function of vessels following ischemia-reperfusion brain injury as a primary event in the pathogenesis of the recurrent cerebral infarction.

  2. Microvascular Endothelial Dysfunction in Sedentary, Obese Humans is mediated by NADPH Oxidase; Influence of Exercise Training

    Science.gov (United States)

    La Favor, Justin D.; Dubis, Gabriel S.; Yan, Huimin; White, Joseph D.; Nelson, Margaret A.M.; Anderson, Ethan J.; Hickner, Robert C.

    2016-01-01

    Objective The objectives of this study were to determine the impact of in vivo reactive oxygen species (ROS) on microvascular endothelial function in obese human subjects and to determine the efficacy of an aerobic exercise intervention on alleviating obesity-associated dysfunctionality. Approach and Results Young, sedentary men and women were divided into lean (BMI 18–25; n=14), intermediate (BMI 28–32.5; n=13), and obese (BMI 33–40; n=15) groups. A novel microdialysis technique was utilized to detect elevated interstitial hydrogen peroxide (H2O2) and superoxide levels in the vastus lateralis of obese compared to both lean and intermediate subjects. Nutritive blood flow was monitored in the vastus lateralis via the microdialysis-ethanol technique. A decrement in acetylcholine-stimulated blood flow revealed impaired microvascular endothelial function in the obese subjects. Perfusion of apocynin, an NADPH oxidase (Nox) inhibitor, lowered (normalized) H2O2 and superoxide levels and reversed microvascular endothelial dysfunction in obese subjects. Following 8-weeks of exercise, H2O2 levels were decreased in the obese subjects and microvascular endothelial function in these subjects was restored to levels similar to lean subjects. Skeletal muscle protein expression of the Nox subunits p22phox, p47phox, and p67phox were increased in obese relative to lean subjects, where p22phox and p67phox expression was attenuated by exercise training in obese subjects. Conclusions This study implicates Nox as a source of excessive ROS production in skeletal muscle of obese individuals, and links excessive Nox derived ROS to microvascular endothelial dysfunction in obesity. Furthermore, aerobic exercise training proved to be an effective strategy for alleviating these maladies. PMID:27765769

  3. Impact of an endothelial progenitor cell capturing stent on coronary microvascular function: comparison with drug-eluting stents.

    Science.gov (United States)

    Choi, Woong Gil; Kim, Soo Hyun; Yoon, Hyung Seok; Lee, Eun Joo; Kim, Dong Woon

    2015-01-01

    Although drug-eluting stents (DESs) effectively reduce restenosis following percutaneous coronary intervention (PCI), they also delay re-endothelialization and impair microvascular function, resulting in adverse clinical outcomes. Endothelial progenitor cell (EPC) capturing stents, by providing a functional endothelial layer on the stent, have beneficial effects on microvascular function. However, data on coronary microvascular function in patients with EPC stents versus DESs are lacking. Seventy-four patients who previously underwent PCI were enrolled in this study. Microvascular function was evaluated 6 months after PCI based on the index of microvascular resistance (IMR) and the coronary flow reserve (CFR). IMR was calculated as the ratio of the mean distal coronary pressure at maximal hyperemia to the inverse of the hyperemic mean transit time (hTmn). The CFR was calculated by dividing the hTmn by the baseline mean transit time. Twenty-one patients (age, 67.2 ± 9.6 years; male:female, 15:6) with an EPC stent and 53 patients (age, 61.5 ± 14.7 years; male:female, 40:13) with second-generation DESs were included in the study. There were no significant differences in the baseline clinical and angiographic characteristics of the two groups. Angiography performed 6 months postoperatively did not show significant differences in their CFR values. However, patients with the EPC stent had a significantly lower IMR than patients with second-generation DESs (median, 25.5 [interquartile range, 12.85 to 28.18] vs. 29.0 [interquartile range, 15.42 to 39.23]; p = 0.043). Microvascular dysfunction was significantly improved after 6 months in patients with EPC stents compared to those with DESs. The complete re-endothelialization achieved with the EPC stent may provide clinical benefits over DESs, especially in patients with microvascular dysfunction.

  4. Activation of melatonin receptor (MT1/2) promotes P-gp transporter in methamphetamine-induced toxicity on primary rat brain microvascular endothelial cells.

    Science.gov (United States)

    Jumnongprakhon, Pichaya; Sivasinprasasn, Sivanan; Govitrapong, Piyarat; Tocharus, Chainarong; Tocharus, Jiraporn

    2017-06-01

    Melatonin has been known as a neuroprotective agent for the central nervous system (CNS) and the blood-brain barrier (BBB), which is the primary structure that comes into contact with several neurotoxins including methamphetamine (METH). Previous studies have reported that the activation of melatonin receptors (MT1/2) by melatonin could protect against METH-induced toxicity in brain endothelial cells via several mechanisms. However, its effects on the P-glycoprotein (P-gp) transporter, the active efflux pump involved in cell homeostasis, are still unclear. Thus, this study investigated the role of melatonin and its receptors on the METH-impaired P-gp transporter in primary rat brain microvascular endothelial cells (BMVECs). The results showed that METH impaired the function of the P-gp transporter, significantly decreasing the efflux of Rho123 and P-gp expression, which caused a significant increase in the intracellular accumulation of Rho123, and these responses were reversed by the interaction of melatonin with its receptors. Blockade of the P-gp transporter by verapamil caused oxidative stress, apoptosis, and cell integrity impairment after METH treatment, and these effects could be reversed by melatonin. Our results, together with previous findings, suggest that the interaction of melatonin with its receptors protects against the effects of the METH-impaired P-gp transporter and that the protective role in METH-induced toxicity was at least partially mediated by the regulation of the P-gp transporter. Thus, melatonin and its receptors (MT1/2) are essential for protecting against BBB impairment caused by METH. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Effective plasmid DNA and small interfering RNA delivery to diseased human brain microvascular endothelial cells.

    Science.gov (United States)

    Slanina, H; Schmutzler, M; Christodoulides, M; Kim, K S; Schubert-Unkmeir, A

    2012-01-01

    Expression of exogenous DNA or small interfering RNA (siRNA) in vitro is significantly affected by the particular delivery system utilized. In this study, we evaluated the transfection efficiency of plasmid DNA and siRNA into human brain microvascular endothelial cells (HBMEC) and meningioma cells, which constitute the blood-cerebrospinal fluid barrier, a target of meningitis-causing pathogens. Chemical transfection methods and various lipofection reagents including Lipofectamin™, FuGene™, or jetPRIME®, as well as physical transfection methods and electroporation techniques were applied. To monitor the transfection efficiencies, HBMEC and meningioma cells were transfected with the reporter plasmid pTagGFP2-actin vector, and efficiency of transfection was estimated by fluorescence microscopy and flow cytometry. We established protocols based on electroporation using Cell Line Nucleofector® Kit V with the Amaxa® Nucleofector® II system from Lonza and the Neon® Transfection system from Invitrogen resulting in up to 41 and 82% green fluorescent protein-positive HBMEC, respectively. Optimal transfection solutions, pulse programs and length were evaluated. We furthermore demonstrated that lipofection is an efficient method to transfect meningioma cells with a transfection efficiency of about 81%. Finally, we applied the successful electroporation protocols to deliver synthetic siRNA to HBMEC and analyzed the role of the actin-binding protein cortactin in Neisseria meningitidis pathogenesis. Copyright © 2012 S. Karger AG, Basel.

  6. Zika Virus Persistently Infects and Is Basolaterally Released from Primary Human Brain Microvascular Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Megan C. Mladinich

    2017-07-01

    Full Text Available Zika virus (ZIKV is a mosquito-borne Flavivirus that has emerged as the cause of encephalitis and fetal microencephaly in the Americas. ZIKV uniquely persists in human bodily fluids for up to 6 months, is sexually transmitted, and traverses the placenta and the blood-brain barrier (BBB to damage neurons. Cells that support persistent ZIKV replication and mechanisms by which ZIKV establishes persistence remain enigmatic but central to ZIKV entry into protected neuronal compartments. The endothelial cell (EC lining of capillaries normally constrains transplacental transmission and forms the BBB, which selectively restricts access of blood constituents to neurons. We found that ZIKV (strain PRVABC59 persistently infects and continuously replicates in primary human brain microvascular ECs (hBMECs, without cytopathology, for >9 days and following hBMEC passage. ZIKV did not permeabilize hBMECs but was released basolaterally from polarized hBMECs, suggesting a direct mechanism for ZIKV to cross the BBB. ZIKV-infected hBMECs were rapidly resistant to alpha interferon (IFN-α and transiently induced, but failed to secrete, IFN-β and IFN-λ. Global transcriptome analysis determined that ZIKV constitutively induced IFN regulatory factor 7 (IRF7, IRF9, and IFN-stimulated genes (ISGs 1 to 9 days postinfection, despite persistently replicating in hBMECs. ZIKV constitutively induced ISG15, HERC5, and USP18, which are linked to hepatitis C virus (HCV persistence and IFN regulation, chemokine CCL5, which is associated with immunopathogenesis, as well as cell survival factors. Our results reveal that hBMECs act as a reservoir of persistent ZIKV replication, suggest routes for ZIKV to cross hBMECs into neuronal compartments, and define novel mechanisms of ZIKV persistence that can be targeted to restrict ZIKV spread.

  7. Galantamine and carbon monoxide protect brain microvascular endothelial cells by heme oxygenase-1 induction

    International Nuclear Information System (INIS)

    Nakao, Atsunori; Kaczorowski, David J.; Zuckerbraun, Brian S.; Lei Jing; Faleo, Gaetano; Deguchi, Kentaro; McCurry, Kenneth R.; Billiar, Timothy R.; Kanno, Shinichi

    2008-01-01

    Galantamine, a reversible inhibitor of acetylcholine esterase (AChE), is a novel drug treatment for mild to moderate Alzheimer's disease and vascular dementia. Interestingly, it has been suggested that galantamine treatment is associated with more clinical benefit in patients with mild-to-moderate Alzheimer disease compared to other AChE inhibitors. We hypothesized that the protective effects of galantamine would involve induction of the protective gene, heme oxygenase-1 (HO-1), in addition to enhancement of the cholinergic system. Brain microvascular endothelial cells (mvECs) were isolated from spontaneous hypertensive rats. Galantamine significantly reduced H 2 O 2 -induced cell death of mvECs in association with HO-1 induction. These protective effects were completely reversed by nuclear factor-κB (NF-κB) inhibition or HO inhibition. Furthermore, galantamine failed to induce HO-1 in mvECs which lack inducible nitric oxide synthase (iNOS), supplementation of a nitric oxide (NO) donor or iNOS gene transfection on iNOS-deficient mvECs resulted in HO-1 induction with galantamine. These data suggest that the protective effects of galantamine require NF-κB activation and iNOS expression, in addition to HO-1. Likewise, carbon monoxide (CO), one of the byproducts of HO, up-regulated HO-1 and protected mvECs from oxidative stress in a similar manner. Our data demonstrate that galantamine mediates cytoprotective effects on mvECs through induction HO-1. This pharmacological action of galantamine may, at least in part, account for the superior clinical efficacy of galantamine in vascular dementia and Alzheimer disease

  8. M3 receptor is involved in the effect of penehyclidine hydrochloride reduced endothelial injury in LPS-stimulated human pulmonary microvascular endothelial cell.

    Science.gov (United States)

    Yuan, Qinghong; Xiao, Fei; Liu, Qiangsheng; Zheng, Fei; Shen, Shiwen; He, Qianwen; Chen, Kai; Wang, Yanlin; Zhang, Zongze; Zhan, Jia

    2018-02-01

    LPS has been recently shown to induce muscarinic acetylcholine 3 receptor (M 3 receptor) expression and penehyclidine hydrochloride (PHC) is an anticholinergic drug which could block the expression of M 3 receptor. PHC has been demonstrated to perform protective effect on cell injury. This study is to investigate whether the effect of PHC on microvascular endothelial injury is related to its inhibition of M 3 receptor or not. HPMVECs were treated with specific M 3 receptor shRNA or PBS, and randomly divided into LPS group (A group), LPS+PHC group (B group), LPS + M 3 shRNA group (C group) and LPS + PHC + M 3 shRNA group (D group). Cells were collected at 60 min after LPS treatment to measure levels of LDH, endothelial permeability, TNF-α and IL-6 levels, NF-κB p65 activation, I-κB protein expression, p38MAPK, and ERK1/2 activations as well as M 3 mRNA expression. PHC could decrease LDH levels, cell permeability, TNF-α and IL-6 levels, p38 MAPK, ERK1/2, NF-κB p65 activations and M 3 mRNA expressions compared with LPS group. When M 3 receptor was silence, the changes of these indices were much more obvious. These findings suggest that M 3 receptor plays an important role in LPS-induced pulmonary microvascular endothelial injury, which is regulated through NF-κB p65 and MAPK activation. And knockout of M 3 receptor could attenuate LPS-induced pulmonary microvascular endothelial injury. Regulative effects of PHC on pulmonary microvascular permeability and NF-κB p65 as well as MAPK activations are including but not limited to inhibition of M 3 receptor. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. In vitro analysis of human periodontal microvascular endothelial cells.

    Science.gov (United States)

    Tsubokawa, Mizuki; Sato, Soh

    2014-08-01

    Endothelial cells (ECs) participate in key aspects of vascular biology, such as maintenance of capillary permeability, initiation of coagulation, and regulation of inflammation. According to previous reports, ECs have revealed highly specific characteristics depending on the organs and tissues. However, some reports have described the characteristics of the capillaries formed by human periodontal ECs. Therefore, the aim of the present study is to examine the functional characteristics of the periodontal microvascular ECs in vitro. Human periodontal ligament-endothelial cells (HPDL-ECs) and human gingiva-endothelial cells (HG-ECs) were isolated by immunoprecipitation with magnetic beads conjugated to a monoclonal anti-CD31 antibody. The isolated HPDL-ECs and HG-ECs were characterized to definitively demonstrate that these cell cultures represented pure ECs. Human umbilical-vein ECs and human dermal microvascular ECs were used for comparison. These cells were compared according to the proliferation potential, the formation of capillary-like tubes, the transendothelial electric resistance (TEER), and the expression of tight junction proteins. HPDL-ECs and HG-ECs with characteristic cobblestone monolayer morphology were obtained, as determined by light microscopy at confluence. Furthermore, the HPDL-ECs and HG-ECs expressed the EC markers platelet endothelial cell adhesion molecule-1 (also known as CD31), von Willebrand factor, and Ulex europaeus agglutinin 1, and the cells stained strongly positive for CD31 and CD309. In addition, the HPDL-ECs and HG-ECs were observed to form capillary-like tubes, and they demonstrated uptake of acetylated low-density lipoprotein. Functional analyses of the HPDL-ECs and HG-ECs showed that, compared to the control cells, tube formation persisted for only a brief period of time, and TEER was substantially reduced at confluence. Furthermore, the cells exhibited delocalization of zonula occludens-1 and occludin at cell-cell contact sites

  10. Mechanical injury induces brain endothelial-derived microvesicle release: Implications for cerebral vascular injury during traumatic brain injury

    Directory of Open Access Journals (Sweden)

    Allison M. Andrews

    2016-02-01

    Full Text Available It is well established that the endothelium responds to mechanical forces induced by changes in shear stress and mechanotransduction. However, our understanding of vascular remodeling following traumatic brain injury (TBI remains incomplete. Recently published studies have revealed that lung and umbilical endothelial cells produce extracellular microvesicles (eMVs, such as microparticles, in response to changes in mechanical forces (blood flow and mechanical injury. Yet, to date, no studies have shown whether brain endothelial cells produce eMVs following TBI. The brain endothelium is highly specialized and forms the blood-brain barrier (BBB, which regulates diffusion and transport of solutes into the brain. This specialization is largely due to the presence of tight junction proteins (TJPs between neighboring endothelial cells. Following TBI, a breakdown in tight junction complexes at the BBB leads to increased permeability, which greatly contributes to the secondary phase of injury. We have therefore tested the hypothesis that brain endothelium responds to mechanical injury, by producing eMVs that contain brain endothelial proteins, specifically TJPs. In our study, primary human adult brain microvascular endothelial cells (BMVEC were subjected to rapid mechanical injury to simulate the abrupt endothelial disruption that can occur in the primary injury phase of TBI. eMVs were isolated from the media following injury at 2, 6, 24 and 48 hrs. Western blot analysis of eMVs demonstrated a time-dependent increase in TJP occludin, PECAM-1 and ICAM-1 following mechanical injury. In addition, activation of ARF6, a small GTPase linked to extracellular vesicle production, was increased after injury. To confirm these results in vivo, mice were subjected to sham surgery or TBI and blood plasma was collected 24 hrs post-injury. Isolation and analysis of eMVs from blood plasma using cryo-EM and flow cytometry revealed elevated levels of vesicles containing

  11. Mechanical Injury Induces Brain Endothelial-Derived Microvesicle Release: Implications for Cerebral Vascular Injury during Traumatic Brain Injury.

    Science.gov (United States)

    Andrews, Allison M; Lutton, Evan M; Merkel, Steven F; Razmpour, Roshanak; Ramirez, Servio H

    2016-01-01

    It is well established that the endothelium responds to mechanical forces induced by changes in shear stress and strain. However, our understanding of vascular remodeling following traumatic brain injury (TBI) remains incomplete. Recently published studies have revealed that lung and umbilical endothelial cells produce extracellular microvesicles (eMVs), such as microparticles, in response to changes in mechanical forces (blood flow and mechanical injury). Yet, to date, no studies have shown whether brain endothelial cells produce eMVs following TBI. The brain endothelium is highly specialized and forms the blood-brain barrier (BBB), which regulates diffusion and transport of solutes into the brain. This specialization is largely due to the presence of tight junction proteins (TJPs) between neighboring endothelial cells. Following TBI, a breakdown in tight junction complexes at the BBB leads to increased permeability, which greatly contributes to the secondary phase of injury. We have therefore tested the hypothesis that brain endothelium responds to mechanical injury, by producing eMVs that contain brain endothelial proteins, specifically TJPs. In our study, primary human adult brain microvascular endothelial cells (BMVEC) were subjected to rapid mechanical injury to simulate the abrupt endothelial disruption that can occur in the primary injury phase of TBI. eMVs were isolated from the media following injury at 2, 6, 24, and 48 h. Western blot analysis of eMVs demonstrated a time-dependent increase in TJP occludin, PECAM-1 and ICAM-1 following mechanical injury. In addition, activation of ARF6, a small GTPase linked to extracellular vesicle production, was increased after injury. To confirm these results in vivo, mice were subjected to sham surgery or TBI and blood plasma was collected 24 h post-injury. Isolation and analysis of eMVs from blood plasma using cryo-EM and flow cytometry revealed elevated levels of vesicles containing occludin following brain trauma

  12. Transcriptome of E. coli K1 bound to human brain microvascular endothelial cells

    OpenAIRE

    Xie, Yi; Parthasarathy, Geetha; Di Cello, Francescopaolo; Teng, Ching-Hao; Paul-Satyaseela, Maneesh; Kim, Kwang Sik

    2007-01-01

    Escherichia coli K1 is the most common Gram-negative organism causing neonatal meningitis. Binding to human brain microvascdular endothelial cells (HBMEC) is an essential step for E. coli K1 traversal of the blood-brain barrier. In this study, we examined expression profiles of E. coli K1 strain RS218 during its binding to HBMEC. Comparison of HBMEC-bound E. coli K1 with collagen-bound E. coli revealed more than one hundred genes whose expression patterns were significantly changed in HBMEC-b...

  13. Comparative study of expression and activity of glucose transporters between stem cell-derived brain microvascular endothelial cells and hCMEC/D3 cells.

    Science.gov (United States)

    Al-Ahmad, Abraham J

    2017-10-01

    Glucose constitutes a major source of energy of mammalian brains. Glucose uptake at the blood-brain barrier (BBB) occurs through a facilitated glucose transport, through glucose transporter 1 (GLUT1), although other isoforms have been described at the BBB. Mutations in GLUT1 are associated with the GLUT1 deficiency syndrome, yet none of the current in vitro models of the human BBB maybe suited for modeling such a disorder. In this study, we investigated the expression of glucose transporters and glucose diffusion across brain microvascular endothelial cells (BMECs) derived from healthy patient-derived induced pluripotent stem cells (iPSCs). We investigated the expression of different glucose transporters at the BBB using immunocytochemistry and flow cytometry and measured glucose uptake and diffusion across BMEC monolayers obtained from two iPSC lines and from hCMEC/D3 cells. BMEC monolayers showed expression of several glucose transporters, in particular GLUT1, GLUT3, and GLUT4. Diffusion of glucose across the monolayers was mediated via a saturable transcellular mechanism and partially inhibited by pharmacological inhibitors. Taken together, our study suggests the presence of several glucose transporters isoforms at the human BBB and demonstrates the feasibility of modeling glucose across the BBB using patient-derived stem cells. Copyright © 2017 the American Physiological Society.

  14. Cyclic adenosine monophosphate levels and the function of skin microvascular endothelial cells.

    Science.gov (United States)

    Tuder, R M; Karasek, M A; Bensch, K G

    1990-02-01

    The maintenance of the normal epithelioid morphology of human dermal microvascular endothelial cells (MEC) grown in vitro depends strongly on the presence of factors that increase intracellular levels of cyclic AMP. Complete removal of dibutyryl cAMP and isobutylmethylxanthine (IMX) from the growth medium results in a progressive transition from an epithelioid to a spindle-shaped cell line. This transition cannot be reversed by the readdition of dibutyryl cAMP and IMX to the growth medium or by addition of agonists that increase cAMP levels. Spindle-shaped MEC lose the ability to express Factor VIII rAG and DR antigens and to bind peripheral blood mononuclear leukocyte (PBML). Ultrastructural analyses of transitional cells and spindle-shaped cells show decreased numbers of Weibel-Palade bodies in transitional cells and their complete absence in spindle-shaped cells. Interferon-gamma alters several functional properties of both epithelioid and spindle-shaped cells. In the absence of dibutyryl cAMP it accelerates the transition from epithelial to spindle-shaped cells, whereas in the presence of cyclic AMP interferon-gamma increases the binding of PBMLs to both epithelioid and spindle-shaped MEC and the endocytic activity of the endothelial cells. These results suggest that cyclic AMP is an important second messenger in the maintenance of several key functions of microvascular endothelial cells. Factors that influence the levels of this messenger in vivo can be expected to influence the angiogenic and immunologic functions of the microvasculature.

  15. Upcyte® Microvascular Endothelial Cells Repopulate Decellularized Scaffold

    Science.gov (United States)

    Dally, Iris; Hartmann, Nadja; Münst, Bernhard; Braspenning, Joris; Walles, Heike

    2013-01-01

    A general problem in tissue engineering is the poor and insufficient blood supply to guarantee tissue cell survival as well as physiological tissue function. To address this limitation, we have developed an in vitro vascularization model in which a decellularized porcine small bowl segment, representing a capillary network within a collagen matrix (biological vascularized scaffold [BioVaSc]), is reseeded with microvascular endothelial cells (mvECs). However, since the supply of mvECs is limited, in general, and as these cells rapidly dedifferentiate, we have applied a novel technology, which allows the generation of large batches of quasi-primary cells with the ability to proliferate, whilst maintaining their differentiated functionality. These so called upcyte mvECs grew for an additional 15 population doublings (PDs) compared to primary cells. Upcyte mvECs retained endothelial characteristics, such as von Willebrandt Factor (vWF), CD31 and endothelial nitric oxide synthase (eNOS) expression, as well as positive Ulex europaeus agglutinin I staining. Upcyte mvECs also retained biological functionality such as tube formation, cell migration, and low density lipoprotein (LDL) uptake, which were still evident after PD27. Initial experiments using MTT and Live/Dead staining indicate that upcyte mvECs repopulate the BioVaSc Scaffold. As with conventional cultures, these cells also express key endothelial molecules (vWF, CD31, and eNOS) in a custom-made bioreactor system even after a prolonged period of 14 days. The combination of upcyte mvECs and the BioVaSc represents a novel and promising approach toward vascularizing bioreactor models which can better reflect organs, such as the liver. PMID:22799502

  16. Perfluorooctane sulfonate (PFOS) induces reactive oxygen species (ROS) production in human microvascular endothelial cells: role in endothelial permeability.

    Science.gov (United States)

    Qian, Yong; Ducatman, Alan; Ward, Rebecca; Leonard, Steve; Bukowski, Valerie; Lan Guo, Nancy; Shi, Xianglin; Vallyathan, Val; Castranova, Vincent

    2010-01-01

    Perfluorooctane sulfonate (PFOS) is a member of the perfluoroalkyl acids (PFAA) containing an eight-carbon backbone. PFOS is a man-made chemical with carbon-fluorine bonds that are among the strongest in organic chemistry, and PFOS is widely used in industry. Human occupational and environmental exposure to PFOS occurs globally. PFOS is non-biodegradable and is persistent in the human body and environment. In this study, data demonstrated that exposure of human microvascular endothelial cells (HMVEC) to PFOS induced the production of reactive oxygen species (ROS) at both high and low concentrations. Morphologically, it was found that exposure to PFOS induced actin filament remodeling and endothelial permeability changes in HMVEC. Furthermore, data demonstrated that the production of ROS plays a regulatory role in PFOS-induced actin filament remodeling and the increase in endothelial permeability. Our results indicate that the generation of ROS may play a role in PFOS-induced aberrations of the endothelial permeability barrier. The results generated from this study may provide a new insight into the potential adverse effects of PFOS exposure on humans at the cellular level.

  17. Uptake of Single-Walled Carbon Nanotubes Conjugated with DNA by Microvascular Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Joseph Harvey

    2012-01-01

    Full Text Available Single-walled carbon nanotubes (SWCNTs have been proposed to have great therapeutic potential. SWCNTs conjugated with drugs or genes travel in the systemic circulation to reach target cells or tissues following extravasation from microvessels although the interaction between SWCNT conjugates and the microvascular endothelial cells (ECs remains unknown. We hypothesized that SWCNT-DNA conjugates would be taken up by microvascular ECs and that this process would be facilitated by SWCNTs compared to facilitation by DNA alone. ECs were treated with various concentrations of SWCNT-DNA-FITC conjugates, and the uptake and intracellular distribution of these conjugates were determined by a confocal microscope imaging system followed by quantitative analysis of fluorescence intensity. The uptake of SWCNT-DNA-FITC conjugates (2 μg/mL by microvascular ECs was significantly greater than that of DNA-FITC (2 μg/mL, observed at 6 hrs after treatment. For the intracellular distribution, SWCNT-DNA-FITC conjugates were detected in the nucleus of ECs, while DNA-FITC was restricted to the cytoplasm. The fluorescence intensity and distribution of SWCNTs were concentration and time independent. The findings demonstrate that SWCNTs facilitate DNA delivery into microvascular ECs, thus suggesting that SWCNTs serving as drug and gene vehicles have therapeutic potential.

  18. Ebola virus glycoprotein-mediated anoikis of primary human cardiac microvascular endothelial cells

    International Nuclear Information System (INIS)

    Ray, Ratna B.; Basu, Arnab; Steele, Robert; Beyene, Aster; McHowat, Jane; Meyer, Keith; Ghosh, Asish K.; Ray, Ranjit

    2004-01-01

    Ebola virus glycoprotein (EGP) has been implicated for the induction of cytotoxicity and injury in vascular cells. On the other hand, EGP has also been suggested to induce massive cell rounding and detachment from the plastic surface by downregulating cell adhesion molecules without causing cytotoxicity. In this study, we have examined the cytotoxic role of EGP in primary endothelial cells by transduction with a replication-deficient recombinant adenovirus expressing EGP (Ad-EGP). Primary human cardiac microvascular endothelial cells (HCMECs) transduced with Ad-EGP displayed loss of cell adhesion from the plastic surface followed by cell death. Transfer of conditioned medium from EGP-transduced HCMEC into naive cells did not induce loss of adhesion or cell death, suggesting that EGP needs to be expressed intracellularly to exert its cytotoxic effect. Subsequent studies suggested that HCMEC death occurred through apoptosis. Results from this study shed light on the EGP-induced anoikis in primary human cardiac endothelial cells, which may have significant pathological consequences

  19. Interactions of Neuropathogenic Escherichia coli K1 (RS218) and Its Derivatives Lacking Genomic Islands with Phagocytic Acanthamoeba castellanii and Nonphagocytic Brain Endothelial Cells

    Science.gov (United States)

    Yousuf, Farzana Abubakar; Yousuf, Zuhair; Iqbal, Junaid; Siddiqui, Ruqaiyyah; Khan, Hafsa; Khan, Naveed Ahmed

    2014-01-01

    Here we determined the role of various genomic islands in E. coli K1 interactions with phagocytic A. castellanii and nonphagocytic brain microvascular endothelial cells. The findings revealed that the genomic islands deletion mutants of RS218 related to toxins (peptide toxin, α-hemolysin), adhesins (P fimbriae, F17-like fimbriae, nonfimbrial adhesins, Hek, and hemagglutinin), protein secretion system (T1SS for hemolysin), invasins (IbeA, CNF1), metabolism (D-serine catabolism, dihydroxyacetone, glycerol, and glyoxylate metabolism) showed reduced interactions with both A. castellanii and brain microvascular endothelial cells. Interestingly, the deletion of RS218-derived genomic island 21 containing adhesins (P fimbriae, F17-like fimbriae, nonfimbrial adhesins, Hek, and hemagglutinin), protein secretion system (T1SS for hemolysin), invasins (CNF1), metabolism (D-serine catabolism) abolished E. coli K1-mediated HBMEC cytotoxicity in a CNF1-independent manner. Therefore, the characterization of these genomic islands should reveal mechanisms of evolutionary gain for E. coli K1 pathogenicity. PMID:24818136

  20. Interactions of Neuropathogenic Escherichia coli K1 (RS218 and Its Derivatives Lacking Genomic Islands with Phagocytic Acanthamoeba castellanii and Nonphagocytic Brain Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Farzana Abubakar Yousuf

    2014-01-01

    Full Text Available Here we determined the role of various genomic islands in E. coli K1 interactions with phagocytic A. castellanii and nonphagocytic brain microvascular endothelial cells. The findings revealed that the genomic islands deletion mutants of RS218 related to toxins (peptide toxin, α-hemolysin, adhesins (P fimbriae, F17-like fimbriae, nonfimbrial adhesins, Hek, and hemagglutinin, protein secretion system (T1SS for hemolysin, invasins (IbeA, CNF1, metabolism (D-serine catabolism, dihydroxyacetone, glycerol, and glyoxylate metabolism showed reduced interactions with both A. castellanii and brain microvascular endothelial cells. Interestingly, the deletion of RS218-derived genomic island 21 containing adhesins (P fimbriae, F17-like fimbriae, nonfimbrial adhesins, Hek, and hemagglutinin, protein secretion system (T1SS for hemolysin, invasins (CNF1, metabolism (D-serine catabolism abolished E. coli K1-mediated HBMEC cytotoxicity in a CNF1-independent manner. Therefore, the characterization of these genomic islands should reveal mechanisms of evolutionary gain for E. coli K1 pathogenicity.

  1. Transcriptional profiling of human brain endothelial cells reveals key properties crucial for predictive in vitro blood-brain barrier models.

    Directory of Open Access Journals (Sweden)

    Eduard Urich

    Full Text Available Brain microvascular endothelial cells (BEC constitute the blood-brain barrier (BBB which forms a dynamic interface between the blood and the central nervous system (CNS. This highly specialized interface restricts paracellular diffusion of fluids and solutes including chemicals, toxins and drugs from entering the brain. In this study we compared the transcriptome profiles of the human immortalized brain endothelial cell line hCMEC/D3 and human primary BEC. We identified transcriptional differences in immune response genes which are directly related to the immortalization procedure of the hCMEC/D3 cells. Interestingly, astrocytic co-culturing reduced cell adhesion and migration molecules in both BECs, which possibly could be related to regulation of immune surveillance of the CNS controlled by astrocytic cells within the neurovascular unit. By matching the transcriptome data from these two cell lines with published transcriptional data from freshly isolated mouse BECs, we discovered striking differences that could explain some of the limitations of using cultured BECs to study BBB properties. Key protein classes such as tight junction proteins, transporters and cell surface receptors show differing expression profiles. For example, the claudin-5, occludin and JAM2 expression is dramatically reduced in the two human BEC lines, which likely explains their low transcellular electric resistance and paracellular leakiness. In addition, the human BEC lines express low levels of unique brain endothelial transporters such as Glut1 and Pgp. Cell surface receptors such as LRP1, RAGE and the insulin receptor that are involved in receptor-mediated transport are also expressed at very low levels. Taken together, these data illustrate that BECs lose their unique protein expression pattern outside of their native environment and display a more generic endothelial cell phenotype. A collection of key genes that seems to be highly regulated by the local

  2. Edaravone Protected Human Brain Microvascular Endothelial Cells from Methylglyoxal-Induced Injury by Inhibiting AGEs/RAGE/Oxidative Stress

    Science.gov (United States)

    Li, Wenlu; Xu, Hongjiao; Hu, Yangmin; He, Ping; Ni, Zhenzhen; Xu, Huimin; Zhang, Zhongmiao; Dai, Haibin

    2013-01-01

    Subjects with diabetes experience an increased risk of cerebrovascular disease and stroke compared with nondiabetic age-matched individuals. Increased formation of reactive physiological dicarbonyl compound methylglyoxal (MGO) seems to be implicated in the development of diabetic vascular complication due to its protein glycation and oxidative stress effect. Edaravone, a novel radical scavenger, has been reported to display the advantageous effects on ischemic stroke both in animals and clinical trials; however, little is known about whether edaravone has protective effects on diabetic cerebrovascular injury. Using cultured human brain microvascular endothelial cells (HBMEC), protective effects of edaravone on MGO and MGO enhancing oxygen-glucose deprivation (OGD) induced injury were investigated. Cell injury was measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) formation, cell account, lactate dehydrogenase (LDH) release and Rhodamine 123 staining. Advanced glycation end-products (AGEs) formation and receptor for advanced glycation end-products (RAGE) expression were measured by western blotting. Cellular oxidative stress was measured by reactive oxygen species (ROS) release. Treatment of MGO for 24 h significantly induced HBMEC injury, which was inhibited by pretreatment of edaravone from 10–100 µmol/l. What’s more, treatment of MGO enhanced AGEs accumulation, RAGE expression and ROS release in the cultured HBMEC, which were inhibited by 100 µmol/l edaravone. Finally, treatment of MGO for 24 h and then followed by 3 h OGD insult significantly enhanced cell injury when compared with OGD insult only, which was also protected by 100 µmol/l edaravone. Thus, edaravone protected HBMEC from MGO and MGO enhancing OGD-induced injury by inhibiting AGEs/RAGE/oxidative stress. PMID:24098758

  3. Edaravone protected human brain microvascular endothelial cells from methylglyoxal-induced injury by inhibiting AGEs/RAGE/oxidative stress.

    Directory of Open Access Journals (Sweden)

    Wenlu Li

    Full Text Available Subjects with diabetes experience an increased risk of cerebrovascular disease and stroke compared with nondiabetic age-matched individuals. Increased formation of reactive physiological dicarbonyl compound methylglyoxal (MGO seems to be implicated in the development of diabetic vascular complication due to its protein glycation and oxidative stress effect. Edaravone, a novel radical scavenger, has been reported to display the advantageous effects on ischemic stroke both in animals and clinical trials; however, little is known about whether edaravone has protective effects on diabetic cerebrovascular injury. Using cultured human brain microvascular endothelial cells (HBMEC, protective effects of edaravone on MGO and MGO enhancing oxygen-glucose deprivation (OGD induced injury were investigated. Cell injury was measured by 3-(4,5-Dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT formation, cell account, lactate dehydrogenase (LDH release and Rhodamine 123 staining. Advanced glycation end-products (AGEs formation and receptor for advanced glycation end-products (RAGE expression were measured by western blotting. Cellular oxidative stress was measured by reactive oxygen species (ROS release. Treatment of MGO for 24 h significantly induced HBMEC injury, which was inhibited by pretreatment of edaravone from 10-100 µmol/l. What's more, treatment of MGO enhanced AGEs accumulation, RAGE expression and ROS release in the cultured HBMEC, which were inhibited by 100 µmol/l edaravone. Finally, treatment of MGO for 24 h and then followed by 3 h OGD insult significantly enhanced cell injury when compared with OGD insult only, which was also protected by 100 µmol/l edaravone. Thus, edaravone protected HBMEC from MGO and MGO enhancing OGD-induced injury by inhibiting AGEs/RAGE/oxidative stress.

  4. Endothelial Proliferation and Increased Blood - Brain Barrier Permeability in the Basal Ganglia in a Rat Model of 3,4-Dihydrozyphenyl-L-Alanine-Induced Dyskinesia

    DEFF Research Database (Denmark)

    Westin, Jenny E.; Lindgren, Hanna S.; Gardi, Jonathan Eyal

    2006-01-01

    3,4-Dihydroxyphenyl-L-alanine (L-DOPA)-induced dyskinesia is associated with molecular and synaptic plasticity in the basal ganglia, but the occurrence of structural remodeling through cell genesis has not been explored. In this study, rats with 6-hydroxydopamine lesions received injections of th...... of angiogenesis and blood-brain barrier dysfunction in an experimental model of L-DOPA-induced dyskinesia. These microvascular changes are likely to affect the kinetics of L-DOPA entry into the brain, favoring the occurrence of motor complications....... dyskinesia. The vast majority (60-80%) of the newborn cells stained positively for endothelial markers. This endothelial proliferation was associated with an upregulation of immature endothelial markers (nestin) and a downregulation of endothelial barrier antigen on blood vessel walls. In addition......, dyskinetic rats exhibited a significant increase in total blood vessel length and a visible extravasation of serum albumin in the two structures in which endothelial proliferation was most pronounced (substantia nigra pars reticulata and entopeduncular nucleus). The present study provides the first evidence...

  5. Blood-based biomarkers of microvascular pathology in Alzheimer's disease.

    LENUS (Irish Health Repository)

    Ewers, Michael

    2012-02-01

    Sporadic Alzheimer\\'s disease (AD) is a genetically complex and chronically progressive neurodegenerative disorder with molecular mechanisms and neuropathologies centering around the amyloidogenic pathway, hyperphosphorylation and aggregation of tau protein, and neurofibrillary degeneration. While cerebrovascular changes have not been traditionally considered to be a central part of AD pathology, a growing body of evidence demonstrates that they may, in fact, be a characteristic feature of the AD brain as well. In particular, microvascular abnormalities within the brain have been associated with pathological AD hallmarks and may precede neurodegeneration. In vivo assessment of microvascular pathology provides a promising approach to develop useful biological markers for early detection and pathological characterization of AD. This review focuses on established blood-based biological marker candidates of microvascular pathology in AD. These candidates include plasma concentration of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) that are increased in AD. Measures of endothelial vasodilatory function including endothelin (ET-1), adrenomedullin (ADM), and atrial natriuretic peptide (ANP), as well as sphingolipids are significantly altered in mild AD or during the predementia stage of mild cognitive impairment (MCI), suggesting sensitivity of these biomarkers for early detection and diagnosis. In conclusion, the emerging clinical diagnostic evidence for the value of blood-based microvascular biomarkers in AD is promising, however, still requires validation in phase II and III diagnostic trials. Moreover, it is still unclear whether the described protein dysbalances are early or downstream pathological events and how the detected systemic microvascular alterations relate to cerebrovascular and neuronal pathologies in the AD brain.

  6. Protein kinase C-α signals P115RhoGEF phosphorylation and RhoA activation in TNF-α-induced mouse brain microvascular endothelial cell barrier dysfunction

    Directory of Open Access Journals (Sweden)

    Deng Xiaolu

    2011-04-01

    Full Text Available Abstract Background Tumor necrosis factor-α (TNF-α, a proinflammatory cytokine, is capable of activating the small GTPase RhoA, which in turn contributes to endothelial barrier dysfunction. However, the underlying signaling mechanisms remained undefined. Therefore, we aimed to determine the role of protein kinase C (PKC isozymes in the mechanism of RhoA activation and in signaling TNF-α-induced mouse brain microvascular endothelial cell (BMEC barrier dysfunction. Methods Bend.3 cells, an immortalized mouse brain endothelial cell line, were exposed to TNF-α (10 ng/mL. RhoA activity was assessed by pull down assay. PKC-α activity was measured using enzyme assasy. BMEC barrier function was measured by transendothelial electrical resistance (TER. p115RhoGEF phosphorylation was detected by autoradiography followed by western blotting. F-actin organization was observed by rhodamine-phalloidin staining. Both pharmacological inhibitors and knockdown approaches were employed to investigate the role of PKC and p115RhoGEF in TNF-α-induced RhoA activation and BMEC permeability. Results We observed that TNF-α induces a rapid phosphorylation of p115RhoGEF, activation of PKC and RhoA in BMECs. Inhibition of conventional PKC by Gö6976 mitigated the TNF-α-induced p115RhoGEF phosphorylation and RhoA activation. Subsequently, we found that these events are regulated by PKC-α rather than PKC-β by using shRNA. In addition, P115-shRNA and n19RhoA (dominant negative mutant of RhoA transfections had no effect on mediating TNF-α-induced PKC-α activation. These data suggest that PKC-α but not PKC-β acts as an upstream regulator of p115RhoGEF phosphorylation and RhoA activation in response to TNF-α. Moreover, depletion of PKC-α, of p115RhoGEF, and inhibition of RhoA activation also prevented TNF-α-induced stress fiber formation and a decrease in TER. Conclusions Taken together, our results show that PKC-α phosphorylation of p115RhoGEF mediates TNF

  7. Analysis of correlations between selected endothelial cell activation markers, disease activity, and nailfold capillaroscopy microvascular changes in systemic lupus erythematosus patients.

    Science.gov (United States)

    Ciołkiewicz, Mariusz; Kuryliszyn-Moskal, Anna; Klimiuk, Piotr Adrian

    2010-02-01

    The aim of the study was to evaluate the correlation between selected serum endothelial cell activation markers such as vascular endothelial growth factor (VEGF), endothelin-1 (ET-1), soluble thrombomodulin (sTM), soluble E-selectin (sE-selectin), disease activity, and microvascular changes determined by nailfold capillaroscopy in patients with systemic lupus erythematosus (SLE). Serum levels of VEGF, ET-1, sTM, and sE-selectin were determined by an enzyme-linked immunosorbent assay in 80 SLE patients. The disease activity was measured with Systemic Lupus Erythematosus Disease Activity Index score. Nailfold capillaroscopy was performed in all patients. Positive correlation was found between VEGF and both ET-1 (r = 0.294, p nailfold capillaroscopy (r = 0.458, p nailfold capillaroscopy. The relationship between changes in nailfold capillaroscopy, endothelial cell activation markers, and the clinical activity of SLE points to an important role of microvascular abnormalities in the clinical manifestation of the disease.

  8. Brain microvascular pericytes are immunoactive in culture: cytokine, chemokine, nitric oxide, and LRP-1 expression in response to lipopolysaccharide

    Directory of Open Access Journals (Sweden)

    Erickson Michelle A

    2011-10-01

    Full Text Available Abstract Background Brain microvascular pericytes are important constituents of the neurovascular unit. These cells are physically the closest cells to the microvascular endothelial cells in brain capillaries. They significantly contribute to the induction and maintenance of the barrier functions of the blood-brain barrier. However, very little is known about their immune activities or their roles in neuroinflammation. Here, we focused on the immunological profile of brain pericytes in culture in the quiescent and immune-challenged state by studying their production of immune mediators such as nitric oxide (NO, cytokines, and chemokines. We also examined the effects of immune challenge on pericyte expression of low density lipoprotein receptor-related protein-1 (LRP-1, a protein involved in the processing of amyloid precursor protein and the brain-to-blood efflux of amyloid-β peptide. Methods Supernatants were collected from primary cultures of mouse brain pericytes. Release of nitric oxide (NO was measured by the Griess reaction and the level of S-nitrosylation of pericyte proteins measured with a modified "biotin-switch" method. Specific mitogen-activated protein kinase (MAPK pathway inhibitors were used to determine involvement of these pathways on NO production. Cytokines and chemokines were analyzed by multianalyte technology. The expression of both subunits of LRP-1 was analyzed by western blot. Results Lipopolysaccharide (LPS induced release of NO by pericytes in a dose-dependent manner that was mediated through MAPK pathways. Nitrative stress resulted in S-nitrosylation of cellular proteins. Eighteen of twenty-three cytokines measured were released constitutively by pericytes or with stimulation by LPS, including interleukin (IL-12, IL-13, IL-9, IL-10, granulocyte-colony stimulating factor, granulocyte macrophage-colony stimulating factor, eotaxin, chemokine (C-C motif ligand (CCL-3, and CCL-4. Pericyte expressions of both subunits of

  9. A subset of group A-like var genes encodes the malaria parasite ligands for binding to human brain endothelial cells

    DEFF Research Database (Denmark)

    Claessens, Antoine; Adams, Yvonne; Ghumra, Ashfaq

    2012-01-01

    Cerebral malaria is the most deadly manifestation of infection with Plasmodium falciparum. The pathology of cerebral malaria is characterized by the accumulation of infected erythrocytes (IEs) in the microvasculature of the brain caused by parasite adhesins on the surface of IEs binding to human...... receptors on microvascular endothelial cells. The parasite and host molecules involved in this interaction are unknown. We selected three P. falciparum strains (HB3, 3D7, and IT/FCR3) for binding to a human brain endothelial cell line (HBEC-5i). The whole transcriptome of isogenic pairs of selected.......029) but not by antibodies from controls with uncomplicated malaria (Mann-Whitney test, P = 0.58). This work describes a binding phenotype for virulence-associated group A P. falciparum erythrocyte membrane protein 1 variants and identifies targets for interventions to treat or prevent cerebral malaria....

  10. Renal microvascular disease in an aging population: a reversible process?

    Science.gov (United States)

    Futrakul, Narisa; Futrakul, Prasit

    2008-01-01

    Renal microvascular disease and tubulointerstitial fibrosis are usually demonstrated in aging in humans and animals. It has recently been proposed that renal microvascular disease is the crucial determinant of tubulointerstitial disease or fibrosis. Enhanced circulating endothelial cell loss is a biomarker that reflects glomerular endothelial injury or renal microvascular disease, and fractional excretion of magnesium (FE Mg) is a sensitive biomarker that reflects an early stage of tubulointerstitial fibrosis. In aging in humans, both of these biomarkers are abnormally elevated. In addition, a glomerular endothelial dysfunction determined by altered hemodynamics associated with peritubular capillary flow reduction is substantiated. A correction of such hemodynamic alteration with vasodilators can effectively improve renal perfusion and restore renal function. Thus, anti-aging therapy can reverse the renal microvascular disease and dysfunction associated with the aging process.

  11. Long-term effects of bariatric surgery on peripheral endothelial function and coronary microvascular function.

    Science.gov (United States)

    Tarzia, Pierpaolo; Lanza, Gaetano A; Sestito, Alfonso; Villano, Angelo; Russo, Giulio; Figliozzi, Stefano; Lamendola, Priscilla; De Vita, Antonio; Crea, Filippo

    We previously demonstrated that bariatric surgery (BS) leads to a short-term significant improvement of endothelial function and coronary microvascular function. In this study we assessed whether BS maintains its beneficial effect at long-term follow up. We studied 19 morbidly obese patients (age 43±9years, 12 women) without any evidence of cardiovascular disease who underwent BS. Patients were studied before BS, at 3 months and at 4.0±1.5years follow up. Peripheral vascular function was assessed by flow-mediated dilation (FMD) and nitrate-mediated dilation (NMD), i.e., brachial artery diameter changes in response to post-ischemic forearm hyperhaemia and to nitroglycerin administration, respectively. Coronary microvascular function was assessed by measuring coronary blood flow (CBF) response to intravenous adenosine and to cold pressor test (CPT) in the left anterior descending coronary artery. Together with improvement of anthropometric and metabolic profile, at long-term follow-up patients showed a significant improvement of FMD (6.43±2.88 vs. 8.21±1.73%, p=0.018), and CBF response to both adenosine (1.73±0.48 vs. 2.58±0.54; pfunction and on coronary microvascular dilator function. Copyright © 2016 Asia Oceania Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.

  12. α-Klotho expression determines nitric oxide synthesis in response to FGF-23 in human aortic endothelial cells.

    Directory of Open Access Journals (Sweden)

    Chih-Ping Chung

    Full Text Available Endothelial cells (ECs express fibroblast growth factor (FGF receptors and are metabolically active after treatment with FGF-23. It is not known if this effect is α-Klotho independent or mediated by humoral or endogenous endothelial α-Klotho. In the present study, we aimed to characterize EC α-Klotho expression within the human vascular tree and to investigate the potential role of α-Klotho in determining FGF-23 mediated EC regulation. Human tissue and ECs from various organs were used for immunohistochemistry and Western blot. Primary cultures of human aortic endothelial cells (HAECs and human brain microvascular endothelial cells (HBMECs were used to generate in vitro cell models. We found endogenous α-Klotho expression in ECs from various organs except in microvascular ECs from human brain. Furthermore, FGF-23 stimulated endothelial nitric oxide synthase (eNOS expression, nitric oxide (NO production, and cell proliferation in HAECs. Interestingly, these effects were not observed in our HBMEC model in vitro. High phosphate treatment and endothelial α-Klotho knockdown mitigated FGF-23 mediated eNOS induction, NO production, and cell proliferation in HAECs. Rescue treatment with soluble α-Klotho did not reverse endothelial FGF-23 resistance caused by reduced or absent α-Klotho expression in HAECs. These novel observations provide evidence for differential α-Klotho functional expression in the human endothelium and its presence may play a role in determining the response to FGF-23 in the vascular tree. α-Klotho was not detected in cerebral microvascular ECs and its absence may render these cells nonresponsive to FGF-23.

  13. HIF-2α Expression Regulates Sprout Formation into 3D Fibrin Matrices in Prolonged Hypoxia in Human Microvascular Endothelial Cells.

    Science.gov (United States)

    Nauta, Tessa D; Duyndam, Monique C A; Weijers, Ester M; van Hinsbergh, Victor M W; Koolwijk, Pieter

    2016-01-01

    During short-term hypoxia, Hypoxia Inducible Factors (particular their subunits HIF-1α and HIF-2α) regulate the expression of many genes including the potent angiogenesis stimulator VEGF. However, in some pathological conditions chronic hypoxia occurs and is accompanied by reduced angiogenesis. We investigated the effect of prolonged hypoxia on the proliferation and sprouting ability of human microvascular endothelial cells and the involvement of the HIFs and Dll4/Notch signaling. Human microvascular endothelial cells (hMVECs), cultured at 20% oxygen for 14 days and seeded on top of 3D fibrin matrices, formed sprouts when stimulated with VEGF-A/TNFα. In contrast, hMVECs precultured at 1% oxygen for 14 days were viable and proliferative, but did not form sprouts into fibrin upon VEGF-A/TNFα stimulation at 1% oxygen. Silencing of HIF-2α with si-RNA partially restored the inhibition of endothelial sprouting, whereas HIF-1α or HIF-3α by si-RNA had no effect. No involvement of Dll4/Notch pathway in the inhibitory effect on endothelial sprouting by prolonged hypoxia was found. In addition, hypoxia decreased the production of urokinase-type plasminogen activator (uPA), needed for migration and invasion, without a significant effect on its inhibitor PAI-1. This was independent of HIF-2α, as si-HIF-2α did not counteract uPA reduction. Prolonged culturing of hMVECs at 1% oxygen inhibited endothelial sprouting into fibrin. Two independent mechanisms contribute. Silencing of HIF-2α with si-RNA partially restored the inhibition of endothelial sprouting pointing to a HIF-2α-dependent mechanism. In addition, reduction of uPA contributed to reduced endothelial tube formation in a fibrin matrix during prolonged hypoxia.

  14. Platelet lysate gel and endothelial progenitors stimulate microvascular network formation in vitro: tissue engineering implications.

    Science.gov (United States)

    Fortunato, Tiago M; Beltrami, Cristina; Emanueli, Costanza; De Bank, Paul A; Pula, Giordano

    2016-05-04

    Revascularisation is a key step for tissue regeneration and complete organ engineering. We describe the generation of human platelet lysate gel (hPLG), an extracellular matrix preparation from human platelets able to support the proliferation of endothelial colony forming cells (ECFCs) in 2D cultures and the formation of a complete microvascular network in vitro in 3D cultures. Existing extracellular matrix preparations require addition of high concentrations of recombinant growth factors and allow only limited formation of capillary-like structures. Additional advantages of our approach over existing extracellular matrices are the absence of any animal product in the composition hPLG and the possibility of obtaining hPLG from patients to generate homologous scaffolds for re-implantation. This discovery has the potential to accelerate the development of regenerative medicine applications based on implantation of microvascular networks expanded ex vivo or the generation of fully vascularised organs.

  15. Systemic and cerebral vascular endothelial growth factor levels increase in murine cerebral malaria along with increased Calpain and caspase activity and can be reduced by erythropoietin treatment

    DEFF Research Database (Denmark)

    Hempel, Casper; Hoyer, Nils; Kildemoes, Anna

    2014-01-01

    The pathogenesis of cerebral malaria (CM) includes compromised microvascular perfusion, increased inflammation, cytoadhesion, and endothelial activation. These events cause blood-brain barrier disruption and neuropathology and associations with the vascular endothelial growth factor (VEGF) signal...

  16. Hydrogen-Rich Saline Attenuates Brain Injury Induced by Cardiopulmonary Bypass and Inhibits Microvascular Endothelial Cell Apoptosis Via the PI3K/Akt/GSK3β Signaling Pathway in Rats

    Directory of Open Access Journals (Sweden)

    Keyan Chen

    2017-10-01

    Full Text Available Background/Aims: Cardiopulmonary bypass (CPB is prone to inducing brain injury during open heart surgery. A hydrogen-rich solution (HRS can prevent oxidation and apoptosis, and inhibit inflammation. This study investigated effects of HRS on brain injury induced by CPB and regulatory mechanisms of the PI3K/Akt/GSK3β signaling pathway. Methods: A rat CPB model and an in vitro cell hypoxia model were established. After HRS treatment, Rat behavior was measured using neurological deficit score; Evans blue (EB was used to assess permeability of the blood-brain barrier (BBB; HE staining was used to observe pathological changes; Inflammatory factors and brain injury markers were detected by ELISA; the PI3K/Akt/GSK3β pathway-related proteins and apoptosis were assessed by western blot, immunohistochemistry and qRT –PCR analyses of brain tissue and neurons. Results: After CPB, brain tissue anatomy was disordered, and cell structure was abnormal. Brain tissue EB content increased. There was an increase in the number of apoptotic cells, an increase in expression of Bax and caspase-3, a decrease in expression of Bcl2, and increases in levels of Akt, GSK3β, P-Akt, and P-GSK3β in brain tissue. HRS treatment attenuated the inflammatory reaction ,brain tissue EB content was significantly reduced and significantly decreased expression levels of Bax, caspase-3, Akt, GSK3β, P-Akt, and P-GSK3β in the brain. After adding the PI3K signaling pathway inhibitor, LY294002, to rat cerebral microvascular endothelial cells (CMECs, HRS could reduce activated Akt expression and downstream regulatory gene phosphorylation of GSK3β expression, and inhibit CMEC apoptosis. Conclusion: The PI3K/Akt/GSK3β signaling pathway plays an important role in the mechanism of CPB-induced brain injury. HRS can reduce CPB-induced brain injury and inhibit CMEC apoptosis through the PI3K/Akt/GSK3β signaling pathway.

  17. (−-Epigallocatechin gallate inhibits endotoxin-induced expression of inflammatory cytokines in human cerebral microvascular endothelial cells

    Directory of Open Access Journals (Sweden)

    Li Jieliang

    2012-07-01

    Full Text Available Abstract Background (−-Epigallocatechin gallate (EGCG is a major polyphenol component of green tea that has antioxidant activities. Lipopolysaccharide (LPS induces inflammatory cytokine production and impairs blood–brain barrier (BBB integrity. We examined the effect of EGCG on LPS-induced expression of the inflammatory cytokines in human cerebral microvascular endothelial cells (hCMECs and BBB permeability. Methods The expression of TNF-α, IL-1β and monocyte chemotactic protein-1 (MCP-1/CCL2 was determined by quantitative real time PCR (qRT-PCR and ELISA. Intercellular adhesion molecule 1 (ICAM-1 and vascular cell adhesion molecule (VCAM in hCMECs were examined by qRT-PCR and Western blotting. Monocytes that adhered to LPS-stimulated endothelial cells were measured by monocyte adhesion assay. Tight junctional factors were detected by qRT-PCR (Claudin 5 and Occludin and immunofluorescence staining (Claudin 5 and ZO-1. The permeability of the hCMEC monolayer was determined by fluorescence spectrophotometry of transmembrane fluorescin and transendothelial electrical resistance (TEER. NF-kB activation was measured by luciferase assay. Results EGCG significantly suppressed the LPS-induced expression of IL-1β and TNF-α in hCMECs. EGCG also inhibited the expression of MCP-1/CCL2, VCAM-1 and ICAM-1. Functional analysis showed that EGCG induced the expression of tight junction proteins (Occludin and Claudin-5 in hCMECs. Investigation of the mechanism showed that EGCG had the ability to inhibit LPS-mediated NF-κB activation. In addition, 67-kD laminin receptor was involved in the anti-inflammatory effect of EGCG. Conclusions Our results demonstrated that LPS induced inflammatory cytokine production in hCMECs, which could be attenuated by EGCG. These data indicate that EGCG has a therapeutic potential for endotoxin-mediated endothelial inflammation.

  18. Regulation of human cerebro-microvascular endothelial baso-lateral adhesion and barrier function by S1P through dual involvement of S1P1 and S1P2 receptors.

    Science.gov (United States)

    Wiltshire, Rachael; Nelson, Vicky; Kho, Dan Ting; Angel, Catherine E; O'Carroll, Simon J; Graham, E Scott

    2016-01-27

    Herein we show that S1P rapidly and acutely reduces the focal adhesion strength and barrier tightness of brain endothelial cells. xCELLigence biosensor technology was used to measure focal adhesion, which was reduced by S1P acutely and this response was mediated through both S1P1 and S1P2 receptors. S1P increased secretion of several pro-inflammatory mediators from brain endothelial cells. However, the magnitude of this response was small in comparison to that mediated by TNFα or IL-1β. Furthermore, S1P did not significantly increase cell-surface expression of any key cell adhesion molecules involved in leukocyte recruitment, included ICAM-1 and VCAM-1. Finally, we reveal that S1P acutely and dynamically regulates microvascular endothelial barrier tightness in a manner consistent with regulated rapid opening followed by closing and strengthening of the barrier. We hypothesise that the role of the S1P receptors in this process is not to cause barrier dysfunction, but is related to controlled opening of the endothelial junctions. This was revealed using real-time measurement of barrier integrity using ECIS ZΘ TEER technology and endothelial viability using xCELLigence technology. Finally, we show that these responses do not occur simply though the pharmacology of a single S1P receptor but involves coordinated action of S1P1 and S1P2 receptors.

  19. The brain microvascular endothelium supports T cell proliferation and has potential for alloantigen presentation.

    Directory of Open Access Journals (Sweden)

    Julie Wheway

    Full Text Available Endothelial cells (EC form the inner lining of blood vessels and are positioned between circulating lymphocytes and tissues. Hypotheses have formed that EC may act as antigen presenting cells based on the intimate interactions with T cells, which are seen in diseases like multiple sclerosis, cerebral malaria (CM and viral neuropathologies. Here, we investigated how human brain microvascular EC (HBEC interact with and support the proliferation of T cells. We found HBEC to express MHC II, CD40 and ICOSL, key molecules for antigen presentation and co-stimulation and to take up fluorescently labeled antigens via macropinocytosis. In co-cultures, we showed that HBEC support and promote the proliferation of CD4(+ and CD8(+ T cells, which both are key in CM pathogenesis, particularly following T cell receptor activation and co-stimulation. Our findings provide novel evidence that HBEC can trigger T cell activation, thereby providing a novel mechanism for neuroimmunological complications of infectious diseases.

  20. Inhibition of dipeptidyl peptidase 4 regulates microvascular endothelial growth induced by inflammatory cytokines

    International Nuclear Information System (INIS)

    Takasawa, Wataru; Ohnuma, Kei; Hatano, Ryo; Endo, Yuko; Dang, Nam H.; Morimoto, Chikao

    2010-01-01

    Research highlights: → TNF-α or IL-1β induces EC proliferation with reduction of CD26 expression. → CD26 siRNA or DPP-4 inhibition enhances TNF-α or IL-1β-induced EC proliferation. → Loss of CD26/DPP-4 enhances aortic sprouting induced by TNF-α or IL-1β. → Capillary formation induced by TNF-α or IL-1β is enahced in the CD26 -/- mice. -- Abstract: CD26/DPP-4 is abundantly expressed on capillary of inflamed lesion as well as effector T cells. Recently, CD26/dipeptidyl peptidase 4 (DPP-4) inhibition has been used as a novel oral therapeutic approach for patients with type 2 diabetes. While accumulating data indicate that vascular inflammation is a key feature of both micro- and macro-vascular complications in diabetes, the direct role of CD26/DPP-4 in endothelial biology is to be elucidated. We herein showed that proinflammatory cytokines such as tumor necrosis factor or interleukin-1 reduce expression of CD26 on microvascular endothelial cells, and that genetical or pharmacological inhibition of CD26/DPP-4 enhances endothelial growth both in vitro and in vivo. With DPP-4 inhibitors being used widely in the treatment of type 2 diabetes, our data strongly suggest that DPP-4 inhibition plays a pivotal role in endothelial growth and may have a potential role in the recovery of local circulation following diabetic vascular complications.

  1. Osteogenic stimulatory conditions enhance growth and maturation of endothelial cell microvascular networks in culture with mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Torbjorn O Pedersen

    2012-12-01

    Full Text Available To optimize culture conditions for in vitro prevascularization of tissue-engineered bone constructs, the development of organotypic blood vessels under osteogenic stimulatory conditions (OM was investigated. Coculture of endothelial cells and mesenchymal stem cells was used to assess proangiogenic effects of mesenchymal stem cells on endothelial cells. Four different culture conditions were evaluated for their effect on development of microvascular endothelial cell networks. Mineralization, deposition of extracellular matrix, and perivascular gene expression were studied in OM. After 3 days, endothelial cells established elongated capillary-like networks, and upregulated expression of vascular markers was seen. After 15 days, all parameters evaluated were significantly increased for cultures in OM. Mature networks developed in OM presented lumens enveloped by basement membrane-like collagen IV, with obvious mineralization and upregulated perivascular gene expression from mesenchymal stem cells. Our results suggest osteogenic stimulatory conditions to be appropriate for in vitro development of vascularized bone implants for tissue engineering.

  2. Reduction in cardiolipin decreases mitochondrial spare respiratory capacity and increases glucose transport into and across human brain cerebral microvascular endothelial cells.

    Science.gov (United States)

    Nguyen, Hieu M; Mejia, Edgard M; Chang, Wenguang; Wang, Ying; Watson, Emily; On, Ngoc; Miller, Donald W; Hatch, Grant M

    2016-10-01

    Microvessel endothelial cells form part of the blood-brain barrier, a restrictively permeable interface that allows transport of only specific compounds into the brain. Cardiolipin is a mitochondrial phospholipid required for function of the electron transport chain and ATP generation. We examined the role of cardiolipin in maintaining mitochondrial function necessary to support barrier properties of brain microvessel endothelial cells. Knockdown of the terminal enzyme of cardiolipin synthesis, cardiolipin synthase, in hCMEC/D3 cells resulted in decreased cellular cardiolipin levels compared to controls. The reduction in cardiolipin resulted in decreased mitochondrial spare respiratory capacity, increased pyruvate kinase activity, and increased 2-deoxy-[(3) H]glucose uptake and glucose transporter-1 expression and localization to membranes in hCMEC/D3 cells compared to controls. The mechanism for the increase in glucose uptake was an increase in adenosine-5'-monophosphate kinase and protein kinase B activity and decreased glycogen synthase kinase 3 beta activity. Knockdown of cardiolipin synthase did not affect permeability of fluorescent dextran across confluent hCMEC/D3 monolayers grown on Transwell(®) inserts. In contrast, knockdown of cardiolipin synthase resulted in an increase in 2-deoxy-[(3) H]glucose transport across these monolayers compared to controls. The data indicate that in hCMEC/D3 cells, spare respiratory capacity is dependent on cardiolipin. In addition, reduction in cardiolipin in these cells alters their cellular energy status and this results in increased glucose transport into and across hCMEC/D3 monolayers. Microvessel endothelial cells form part of the blood-brain barrier, a restrictively permeable interface that allows transport of only specific compounds into the brain. In human adult brain endothelial cell hCMEC/D3 monolayers cultured on Transwell(®) plates, knockdown of cardiolipin synthase results in decrease in mitochondrial

  3. Aging impairs transcriptional regulation of vascular endothelial growth factor in human microvascular endothelial cells: implications for angiogenesis and cell survival.

    Science.gov (United States)

    Ahluwalia, A; Jones, M K; Szabo, S; Tarnawski, A S

    2014-04-01

    In some tissues, aging impairs angiogenesis and reduces expression of vascular endothelial growth factor A (VEGF), a fundamental regulator of angiogenesis. We previously examined angiogenesis in aging and young gastric mucosa in vivo and in vitro and showed that an imbalance between expressions of VEGF (pro-angiogenic factor) and endostatin (anti-angiogenic protein) results in an aging-related impairment of angiogenesis in rats. However, the human relevance of these findings, and whether these mechanisms apply to endothelial cells derived from other tissues, is not clear. Since P-STAT3 and P-CREB are transcription factors that, in association with HIF-1α, can activate VEGF gene expression in some cells (e.g., liver cancer cells, vascular smooth muscle cells), we examined the expression of these two proteins in human dermal microvascular endothelial cells (HMVECs) derived from aging and neonatal individuals. We examined and quantified in vitro angiogenesis, expression of VEGF, P-STAT3, P-CREB and importin-α in HMVECs isolated from neonates (neonatal) and a 66 year old subject (aging). We also examined the effects of treatment with exogenous VEGF and endostatin on in vitro angiogenesis in these cells. Endothelial cells isolated from aging individuals had impaired angiogenesis (vs. neonatal endothelial cells) and reduced expression of VEGF mRNA and protein. Aged HMVECs also had reduced importin-α expression, and reduced expression and nuclear translocation of P-STAT3 and P-CREB. Reduced VEGF gene expression in aged HMVECs strongly correlated with the decreased levels of P-STAT3, P-CREB and importin-α in these cells. Our study clearly demonstrates that endothelial cells from aging individuals have impaired angiogenesis and reduced expression of VEGF likely due to impaired nuclear transport of P-STAT3 and P-CREB transcription factors in these cells.

  4. Defense at the border : the blood-brain barrier versus bacterial foreigners

    NARCIS (Netherlands)

    van Sorge, Nina M.; Doran, Kelly S.

    Bacterial meningitis is among the top ten causes of infectious disease-related deaths worldwide, with up to half of the survivors left with permanent neurological sequelae. The blood-brain barrier (BBB), composed mainly of specialized brain microvascular endothelial cells, maintains biochemical

  5. Urokinase receptor expression on human microvascular endothelial cells is increased by hypoxia: Implications for capillary-like tube formation in a fibrin matrix

    NARCIS (Netherlands)

    Kroon, M.E.; Koolwijk, P.; Vecht, B. van der; Hinsbergh, V.W.M. van

    2000-01-01

    Hypoxia stimulates angiogenesis, the formation of new blood vessels. This study evaluates the direct effect of hypoxia (1% oxygen) on the angiogenic response of human microvascular endothelial cells (hMVECs) seeded on top of a 3-dimensional fibrin matrix, hMVECs stimulated with fibroblast growth

  6. ABCD1 dysfunction alters white matter microvascular perfusion

    DEFF Research Database (Denmark)

    Lauer, Arne; Da, Xiao; Hansen, Mikkel Bo

    2017-01-01

    Cerebral X-linked adrenoleukodystrophy is a devastating neurodegenerative disorder caused by mutations in the ABCD1 gene, which lead to a rapidly progressive cerebral inflammatory demyelination in up to 60% of affected males. Selective brain endothelial dysfunction and increased permeability...... of the blood–brain barrier suggest that white matter microvascular dysfunction contributes to the conversion to cerebral disease. Applying a vascular model to conventional dynamic susceptibility contrast magnetic reson- ance perfusion imaging, we demonstrate that lack of ABCD1 function causes increased...... capillary flow heterogeneity in asymptom- atic hemizygotes predominantly in the white matter regions and developmental stages with the highest probability for conversion to cerebral disease. In subjects with ongoing inflammatory demyelination we observed a sequence of increased capillary flow hetero...

  7. Phthalimide neovascular factor 1 (PNF1) modulates MT1-MMP activity in human microvascular endothelial cells.

    Science.gov (United States)

    Wieghaus, Kristen A; Gianchandani, Erwin P; Neal, Rebekah A; Paige, Mikell A; Brown, Milton L; Papin, Jason A; Botchwey, Edward A

    2009-07-01

    We are creating synthetic pharmaceuticals with angiogenic activity and potential to promote vascular invasion. We previously demonstrated that one of these molecules, phthalimide neovascular factor 1 (PNF1), significantly expands microvascular networks in vivo following sustained release from poly(lactic-co-glycolic acid) (PLAGA) films. In addition, to probe PNF1 mode of action, we recently applied a novel pathway-based compendium analysis to a multi-timepoint, controlled microarray data set of PNF1-treated (vs. control) human microvascular endothelial cells (HMVECs), and we identified induction of tumor necrosis factor-alpha (TNF-alpha) and, subsequently, transforming growth factor-beta (TGF-beta) signaling networks by PNF1. Here we validate this microarray data set with quantitative real-time polymerase chain reaction (RT-PCR) analysis. Subsequently, we probe this data set and identify three specific TGF-beta-induced genes with regulation by PNF1 conserved over multiple timepoints-amyloid beta (A4) precursor protein (APP), early growth response 1 (EGR-1), and matrix metalloproteinase 14 (MMP14 or MT1-MMP)-that are also implicated in angiogenesis. We further focus on MMP14 given its unique role in angiogenesis, and we validate MT1-MMP modulation by PNF1 with an in vitro fluorescence assay that demonstrates the direct effects that PNF1 exerts on functional metalloproteinase activity. We also utilize endothelial cord formation in collagen gels to show that PNF1-induced stimulation of endothelial cord network formation in vitro is in some way MT1-MMP-dependent. Ultimately, this new network analysis of our transcriptional footprint characterizing PNF1 activity 1-48 h post-supplementation in HMVECs coupled with corresponding validating experiments suggests a key set of a few specific targets that are involved in PNF1 mode of action and important for successful promotion of the neovascularization that we have observed by the drug in vivo.

  8. Microvascular pericytes in healthy and diseased kidneys

    Science.gov (United States)

    Pan, Szu-Yu; Chang, Yu-Ting; Lin, Shuei-Liong

    2014-01-01

    Pericytes are interstitial mesenchymal cells found in many major organs. In the kidney, microvascular pericytes are defined anatomically as extensively branched, collagen-producing cells in close contact with endothelial cells. Although many molecular markers have been proposed, none of them can identify the pericytes with satisfactory specificity or sensitivity. The roles of microvascular pericytes in kidneys were poorly understood in the past. Recently, by using genetic lineage tracing to label collagen-producing cells or mesenchymal cells, the elusive characteristics of the pericytes have been illuminated. The purpose of this article is to review recent advances in the understanding of microvascular pericytes in the kidneys. In healthy kidney, the pericytes are found to take part in the maintenance of microvascular stability. Detachment of the pericytes from the microvasculature and loss of the close contact with endothelial cells have been observed during renal insult. Renal microvascular pericytes have been shown to be the major source of scar-forming myofibroblasts in fibrogenic kidney disease. Targeting the crosstalk between pericytes and neighboring endothelial cells or tubular epithelial cells may inhibit the pericyte–myofibroblast transition, prevent peritubular capillary rarefaction, and attenuate renal fibrosis. In addition, renal pericytes deserve attention for their potential to produce erythropoietin in healthy kidneys as pericytes stand in the front line, sensing the change of oxygenation and hemoglobin concentration. Further delineation of the mechanisms underlying the reduced erythropoietin production occurring during pericyte–myofibroblast transition may be promising for the development of new treatment strategies for anemia in chronic kidney disease. PMID:24465134

  9. Withaferin A promotes proliferation and migration of brain ...

    African Journals Online (AJOL)

    from the literature, we designed an experiment to study the effect of withaferin A on suppression of brain tumor growth in a nude mice model with an attempt to develop potent therapeutic agent. EXPERIMENTAL. Cell line and culture. The mouse brain microvascular endothelial cell line, BALB-5023 was purchased from the.

  10. Long noncoding RNA-MEG3 is involved in diabetes mellitus-related microvascular dysfunction

    Energy Technology Data Exchange (ETDEWEB)

    Qiu, Gui-Zhen [Department of Health, Linyi People' s Hospital, Shandong University, Shandong (China); Tian, Wei [Department of Nursing, Linyi Oncosurgical Hospital, Shandong (China); Fu, Hai-Tao [Department of Ophthalmology, Linyi People' s Hospital, Shandong University, Shandong (China); Li, Chao-Peng, E-mail: lcpcn@163.com [Eye Institute of Xuzhou, Jiangsu (China); Liu, Ban, E-mail: liuban@126.com [Department of Cardiology, Shanghai Tenth People' s Hospital, Tongji University School of Medicine, Shanghai (China)

    2016-02-26

    Microvascular dysfunction is an important characteristic of diabetic retinopathy. Long non-coding RNAs (lncRNAs) play important roles in diverse biological processes. In this study, we investigated the role of lncRNA-MEG3 in diabetes-related microvascular dysfunction. We show that MEG3 expression level is significantly down-regulated in the retinas of STZ-induced diabetic mice, and endothelial cells upon high glucose and oxidative stress. MEG3 knockdown aggravates retinal vessel dysfunction in vivo, as shown by serious capillary degeneration, and increased microvascular leakage and inflammation. MEG3 knockdown also regulates retinal endothelial cell proliferation, migration, and tube formation in vitro. The role of MEG3 in endothelial cell function is mainly mediated by the activation of PI3k/Akt signaling. MEG3 up-regulation may serve as a therapeutic strategy for treating diabetes-related microvascular complications. - Highlights: • LncRNA-MEG3 level is down-regulated upon diabetic stress. • MEG3 knockdown aggravates retinal vascular dysfunction in vivo. • MEG3 regulates retinal endothelial cell function in vitro. • MEG3 regulates endothelial cell function through PI3k/Akt signaling.

  11. Long noncoding RNA-MEG3 is involved in diabetes mellitus-related microvascular dysfunction

    International Nuclear Information System (INIS)

    Qiu, Gui-Zhen; Tian, Wei; Fu, Hai-Tao; Li, Chao-Peng; Liu, Ban

    2016-01-01

    Microvascular dysfunction is an important characteristic of diabetic retinopathy. Long non-coding RNAs (lncRNAs) play important roles in diverse biological processes. In this study, we investigated the role of lncRNA-MEG3 in diabetes-related microvascular dysfunction. We show that MEG3 expression level is significantly down-regulated in the retinas of STZ-induced diabetic mice, and endothelial cells upon high glucose and oxidative stress. MEG3 knockdown aggravates retinal vessel dysfunction in vivo, as shown by serious capillary degeneration, and increased microvascular leakage and inflammation. MEG3 knockdown also regulates retinal endothelial cell proliferation, migration, and tube formation in vitro. The role of MEG3 in endothelial cell function is mainly mediated by the activation of PI3k/Akt signaling. MEG3 up-regulation may serve as a therapeutic strategy for treating diabetes-related microvascular complications. - Highlights: • LncRNA-MEG3 level is down-regulated upon diabetic stress. • MEG3 knockdown aggravates retinal vascular dysfunction in vivo. • MEG3 regulates retinal endothelial cell function in vitro. • MEG3 regulates endothelial cell function through PI3k/Akt signaling.

  12. Astrocyte–endothelial interactions and blood–brain barrier permeability*

    Science.gov (United States)

    Abbott, N Joan

    2002-01-01

    The blood–brain barrier (BBB) is formed by brain endothelial cells lining the cerebral microvasculature, and is an important mechanism for protecting the brain from fluctuations in plasma composition, and from circulating agents such as neurotransmitters and xenobiotics capable of disturbing neural function. The barrier also plays an important role in the homeostatic regulation of the brain microenvironment necessary for the stable and co-ordinated activity of neurones. The BBB phenotype develops under the influence of associated brain cells, especially astrocytic glia, and consists of more complex tight junctions than in other capillary endothelia, and a number of specific transport and enzyme systems which regulate molecular traffic across the endothelial cells. Transporters characteristic of the BBB phenotype include both uptake mechanisms (e.g. GLUT-1 glucose carrier, L1 amino acid transporter) and efflux transporters (e.g. P-glycoprotein). In addition to a role in long-term barrier induction and maintenance, astrocytes and other cells can release chemical factors that modulate endothelial permeability over a time-scale of seconds to minutes. Cell culture models, both primary and cell lines, have been used to investigate aspects of barrier induction and modulation. Conditioned medium taken from growing glial cells can reproduce some of the inductive effects, evidence for involvement of diffusible factors. However, for some features of endothelial differentiation and induction, the extracellular matrix plays an important role. Several candidate molecules have been identified, capable of mimicking aspects of glial-mediated barrier induction of brain endothelium; these include TGFβ, GDNF, bFGF, IL-6 and steroids. In addition, factors secreted by brain endothelial cells including leukaemia inhibitory factor (LIF) have been shown to induce astrocytic differentiation. Thus endothelium and astrocytes are involved in two-way induction. Short-term modulation of brain

  13. Palmitate-induced inflammatory pathways in human adipose microvascular endothelial cells promote monocyte adhesion and impair insulin transcytosis.

    Science.gov (United States)

    Pillon, Nicolas J; Azizi, Paymon M; Li, Yujin E; Liu, Jun; Wang, Changsen; Chan, Kenny L; Hopperton, Kathryn E; Bazinet, Richard P; Heit, Bryan; Bilan, Philip J; Lee, Warren L; Klip, Amira

    2015-07-01

    Obesity is associated with inflammation and immune cell recruitment to adipose tissue, muscle and intima of atherosclerotic blood vessels. Obesity and hyperlipidemia are also associated with tissue insulin resistance and can compromise insulin delivery to muscle. The muscle/fat microvascular endothelium mediates insulin delivery and facilitates monocyte transmigration, yet its contribution to the consequences of hyperlipidemia is poorly understood. Using primary endothelial cells from human adipose tissue microvasculature (HAMEC), we investigated the effects of physiological levels of fatty acids on endothelial inflammation and function. Expression of cytokines and adhesion molecules was measured by RT-qPCR. Signaling pathways were evaluated by pharmacological manipulation and immunoblotting. Surface expression of adhesion molecules was determined by immunohistochemistry. THP1 monocyte interaction with HAMEC was measured by cell adhesion and migration across transwells. Insulin transcytosis was measured by total internal reflection fluorescence microscopy. Palmitate, but not palmitoleate, elevated the expression of IL-6, IL-8, TLR2 (Toll-like receptor 2), and intercellular adhesion molecule 1 (ICAM-1). HAMEC had markedly low fatty acid uptake and oxidation, and CD36 inhibition did not reverse the palmitate-induced expression of adhesion molecules, suggesting that inflammation did not arise from palmitate uptake/metabolism. Instead, inhibition of TLR4 to NF-κB signaling blunted palmitate-induced ICAM-1 expression. Importantly, palmitate-induced surface expression of ICAM-1 promoted monocyte binding and transmigration. Conversely, palmitate reduced insulin transcytosis, an effect reversed by TLR4 inhibition. In summary, palmitate activates inflammatory pathways in primary microvascular endothelial cells, impairing insulin transport and increasing monocyte transmigration. This behavior may contribute in vivo to reduced tissue insulin action and enhanced tissue

  14. Endothelin-1 Mediates Brain Microvascular Dysfunction Leading to Long-Term Cognitive Impairment in a Model of Experimental Cerebral Malaria.

    Directory of Open Access Journals (Sweden)

    Brandi D Freeman

    2016-03-01

    Full Text Available Plasmodium falciparum infection causes a wide spectrum of diseases, including cerebral malaria, a potentially life-threatening encephalopathy. Vasculopathy is thought to contribute to cerebral malaria pathogenesis. The vasoactive compound endothelin-1, a key participant in many inflammatory processes, likely mediates vascular and cognitive dysfunctions in cerebral malaria. We previously demonstrated that C57BL6 mice infected with P. berghei ANKA, our fatal experimental cerebral malaria model, sustained memory loss. Herein, we demonstrate that an endothelin type A receptor (ETA antagonist prevented experimental cerebral malaria-induced neurocognitive impairments and improved survival. ETA antagonism prevented blood-brain barrier disruption and cerebral vasoconstriction during experimental cerebral malaria, and reduced brain endothelial activation, diminishing brain microvascular congestion. Furthermore, exogenous endothelin-1 administration to P. berghei NK65-infected mice, a model generally regarded as a non-cerebral malaria negative control for P. berghei ANKA infection, led to experimental cerebral malaria-like memory deficits. Our data indicate that endothelin-1 is critical in the development of cerebrovascular and cognitive impairments with experimental cerebral malaria. This vasoactive peptide may thus serve as a potential target for adjunctive therapy in the management of cerebral malaria.

  15. Endothelial cell marker PAL-E reactivity in brain tumor, developing brain, and brain disease

    NARCIS (Netherlands)

    Leenstra, S.; Troost, D.; Das, P. K.; Claessen, N.; Becker, A. E.; Bosch, D. A.

    1993-01-01

    The endothelial cell marker PAL-E is not reactive to vessels in the normal brain. The present study concerns the PAL-E reactivity in brain tumors in contrast to normal brain and nonneoplastic brain disease. A total of 122 specimens were examined: brain tumors (n = 94), nonneoplastic brain disease (n

  16. Influenza H5N1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells

    Directory of Open Access Journals (Sweden)

    Yuen Kit M

    2009-10-01

    Full Text Available Abstract Background Highly pathogenic avian influenza (HPAI H5N1 virus is entrenched in poultry in Asia and Africa and continues to infect humans zoonotically causing acute respiratory disease syndrome and death. There is evidence that the virus may sometimes spread beyond respiratory tract to cause disseminated infection. The primary target cell for HPAI H5N1 virus in human lung is the alveolar epithelial cell. Alveolar epithelium and its adjacent lung microvascular endothelium form host barriers to the initiation of infection and dissemination of influenza H5N1 infection in humans. These are polarized cells and the polarity of influenza virus entry and egress as well as the secretion of cytokines and chemokines from the virus infected cells are likely to be central to the pathogenesis of human H5N1 disease. Aim To study influenza A (H5N1 virus replication and host innate immune responses in polarized primary human alveolar epithelial cells and lung microvascular endothelial cells and its relevance to the pathogenesis of human H5N1 disease. Methods We use an in vitro model of polarized primary human alveolar epithelial cells and lung microvascular endothelial cells grown in transwell culture inserts to compare infection with influenza A subtype H1N1 and H5N1 viruses via the apical or basolateral surfaces. Results We demonstrate that both influenza H1N1 and H5N1 viruses efficiently infect alveolar epithelial cells from both apical and basolateral surface of the epithelium but release of newly formed virus is mainly from the apical side of the epithelium. In contrast, influenza H5N1 virus, but not H1N1 virus, efficiently infected polarized microvascular endothelial cells from both apical and basolateral aspects. This provides a mechanistic explanation for how H5N1 virus may infect the lung from systemic circulation. Epidemiological evidence has implicated ingestion of virus-contaminated foods as the source of infection in some instances and our

  17. Label-free quantitative cell division monitoring of endothelial cells by digital holographic microscopy

    Science.gov (United States)

    Kemper, Björn; Bauwens, Andreas; Vollmer, Angelika; Ketelhut, Steffi; Langehanenberg, Patrik; Müthing, Johannes; Karch, Helge; von Bally, Gert

    2010-05-01

    Digital holographic microscopy (DHM) enables quantitative multifocus phase contrast imaging for nondestructive technical inspection and live cell analysis. Time-lapse investigations on human brain microvascular endothelial cells demonstrate the use of DHM for label-free dynamic quantitative monitoring of cell division of mother cells into daughter cells. Cytokinetic DHM analysis provides future applications in toxicology and cancer research.

  18. Effect of penehyclidine hydrochloride on β-arrestin-1 expression in lipopolysaccharide-induced human pulmonary microvascular endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhan, J. [Department of Anesthesiology, Zhongnan Hospital, Wuhan University, Wuhan, Hubei (China); Xiao, F. [Department of Osteology, Pu Ai Hospital, Huazhong University of Science and Technology, Wuhan, Hubei (China); Zhang, Z.Z.; Wang, Y.P.; Chen, K.; Wang, Y.L. [Department of Anesthesiology, Zhongnan Hospital, Wuhan University, Wuhan, Hubei (China)

    2013-12-02

    β-arrestins are expressed proteins that were first described, and are well-known, as negative regulators of G protein-coupled receptor signaling. Penehyclidine hydrochloride (PHC) is a new anti-cholinergic drug that can inhibit biomembrane lipid peroxidation, and decrease cytokines and oxyradicals. However, to date, no reports on the effects of PHC on β-arrestin-1 in cells have been published. The aim of this study was to investigate the effect of PHC on β-arrestin-1 expression in lipopolysaccharide (LPS)-induced human pulmonary microvascular endothelial cells (HPMEC). Cultured HPMEC were pretreated with PHC, followed by LPS treatment. Muscarinic receptor mRNAs were assayed by real-time quantitative PCR. Cell viability was assayed by the methyl thiazolyl tetrazolium (MTT) conversion test. The dose and time effects of PHC on β-arrestin-1 expression in LPS-induced HPMEC were determined by Western blot analysis. Cell malondialdehyde (MDA) level and superoxide dismutase (SOD) activity were measured. It was found that the M{sub 3} receptor was the one most highly expressed, and was activated 5 min after LPS challenge. Furthermore, 2 μg/mL PHC significantly upregulated expression of β-arrestin-1 within 10 to 15 min. Compared with the control group, MDA levels in cells were remarkably increased and SOD activities were significantly decreased in LPS pretreated cells, while PHC markedly decreased MDA levels and increased SOD activities. We conclude that PHC attenuated ROS injury by upregulating β-arrestin-1 expression, thereby implicating a mechanism by which PHC may exert its protective effects against LPS-induced pulmonary microvascular endothelial cell injury.

  19. Effect of penehyclidine hydrochloride on β-arrestin-1 expression in lipopolysaccharide-induced human pulmonary microvascular endothelial cells

    International Nuclear Information System (INIS)

    Zhan, J.; Xiao, F.; Zhang, Z.Z.; Wang, Y.P.; Chen, K.; Wang, Y.L.

    2013-01-01

    β-arrestins are expressed proteins that were first described, and are well-known, as negative regulators of G protein-coupled receptor signaling. Penehyclidine hydrochloride (PHC) is a new anti-cholinergic drug that can inhibit biomembrane lipid peroxidation, and decrease cytokines and oxyradicals. However, to date, no reports on the effects of PHC on β-arrestin-1 in cells have been published. The aim of this study was to investigate the effect of PHC on β-arrestin-1 expression in lipopolysaccharide (LPS)-induced human pulmonary microvascular endothelial cells (HPMEC). Cultured HPMEC were pretreated with PHC, followed by LPS treatment. Muscarinic receptor mRNAs were assayed by real-time quantitative PCR. Cell viability was assayed by the methyl thiazolyl tetrazolium (MTT) conversion test. The dose and time effects of PHC on β-arrestin-1 expression in LPS-induced HPMEC were determined by Western blot analysis. Cell malondialdehyde (MDA) level and superoxide dismutase (SOD) activity were measured. It was found that the M 3 receptor was the one most highly expressed, and was activated 5 min after LPS challenge. Furthermore, 2 μg/mL PHC significantly upregulated expression of β-arrestin-1 within 10 to 15 min. Compared with the control group, MDA levels in cells were remarkably increased and SOD activities were significantly decreased in LPS pretreated cells, while PHC markedly decreased MDA levels and increased SOD activities. We conclude that PHC attenuated ROS injury by upregulating β-arrestin-1 expression, thereby implicating a mechanism by which PHC may exert its protective effects against LPS-induced pulmonary microvascular endothelial cell injury

  20. Increased expression of endothelial antigen PAL-E in human diabetic retinopathy correlates with microvascular leakage

    NARCIS (Netherlands)

    Schlingemann, R. O.; Hofman, P.; Vrensen, G. F.; Blaauwgeers, H. G.

    1999-01-01

    AIMS/HYPOTHESIS: The Pathologische Anatomie Leiden-Endothelium (PAL-E) antigen is a marker for loss of the blood-brain barrier function in brain tumours. It is endothelium specific and is associated with the endothelial plasmalemmal vesicles (caveolae) involved in transcellular transport. To test

  1. Non-invasive detection and quantification of brain microvascular deficits by near-infrared spectroscopy in a rat model of Vascular Cognitive Impairment

    Science.gov (United States)

    Hallacoglu, Bertan; Sassaroli, Angelo M.; Rosenberg, Irwin H.; Troen, Aron; Fantini, Sergio

    2011-02-01

    Structural abnormalities in brain microvasculature are commonly associated with Alzheimer's Disease and other dementias. However, the extent to which structural microvascular abnormalities cause functional impairments in brain circulation and thereby to cognitive impairment is unclear. Non-invasive, near-infrared spectroscopy (NIRS) methods can be used to determine the absolute hemoglobin concentration and saturation in brain tissue, from which additional parameters such as cerebral blood volume (a theoretical correlate of brain microvascular density) can be derived. Validating such NIRS parameters in animal models, and understanding their relationship to cognitive function is an important step in the ultimate application of these methods to humans. To this end we applied a non-invasive multidistance NIRS method to determine the absolute concentration and saturation of cerebral hemoglobin in rat, by separately measuring absorption and reduced scattering coefficients without relying on pre- or post-correction factors. We applied this method to study brain circulation in folate deficient rats, which express brain microvascular pathology1 and which we have shown to develop cognitive impairment.2 We found absolute brain hemoglobin concentration ([HbT]) and oxygen saturation (StO2) to be significantly lower in folate deficient rats (n=6) with respect to control rats (n=5) (for [HbT]: 73+/-10 μM vs. 95+/-14 μM for StO2: 55%+/-7% vs. 66% +/-4%), implicating microvascular pathology and diminished oxygen delivery as a mechanism of cognitive impairment. More generally, our study highlights how noninvasive, absolute NIRS measurements can provide unique insight into the pathophysiology of Vascular Cognitive Impairment. Applying this method to this and other rat models of cognitive impairment will help to validate physiologically meaningful NIRS parameters for the ultimate goal of studying cerebral microvascular disease and cognitive decline in humans.

  2. Evaluating the involvement of cerebral microvascular endothelial Na+/K+-ATPase and Na+-K+-2Cl- co-transporter in electrolyte fluxes in an in vitro blood-brain barrier model of dehydration

    DEFF Research Database (Denmark)

    Lykke, Kasper; Assentoft, Mette; Hørlyck, Sofie

    2018-01-01

    The blood-brain barrier (BBB) is involved in brain water and salt homeostasis. Blood osmolarity increases during dehydration and water is osmotically extracted from the brain. The loss of water is less than expected from pure osmotic forces, due to brain electrolyte accumulation. Although...... dehydration, we employed a tight in vitro co-culture BBB model with primary cultures of brain endothelial cells and astrocytes. The Na+/K+-ATPase and the NKCC1 were both functionally dominant in the abluminal membrane. Exposure of the in vitro BBB model to conditions mimicking systemic dehydration, i...... isozymes. Abluminally expressed endothelial Na+/K+-ATPase, and not NKCC1, may therefore counteract osmotic brain water loss during systemic dehydration by promoting brain Na+ accumulation....

  3. [Recent advances on pericytes in microvascular dysfunction and traditional Chinese medicine prevention].

    Science.gov (United States)

    Liu, Lei; Liu, Jian-Xun; Guo, Hao; Ren, Jian-Xun

    2017-08-01

    Pericytesis a kind of widespread vascular mural cells embedded within the vascular basement membrane of blood microvessels, constituting the barrier of capillaries and tissue spaces together with endothelial cells. Pericytes communicate with microvascular endothelial cells through cell connections or paracrine signals, playing an important role in important physiological processes such as blood flow, vascular permeability and vascular formation. Pericytes dysfunction may participate in some microvascular dysfunction, and also mediate pathological repair process, therefore pericytes attracted more and more attention. Traditional Chinese medicine suggests that microvascular dysfunction belongs to the collaterals disease; Qi stagnation and blood stasis in collaterals result in function imbalance of internal organs. Traditional Chinese medicine (TCM) has shown effects on pericytes in microvascular dysfunction, for example qi reinforcing blood-circulation activating medicines can reduce the damage of retinal pericytes in diabetic retinopathy. However, there are some limitations of research fields, inaccuracy of research techniques and methods, and lack of mechanism elaboration depth in the study of microvascular lesion pericytes. This paper reviewed the biological characteristics of pericytes and pericytes in microvascular dysfunction, as well as the intervention study of TCM on pericytes. The article aims to provide reference for the research of pericytes in microvascular dysfunction and the TCM study on pericytes. Copyright© by the Chinese Pharmaceutical Association.

  4. GM-CSF ameliorates microvascular barrier integrity via pericyte-derived Ang-1 in wound healing.

    Science.gov (United States)

    Yan, Min; Hu, Yange; Yao, Min; Bao, Shisan; Fang, Yong

    2017-11-01

    Skin wound healing involves complex coordinated interactions of cells, tissues, and mediators. Maintaining microvascular barrier integrity is one of the key events for endothelial homeostasis during wound healing. Vasodilation is observed after vasoconstriction, which causes blood vessels to become porous, facilitates leukocyte infiltration and aids angiogenesis at the wound-area, postinjury. Eventually, vessel integrity has to be reestablished for vascular maturation. Numerous studies have found that granulocyte macrophage colony-stimulating factor (GM-CSF) accelerates wound healing by inducing recruitment of repair cells into the injury area and releases of cytokines. However, whether GM-CSF is involving in the maintaining of microvascular barrier integrity and the underlying mechanism remain still unclear. Aim of this study was to investigate the effects of GM-CSF on modulation of microvascular permeability in wound healing and underlying mechanisms. Wound closure and microvascular leakage was investigated using a full-thickness skin wound mouse model after GM-CSF intervention. The endothelial permeability was measured by Evans blue assay in vivo and in vitro endothelium/pericyte co-culture system using a FITC-Dextran permeability assay. To identify the source of angiopoietin-1 (Ang-1), double staining is used in vivo and ELISA and qPCR are used in vitro. To determine the specific effect of Ang-1 on GM-CSF maintaining microvascular stabilization, Ang-1 siRNA was applied to inhibit Ang-1 production in vivo and in vitro. Wound closure was significantly accelerated and microvascular leakage was ameliorated after GM-CSF treatment in mouse wound sites. GM-CSF decreased endothelial permeability through tightening endothelial junctions and increased Ang-1 protein level that was derived by perictye. Furthermore, applications of siRNAAng-1 inhibited GM-CSF mediated protection of microvascular barrier integrity both in vivo and in vitro. Our data indicate that GM

  5. Gliovascular and cytokine interactions modulate brain endothelial barrier in vitro.

    Science.gov (United States)

    Chaitanya, Ganta V; Cromer, Walter E; Wells, Shannon R; Jennings, Merilyn H; Couraud, P Olivier; Romero, Ignacio A; Weksler, Babette; Erdreich-Epstein, Anat; Mathis, J Michael; Minagar, Alireza; Alexander, J Steven

    2011-11-23

    The glio-vascular unit (G-unit) plays a prominent role in maintaining homeostasis of the blood-brain barrier (BBB) and disturbances in cells forming this unit may seriously dysregulate BBB. The direct and indirect effects of cytokines on cellular components of the BBB are not yet unclear. The present study compares the effects of cytokines and cytokine-treated astrocytes on brain endothelial barrier. 3-dimensional transwell co-cultures of brain endothelium and related-barrier forming cells with astrocytes were used to investigate gliovascular barrier responses to cytokines during pathological stresses. Gliovascular barrier was measured using trans-endothelial electrical resistance (TEER), a sensitive index of in vitro barrier integrity. We found that neither TNF-α, IL-1β or IFN-γ directly reduced barrier in human or mouse brain endothelial cells or ECV-304 barrier (independent of cell viability/metabolism), but found that astrocyte exposure to cytokines in co-culture significantly reduced endothelial (and ECV-304) barrier. These results indicate that the barrier established by human and mouse brain endothelial cells (and other cells) may respond positively to cytokines alone, but that during pathological conditions, cytokines dysregulate the barrier forming cells indirectly through astrocyte activation involving reorganization of junctions, matrix, focal adhesion or release of barrier modulating factors (e.g. oxidants, MMPs). © 2011 Chaitanya et al; licensee BioMed Central Ltd.

  6. Cardiac microvascular endothelial cells express a functional Ca+ -sensing receptor.

    Science.gov (United States)

    Berra Romani, Roberto; Raqeeb, Abdul; Laforenza, Umberto; Scaffino, Manuela Federica; Moccia, Francesco; Avelino-Cruz, Josè Everardo; Oldani, Amanda; Coltrini, Daniela; Milesi, Veronica; Taglietti, Vanni; Tanzi, Franco

    2009-01-01

    The mechanism whereby extracellular Ca(2+) exerts the endothelium-dependent control of vascular tone is still unclear. In this study, we assessed whether cardiac microvascular endothelial cells (CMEC) express a functional extracellular Ca(2+)-sensing receptor (CaSR) using a variety of techniques. CaSR mRNA was detected using RT-PCR, and CaSR protein was identified by immunocytochemical analysis. In order to assess the functionality of the receptor, CMEC were loaded with the Ca(2+)-sensitive fluorochrome, Fura-2/AM. A number of CaSR agonists, such as spermine, Gd(3+), La(3+) and neomycin, elicited a heterogeneous intracellular Ca(2+) signal, which was abolished by disruption of inositol 1,4,5-trisphosphate (InsP(3)) signaling and by depletion of intracellular stores with cyclopiazonic acid. The inhibition of the Na(+)/Ca(2+) exchanger upon substitution of extracellular Na(+) unmasked the Ca(2+) signal triggered by an increase in extracellular Ca(2+) levels. Finally, aromatic amino acids, which function as allosteric activators of CaSR, potentiated the Ca(2+) response to the CaSR agonist La(3+). These data provide evidence that CMEC express CaSR, which is able to respond to physiological agonists by mobilizing Ca(2+) from intracellular InsP(3)-sensitive stores. Copyright 2008 S. Karger AG, Basel.

  7. Resveratrol Targeting of Carcinogen-Induced Brain Endothelial Cell Inflammation Biomarkers MMP-9 and COX-2 is Sirt1-Independent

    Directory of Open Access Journals (Sweden)

    Borhane Annabi

    2012-01-01

    Full Text Available The occurrence of a functional relationship between the release of metalloproteinases (MMPs and the expression of cyclooxygenase (COX-2, two inducible pro-inflammatory biomarkers with important pro-angiogenic effects, has recently been inferred. While brain endothelial cells play an essential role as structural and functional components of the blood-brain barrier (BBB, increased BBB breakdown is thought to be linked to neuroinflammation. Chemopreventive mechanisms targeting both MMPs and COX-2 however remain poorly investigated. In this study, we evaluated the pharmacological targeting of Sirt1 by the diet-derived and antiinflammatory polyphenol resveratrol. Total RNA, cell lysates, and conditioned culture media from human brain microvascular endothelial cells (HBMEC were analyzed using qRT-PCR, immunoblotting, and zymography respectively. Tissue scan microarray analysis of grade I–IV brain tumours cDNA revealed increased gene expression of Sirt-1 from grade I–III but surprisingly not in grade IV brain tumours. HBMEC were treated with a combination of resveratrol and phorbol 12-myristate 13-acetate (PMA, a carcinogen known to increase MMP-9 and COX-2 through NF-κB. We found that resveratrol efficiently reversed the PMA-induced MMP-9 secretion and COX-2 expression. Gene silencing of Sirt1, a critical modulator of angiogenesis and putative target of resveratrol, did not lead to significant reversal of MMP-9 and COX-2 inhibition. Decreased resveratrol inhibitory potential of carcinogen-induced IκB phosphorylation in siSirt1-transfected HBMEC was however observed. Our results suggest that resveratrol may prevent BBB disruption during neuroinflammation by inhibiting MMP-9 and COX-2 and act as a pharmacological NF-κB signal transduction inhibitor independent of Sirt1.

  8. Effects of amelogenins on angiogenesis-associated processes of endothelial cells

    DEFF Research Database (Denmark)

    Almqvist, S; Kleinman, H K; Werthén, M

    2011-01-01

    To study the effects of an amelogenin mixture on integrin-dependent adhesion, DNA synthesis and apoptosis of cultured human dermal microvascular endothelial cells and angiogenesis in an organotypic assay.......To study the effects of an amelogenin mixture on integrin-dependent adhesion, DNA synthesis and apoptosis of cultured human dermal microvascular endothelial cells and angiogenesis in an organotypic assay....

  9. Platelets alter gene expression profile in human brain endothelial cells in an in vitro model of cerebral malaria.

    Directory of Open Access Journals (Sweden)

    Mathieu Barbier

    Full Text Available Platelet adhesion to the brain microvasculature has been associated with cerebral malaria (CM in humans, suggesting that platelets play a role in the pathogenesis of this syndrome. In vitro co-cultures have shown that platelets can act as a bridge between Plasmodium falciparum-infected red blood cells (pRBC and human brain microvascular endothelial cells (HBEC and potentiate HBEC apoptosis. Using cDNA microarray technology, we analyzed transcriptional changes of HBEC in response to platelets in the presence or the absence of tumor necrosis factor (TNF and pRBC, which have been reported to alter gene expression in endothelial cells. Using a rigorous statistical approach with multiple test corrections, we showed a significant effect of platelets on gene expression in HBEC. We also detected a strong effect of TNF, whereas there was no transcriptional change induced specifically by pRBC. Nevertheless, a global ANOVA and a two-way ANOVA suggested that pRBC acted in interaction with platelets and TNF to alter gene expression in HBEC. The expression of selected genes was validated by RT-qPCR. The analysis of gene functional annotation indicated that platelets induce the expression of genes involved in inflammation and apoptosis, such as genes involved in chemokine-, TREM1-, cytokine-, IL10-, TGFβ-, death-receptor-, and apoptosis-signaling. Overall, our results support the hypothesis that platelets play a pathogenic role in CM.

  10. Internalization of targeted quantum dots by brain capillary endothelial cells in vivo.

    Science.gov (United States)

    Paris-Robidas, Sarah; Brouard, Danny; Emond, Vincent; Parent, Martin; Calon, Frédéric

    2016-04-01

    Receptors located on brain capillary endothelial cells forming the blood-brain barrier are the target of most brain drug delivery approaches. Yet, direct subcellular evidence of vectorized transport of nanoformulations into the brain is lacking. To resolve this question, quantum dots were conjugated to monoclonal antibodies (Ri7) targeting the murine transferrin receptor. Specific transferrin receptor-mediated endocytosis of Ri7-quantum dots was first confirmed in N2A and bEnd5 cells. After intravenous injection in mice, Ri7-quantum dots exhibited a fourfold higher volume of distribution in brain tissues, compared to controls. Immunofluorescence analysis showed that Ri7-quantum dots were sequestered throughout the cerebral vasculature 30 min, 1 h, and 4 h post injection, with a decline of signal intensity after 24 h. Transmission electron microscopic studies confirmed that Ri7-quantum dots were massively internalized by brain capillary endothelial cells, averaging 37 ± 4 Ri7-quantum dots/cell 1 h after injection. Most quantum dots within brain capillary endothelial cells were observed in small vesicles (58%), with a smaller proportion detected in tubular structures or in multivesicular bodies. Parenchymal penetration of Ri7-quantum dots was extremely low and comparable to control IgG. Our results show that systemically administered Ri7-quantum dots complexes undergo extensive endocytosis by brain capillary endothelial cells and open the door for novel therapeutic approaches based on brain endothelial cell drug delivery. © The Author(s) 2015.

  11. Tumor Response to Radiotherapy Regulated by Endothelial Cell Apoptosis

    Science.gov (United States)

    Garcia-Barros, Monica; Paris, Francois; Cordon-Cardo, Carlos; Lyden, David; Rafii, Shahin; Haimovitz-Friedman, Adriana; Fuks, Zvi; Kolesnick, Richard

    2003-05-01

    About 50% of cancer patients receive radiation therapy. Here we investigated the hypothesis that tumor response to radiation is determined not only by tumor cell phenotype but also by microvascular sensitivity. MCA/129 fibrosarcomas and B16F1 melanomas grown in apoptosis-resistant acid sphingomyelinase (asmase)-deficient or Bax-deficient mice displayed markedly reduced baseline microvascular endothelial apoptosis and grew 200 to 400% faster than tumors on wild-type microvasculature. Thus, endothelial apoptosis is a homeostatic factor regulating angiogenesis-dependent tumor growth. Moreover, these tumors exhibited reduced endothelial apoptosis upon irradiation and, unlike tumors in wild-type mice, they were resistant to single-dose radiation up to 20 grays (Gy). These studies indicate that microvascular damage regulates tumor cell response to radiation at the clinically relevant dose range.

  12. Human iPSC-Derived Endothelial Cells and Microengineered Organ-Chip Enhance Neuronal Development

    Directory of Open Access Journals (Sweden)

    Samuel Sances

    2018-04-01

    Full Text Available Summary: Human stem cell-derived models of development and neurodegenerative diseases are challenged by cellular immaturity in vitro. Microengineered organ-on-chip (or Organ-Chip systems are designed to emulate microvolume cytoarchitecture and enable co-culture of distinct cell types. Brain microvascular endothelial cells (BMECs share common signaling pathways with neurons early in development, but their contribution to human neuronal maturation is largely unknown. To study this interaction and influence of microculture, we derived both spinal motor neurons and BMECs from human induced pluripotent stem cells and observed increased calcium transient function and Chip-specific gene expression in Organ-Chips compared with 96-well plates. Seeding BMECs in the Organ-Chip led to vascular-neural interaction and specific gene activation that further enhanced neuronal function and in vivo-like signatures. The results show that the vascular system has specific maturation effects on spinal cord neural tissue, and the use of Organ-Chips can move stem cell models closer to an in vivo condition. : Sances et al. combine Organ-Chip technology with human induced pluripotent stem cell-derived spinal motor neurons to study the maturation effects of Organ-Chip culture. By including microvascular cells also derived from the same patient line, the authors show enhancement of neuronal function, reproduction of vascular-neuron pathways, and specific gene activation that resembles in vivo spinal cord development. Keywords: organ-on-chip, spinal cord, iPSC, disease modeling, amyotrophic lateral sclerosis, microphysiological system, brain microvascular endothelial cells, spinal motor neurons, vasculature, microfluidic device

  13. Brain microvascular function during cardiopulmonary bypass

    International Nuclear Information System (INIS)

    Sorensen, H.R.; Husum, B.; Waaben, J.; Andersen, K.; Andersen, L.I.; Gefke, K.; Kaarsen, A.L.; Gjedde, A.

    1987-01-01

    Emboli in the brain microvasculature may inhibit brain activity during cardiopulmonary bypass. Such hypothetical blockade, if confirmed, may be responsible for the reduction of cerebral metabolic rate for glucose observed in animals subjected to cardiopulmonary bypass. In previous studies of cerebral blood flow during bypass, brain microcirculation was not evaluated. In the present study in animals (pigs), reduction of the number of perfused capillaries was estimated by measurements of the capillary diffusion capacity for hydrophilic tracers of low permeability. Capillary diffusion capacity, cerebral blood flow, and cerebral metabolic rate for glucose were measured simultaneously by the integral method, different tracers being used with different circulation times. In eight animals subjected to normothermic cardiopulmonary bypass, and seven subjected to hypothermic bypass, cerebral blood flow, cerebral metabolic rate for glucose, and capillary diffusion capacity decreased significantly: cerebral blood flow from 63 to 43 ml/100 gm/min in normothermia and to 34 ml/100 gm/min in hypothermia and cerebral metabolic rate for glucose from 43.0 to 23.0 mumol/100 gm/min in normothermia and to 14.1 mumol/100 gm/min in hypothermia. The capillary diffusion capacity declined markedly from 0.15 to 0.03 ml/100 gm/min in normothermia but only to 0.08 ml/100 gm/min in hypothermia. We conclude that the decrease of cerebral metabolic rate for glucose during normothermic cardiopulmonary bypass is caused by interruption of blood flow through a part of the capillary bed, possibly by microemboli, and that cerebral blood flow is an inadequate indicator of capillary blood flow. Further studies must clarify why normal microvascular function appears to be preserved during hypothermic cardiopulmonary bypass

  14. Endothelial glycocalyx on brain endothelial cells is lost in experimental cerebral malaria

    DEFF Research Database (Denmark)

    Hempel, Casper; Hyttel, Poul; Kurtzhals, Jørgen Al

    2014-01-01

    We hypothesized that the glycocalyx, which is important for endothelial integrity, is lost in severe malaria. C57BL/6 mice were infected with Plasmodium berghei ANKA, resulting in cerebral malaria, or P. chabaudi AS, resulting in uncomplicated malaria. We visualized the glycocalyx with transmission...... electron microscopy and measured circulating glycosaminoglycans by dot blot and ELISA. The glycocalyx was degraded in brain vasculature in cerebral and to a lesser degree uncomplicated malaria. It was affected on both intact and apoptotic endothelial cells. Circulating glycosaminoglycan levels suggested...

  15. Inhibition of Murine Pulmonary Microvascular Endothelial Cell Apoptosis Promotes Recovery of Barrier Function under Septic Conditions

    Directory of Open Access Journals (Sweden)

    Lefeng Wang

    2017-01-01

    Full Text Available Sepsis is characterized by injury of the pulmonary microvasculature and the pulmonary microvascular endothelial cells (PMVEC, leading to barrier dysfunction and acute respiratory distress syndrome (ARDS. Our recent work identified a strong correlation between PMVEC apoptosis and microvascular leak in septic mice in vivo, but the specific role of apoptosis in septic PMVEC barrier dysfunction remains unclear. Thus, we hypothesize that PMVEC apoptosis is likely required for PMVEC barrier dysfunction under septic conditions in vitro. Septic stimulation (mixture of tumour necrosis factor α, interleukin 1β, and interferon γ [cytomix] of isolated murine PMVEC resulted in a significant loss of barrier function as early as 4 h after stimulation, which persisted until 24 h. PMVEC apoptosis, as reflected by caspase activation, DNA fragmentation, and loss of membrane polarity, was first apparent at 8 h after cytomix. Pretreatment of PMVEC with the pan-caspase inhibitor Q-VD significantly decreased septic PMVEC apoptosis and was associated with reestablishment of PMVEC barrier function at 16 and 24 h after stimulation but had no effect on septic PMVEC barrier dysfunction over the first 8 h. Collectively, our data suggest that early septic murine PMVEC barrier dysfunction driven by proinflammatory cytokines is not mediated through apoptosis, but PMVEC apoptosis contributes to late septic PMVEC barrier dysfunction.

  16. Experimental validation of the predicted binding site of Escherichia coli K1 outer membrane protein A to human brain microvascular endothelial cells: identification of critical mutations that prevent E. coli meningitis.

    Science.gov (United States)

    Pascal, Tod A; Abrol, Ravinder; Mittal, Rahul; Wang, Ying; Prasadarao, Nemani V; Goddard, William A

    2010-11-26

    Escherichia coli K1, the most common cause of meningitis in neonates, has been shown to interact with GlcNAc1-4GlcNAc epitopes of Ecgp96 on human brain microvascular endothelial cells (HBMECs) via OmpA (outer membrane protein A). However, the precise domains of extracellular loops of OmpA interacting with the chitobiose epitopes have not been elucidated. We report the loop-barrel model of these OmpA interactions with the carbohydrate moieties of Ecgp96 predicted from molecular modeling. To test this model experimentally, we generated E. coli K1 strains expressing OmpA with mutations of residues predicted to be critical for interaction with the HBMEC and tested E. coli invasion efficiency. For these same mutations, we predicted the interaction free energies (including explicit calculation of the entropy) from molecular dynamics (MD), finding excellent correlation (R(2) = 90%) with experimental invasion efficiency. Particularly important is that mutating specific residues in loops 1, 2, and 4 to alanines resulted in significant inhibition of E. coli K1 invasion in HBMECs, which is consistent with the complete lack of binding found in the MD simulations for these two cases. These studies suggest that inhibition of the interactions of these residues of Loop 1, 2, and 4 with Ecgp96 could provide a therapeutic strategy to prevent neonatal meningitis due to E. coli K1.

  17. Impact of commercial cigarette smoke condensate on brain tissue co-cultured with astrocytes and blood-brain barrier endothelial cells.

    Science.gov (United States)

    Lee, Seon-Bong; Kim, Ju-Hyeong; Cho, Myung-Haing; Choe, Eun-Sang; Kim, Kwang-Sik; Shim, Soon-Mi

    2017-01-01

    The purpose of the current study was to investigate the effect of two commercial cigarette smoke condensates (CCSC) on oxidative stress and cell cytotoxicity in human brain (T98G) or astrocytes (U-373 MG) in the presence of human brain microvascular endothelial cells (HBMEC). Cell viability of mono-culture of T98G or U-373 MG was markedly decreased in a concentration-dependent manner, and T98G was more susceptible than U-373 MG to CCSC exposure. Cytotoxicity was less prominent when T98G was co-cultured with HBMEC than when T98G was co-cultured with U-373 MG. Significant reduction in trans-epithelial electric resistance (TEER), a biomarker of cellular integrity was noted in HBMEC co-cultured with T98G (HBMEC-T98G co-culture) and U-373 MG co-cultured with T98G (U-373 MG-T98G co-culture) after 24 or 48 hr CCSC exposure, respectively. TEER value of U-373 MG co-cultured with T98G (79-84%) was higher than HBMEC co-cultured with T98G (62-63%) within 120-hr incubation with CCSC. Reactive oxygen species (ROS) generated by CCSC in mono-culture of T98G and U-373 MG reached highest levels at 4 and 16 mg/ml, respectively. ROS production by T98G fell when co-cultured with HBMEC or U-373MG. These findings suggest that adverse consequences of CCSC treatment on brain cells may be protected by blood-brain barrier or astrocytes, but with chronic exposure toxicity may be worsened due to destruction of cellular integrity.

  18. Preserved microvascular endothelial function in young, obese adults with functional loss of nitric oxide signaling

    Directory of Open Access Journals (Sweden)

    John eHarrell

    2015-12-01

    Full Text Available Data indicate endothelium-dependent dilation (EDD may be preserved in the skeletal muscle microcirculation of young, obese adults. Preserved EDD might be mediated by compensatory mechanisms, impeding insight into preclinical vascular dysfunction. We aimed to determine the functional roles of nitric oxide synthase (NOS and cyclooxygenase (COX toward EDD in younger obese adults. We first hypothesized EDD would be preserved in young, obese adults. Further, we hypothesized a reduced contribution of NOS in young, obese adults would be replaced by increased COX signaling. Microvascular EDD was assessed with Doppler ultrasound and brachial artery infusion of acetylcholine (ACh in younger (27±1 yr obese (n=29 and lean (n=46 humans. Individual and combined contributions of NOS and COX were examined with intra-arterial infusions of L-NMMA and ketorolac, respectively. Vasodilation was quantified as an increase in forearm vascular conductance (ΔFVC. Arterial endothelial cell biopsies were analyzed for protein expression of endothelial nitric oxide synthase (eNOS. ΔFVC to ACh was similar between groups. After L-NMMA, ΔFVC to ACh was greater in obese adults (p<0.05. There were no group differences in ΔFVC to ACh with ketorolac. With combined NOS-COX inhibition, ΔFVC was greater in obese adults at the intermediate dose of ACh. Surprisingly, arterial endothelial cell eNOS and phosphorylated eNOS were similar between groups. Younger obese adults exhibit preserved EDD and eNOS expression despite functional dissociation of NOS-mediated vasodilation and similar COX signaling. Compensatory NOS- and COX-independent vasodilatory mechanisms conceal reduced NOS contributions in otherwise healthy obese adults early in life, which may contribute to vascular dysfunction.

  19. Flavonoids targeting of IκB phosphorylation abrogates carcinogen-induced MMP-9 and COX-2 expression in human brain endothelial cells

    Directory of Open Access Journals (Sweden)

    Tahanian E

    2011-05-01

    Full Text Available Elizabeth Tahanian¹, Luis Arguello Sanchez¹, Tze Chieh Shiao², René Roy², Borhane Annabi¹¹Centre de Recherche BioMED, ²Centre de Recherche PharmaQAM, Département de chimie, Université du Québec à Montréal, QC, CanadaAbstract: Brain endothelial cells play an essential role as structural and functional components of the blood–brain barrier (BBB. Increased BBB breakdown and brain injury are associated with neuroinflammation and are thought to trigger mechanisms involving matrix metalloproteinase upregulation. Emerging evidence also indicates that cyclooxygenase (COX inhibition limits BBB disruption, but the mechanisms linking metalloproteinase to COX remain unknown. In this study, we sought to investigate the nuclear factor-kappa B (NF-κB signaling pathway, a common pathway in both the regulation of matrix metalloproteinase-9 (MMP-9 and COX-2 expression, and the inhibitory properties of several chemopreventive flavonoids. Human brain microvascular endothelial cells were treated with a combination of phorbol 12-myristate 13-acetate (PMA, a carcinogen documented to increase MMP-9 and COX-2 through NF-κB, and several naturally occurring flavonoids. Among the molecules tested, we found that fisetin, apigenin, and luteolin specifically and dose-dependently antagonized PMA-induced COX-2 and MMP-9 gene and protein expressions as assessed by qRT-PCR, immunoblotting, and zymography respectively. We further demonstrate that flavonoids impact on IκK-mediated phosphorylation activity as demonstrated by the inhibition of PMA-induced IκB phosphorylation levels. Our results suggest that BBB disruption during neuroinflammation could be pharmacologically reduced by a specific class of flavonoids acting as NF-κB signal transduction inhibitors.Keywords: blood–brain barrier, flavonoids, neuroinflammation, NF-κB signal transduction inhibitors

  20. Anesthetic propofol overdose causes endothelial cytotoxicity in vitro and endothelial barrier dysfunction in vivo

    International Nuclear Information System (INIS)

    Lin, Ming-Chung; Chen, Chia-Ling; Yang, Tsan-Tzu; Choi, Pui-Ching; Hsing, Chung-Hsi; Lin, Chiou-Feng

    2012-01-01

    An overdose and a prolonged treatment of propofol may cause cellular cytotoxicity in multiple organs and tissues such as brain, heart, kidney, skeletal muscle, and immune cells; however, the underlying mechanism remains undocumented, particularly in vascular endothelial cells. Our previous studies showed that the activation of glycogen synthase kinase (GSK)-3 is pro-apoptotic in phagocytes during overdose of propofol treatment. Regarding the intravascular administration of propofol, we therefore hypothesized that propofol overdose also induces endothelial cytotoxicity via GSK-3. Propofol overdose (100 μg/ml) inhibited growth in human arterial and microvascular endothelial cells. After treatment, most of the endothelial cells experienced caspase-independent necrosis-like cell death. The activation of cathepsin D following lysosomal membrane permeabilization (LMP) determined necrosis-like cell death. Furthermore, propofol overdose also induced caspase-dependent apoptosis, at least in part. Caspase-3 was activated and acted downstream of mitochondrial transmembrane potential (MTP) loss; however, lysosomal cathepsins were not required for endothelial cell apoptosis. Notably, activation of GSK-3 was essential for propofol overdose-induced mitochondrial damage and apoptosis, but not necrosis-like cell death. Intraperitoneal administration of a propofol overdose in BALB/c mice caused an increase in peritoneal vascular permeability. These results demonstrate the cytotoxic effects of propofol overdose, including cathepsin D-regulated necrosis-like cell death and GSK-3-regulated mitochondrial apoptosis, on endothelial cells in vitro and the endothelial barrier dysfunction by propofol in vivo. Highlights: ► Propofol overdose causes apoptosis and necrosis in endothelial cells. ► Propofol overdose triggers lysosomal dysfunction independent of autophagy. ► Glycogen synthase kinase-3 facilitates propofol overdose-induced apoptosis. ► Propofol overdose causes an increase

  1. Anesthetic propofol overdose causes endothelial cytotoxicity in vitro and endothelial barrier dysfunction in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Ming-Chung [Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan (China); Department of Anesthesiology, Chi Mei Medical Center, Liouying, Tainan, Taiwan (China); Chen, Chia-Ling [Center of Infectious Disease and Signaling Research, College of Medicine, National Cheng Kung University, Tainan, Taiwan (China); Yang, Tsan-Tzu; Choi, Pui-Ching [Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan (China); Hsing, Chung-Hsi [Department of Anesthesiology, Chi Mei Medical Center, Tainan, Taiwan (China); Department of Anesthesiology, College of Medicine, Taipei Medical University, Taipei, Taiwan (China); Lin, Chiou-Feng, E-mail: cflin@mail.ncku.edu.tw [Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan (China); Center of Infectious Disease and Signaling Research, College of Medicine, National Cheng Kung University, Tainan, Taiwan (China); Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan (China); Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan (China)

    2012-12-01

    An overdose and a prolonged treatment of propofol may cause cellular cytotoxicity in multiple organs and tissues such as brain, heart, kidney, skeletal muscle, and immune cells; however, the underlying mechanism remains undocumented, particularly in vascular endothelial cells. Our previous studies showed that the activation of glycogen synthase kinase (GSK)-3 is pro-apoptotic in phagocytes during overdose of propofol treatment. Regarding the intravascular administration of propofol, we therefore hypothesized that propofol overdose also induces endothelial cytotoxicity via GSK-3. Propofol overdose (100 μg/ml) inhibited growth in human arterial and microvascular endothelial cells. After treatment, most of the endothelial cells experienced caspase-independent necrosis-like cell death. The activation of cathepsin D following lysosomal membrane permeabilization (LMP) determined necrosis-like cell death. Furthermore, propofol overdose also induced caspase-dependent apoptosis, at least in part. Caspase-3 was activated and acted downstream of mitochondrial transmembrane potential (MTP) loss; however, lysosomal cathepsins were not required for endothelial cell apoptosis. Notably, activation of GSK-3 was essential for propofol overdose-induced mitochondrial damage and apoptosis, but not necrosis-like cell death. Intraperitoneal administration of a propofol overdose in BALB/c mice caused an increase in peritoneal vascular permeability. These results demonstrate the cytotoxic effects of propofol overdose, including cathepsin D-regulated necrosis-like cell death and GSK-3-regulated mitochondrial apoptosis, on endothelial cells in vitro and the endothelial barrier dysfunction by propofol in vivo. Highlights: ► Propofol overdose causes apoptosis and necrosis in endothelial cells. ► Propofol overdose triggers lysosomal dysfunction independent of autophagy. ► Glycogen synthase kinase-3 facilitates propofol overdose-induced apoptosis. ► Propofol overdose causes an increase

  2. Cultivation of Human Microvascular Endothelial Cells on Topographical Substrates to Mimic the Human Corneal Endothelium

    Directory of Open Access Journals (Sweden)

    Jie Shi Chua

    2013-03-01

    Full Text Available Human corneal endothelial cells have a limited ability to replicate in vivo and in vitro. Allograft transplantation becomes necessary when an accident or trauma results in excessive cell loss. The reconstruction of the cornea endothelium using autologous cell sources is a promising alternative option for therapeutic or in vitro drug testing applications. The native corneal endothelium rests on the Descemet’s membrane, which has nanotopographies of fibers and pores. The use of synthetic topographies mimics the native environment, and it is hypothesized that this can direct the behavior and growth of human microvascular endothelial cells (HMVECs to resemble the corneal endothelium. In this study, HMVECs are cultivated on substrates with micron and nano-scaled pillar and well topographies. Closely packed HMVEC monolayers with polygonal cells and well-developed tight junctions were formed on the topographical substrates. Sodium/potassium (Na+/K+ adenine triphosphatase (ATPase expression was enhanced on the microwells substrate, which also promotes microvilli formation, while more hexagonal-like cells are found on the micropillars samples. The data obtained suggests that the use of optimized surface patterning, in particular, the microtopographies, can induce HMVECs to adopt a more corneal endothelium-like morphology with similar barrier and pump functions. The mechanism involved in cell contact guidance by the specific topographical features will be of interest for future studies.

  3. Molecular characterization of EG-VEGF-mediated angiogenesis: differential effects on microvascular and macrovascular endothelial cells.

    Science.gov (United States)

    Brouillet, Sophie; Hoffmann, Pascale; Benharouga, Mohamed; Salomon, Aude; Schaal, Jean-Patrick; Feige, Jean-Jacques; Alfaidy, Nadia

    2010-08-15

    Endocrine gland derived vascular endothelial growth factor (EG-VEGF) also called prokineticin (PK1), has been identified and linked to several biological processes including angiogenesis. EG-VEGF is abundantly expressed in the highest vascularized organ, the human placenta. Here we characterized its angiogenic effect using different experimental procedures. Immunohistochemistry was used to localize EG-VEGF receptors (PROKR1 and PROKR2) in placental and umbilical cord tissue. Primary microvascular placental endothelial cell (HPEC) and umbilical vein-derived macrovascular EC (HUVEC) were used to assess its effects on proliferation, migration, cell survival, pseudovascular organization, spheroid sprouting, permeability and paracellular transport. siRNA and neutralizing antibody strategies were used to differentiate PROKR1- from PROKR2-mediated effects. Our results show that 1) HPEC and HUVEC express both types of receptors 2) EG-VEGF stimulates HPEC's proliferation, migration and survival, but increases only survival in HUVECs. and 3) EG-VEGF was more potent than VEGF in stimulating HPEC sprout formation, pseudovascular organization, and it significantly increases HPEC permeability and paracellular transport. More importantly, we demonstrated that PROKR1 mediates EG-VEGF angiogenic effects, whereas PROKR2 mediates cellular permeability. Altogether, these data characterized angiogenic processes mediated by EG-VEGF, depicted a new angiogenic factor in the placenta, and suggest a novel view of the regulation of angiogenesis in placental pathologies.

  4. Microvascular Remodeling and Wound Healing: A Role for Pericytes

    Science.gov (United States)

    Dulmovits, Brian M.; Herman, Ira M.

    2012-01-01

    Physiologic wound healing is highly dependent on the coordinated functions of vascular and non-vascular cells. Resolution of tissue injury involves coagulation, inflammation, formation of granulation tissue, remodeling and scarring. Angiogenesis, the growth of microvessels the size of capillaries, is crucial for these processes, delivering blood-borne cells, nutrients and oxygen to actively remodeling areas. Central to angiogenic induction and regulation is microvascular remodeling, which is dependent upon capillary endothelial cell and pericyte interactions. Despite our growing knowledge of pericyte-endothelial cell crosstalk, it is unclear how the interplay among pericytes, inflammatory cells, glia and connective tissue elements shape microvascular injury response. Here, we consider the relationships that pericytes form with the cellular effectors of healing in normal and diabetic environments, including repair following injury and vascular complications of diabetes, such as diabetic macular edema and proliferative diabetic retinopathy. In addition, pericytes and stem cells possessing “pericyte-like” characteristics are gaining considerable attention in experimental and clinical efforts aimed at promoting healing or eradicating ocular vascular proliferative disorders. As the origin, identification and characterization of microvascular pericyte progenitor populations remains somewhat ambiguous, the molecular markers, structural and functional characteristics of pericytes will be briefly reviewed. PMID:22750474

  5. Laminar shear stress modulates endothelial luminal surface stiffness in a tissue-specific manner.

    Science.gov (United States)

    Merna, Nick; Wong, Andrew K; Barahona, Victor; Llanos, Pierre; Kunar, Balvir; Palikuqi, Brisa; Ginsberg, Michael; Rafii, Shahin; Rabbany, Sina Y

    2018-04-17

    Endothelial cells form vascular beds in all organs and are exposed to a range of mechanical forces that regulate cellular phenotype. We sought to determine the role of endothelial luminal surface stiffness in tissue-specific mechanotransduction of laminar shear stress in microvascular mouse cells and the role of arachidonic acid in mediating this response. Microvascular mouse endothelial cells were subjected to laminar shear stress at 4 dynes/cm 2 for 12 hours in parallel plate flow chambers that enabled real-time optical microscopy and atomic force microscopy measurements of cell stiffness. Lung endothelial cells aligned parallel to flow, while cardiac endothelial cells did not. This rapid alignment was accompanied by increased cell stiffness. The addition of arachidonic acid to cardiac endothelial cells increased alignment and stiffness in response to shear stress. Inhibition of arachidonic acid in lung endothelial cells and embryonic stem cell-derived endothelial cells prevented cellular alignment and decreased cell stiffness. Our findings suggest that increased endothelial luminal surface stiffness in microvascular cells may facilitate mechanotransduction and alignment in response to laminar shear stress. Furthermore, the arachidonic acid pathway may mediate this tissue-specific process. An improved understanding of this response will aid in the treatment of organ-specific vascular disease. © 2018 John Wiley & Sons Ltd.

  6. Microvascular inflammation in atherosclerosis

    Directory of Open Access Journals (Sweden)

    Laura Vitiello

    2014-06-01

    Full Text Available Atherogenesis is the pathogenetic process leading to formation of the atheroma lesion. It is associated to a chronic inflammatory state initially stimulated by an aberrant accumulation of lipid molecules beyond the endothelial barrier. This event triggers a cascade of deleterious events mainly through immune cell stimulation with the consequent liberation of potent pro-inflammatory and tissue damaging mediators. The atherogenetic process implies marked modifications of endothelial cell functions and a radical change in the endothelial–leukocyte interaction pattern. Moreover, accumulating evidence shows an important link between microvascular and inflammatory responses and major cardiovascular risk factors. This review illustrates the current knowledge on the effects of obesity, hypercholesterolemia and diabetes on microcirculation; their pathophysiological implications will be discussed.

  7. A nanoengineered peptidic delivery system with specificity for human brain capillary endothelial cells

    DEFF Research Database (Denmark)

    Wu, Linping; Moghimi, Seyed Moein

    2016-01-01

    , without manipulating the integrity of the BBB. This may be achieved by simultaneous and appropriate nanoparticle surface decoration with polymers that protect nanoparticles against rapid interception by body's defenses and ligands specific for cerebral capillary endothelial cells. To date, the binding...... avidity of the majority of the so-called ‘brain-specific’ nanoparticles to the brain capillary endothelial cells has been poor, even during in vitro conditions. We have addressed this issue and designed a versatile peptidic nanoplatform with high binding avidity to the human cerebral capillary endothelial...... cells. This was achieved by selecting an appropriate phage-derived peptide with high specificity for human brain capillary endothelial cells, which following careful structural modifications spontaneously formed a nanoparticle-fiber network. The peptidic network was characterized fully and its uptake...

  8. Effect of a Tibetan herbal mixture on microvascular endothelial function, heart rate variability and biomarkers of inflammation, clotting and coagulation.

    Science.gov (United States)

    Schäfer, Daniela; Lambrecht, Julia; Radtke, Thomas; Wilhelm, Matthias; Saner, Hugo

    2015-08-01

    In this 6-week prospective, randomized, placebo-controlled and double-blind study, we investigated the effects of a natural herbal remedy based on a recipe from Tibet (Padma® 28), on microvascular endothelial function, heart rate variability and biomarkers of inflammation, clotting and coagulation in 80 coronary artery disease (CAD) patients (age 66 ± 8 years) on guideline-based medication for secondary prevention. We found no significant effects of Padma 28 and conclude that the addition of Padma 28 to guideline-based secondary prevention treatment of CAD did not lead to significant effects on important surrogate markers in elderly male CAD patients. © The European Society of Cardiology 2014.

  9. A porcine astrocyte/endothelial cell co-culture model of the blood-brain barrier.

    Science.gov (United States)

    Jeliazkova-Mecheva, Valentina V; Bobilya, Dennis J

    2003-10-01

    A method for the isolation of porcine atrocytes as a simple extension of a previously described procedure for isolation of brain capillary endothelial cells from adolescent pigs [Methods Cell Sci. 17 (1995) 2] is described. The obtained astroglial culture purified through two passages and by the method of the selective detachment was validated by a phase contrast microscopy and through an immunofluorescent assay for the glial fibrillary acidic protein (GFAP). Porcine astrocytes were co-cultivated with porcine brain capillary endothelial cells (PBCEC) for the development of an in vitro blood-brain barrier (BBB) model. The model was visualized by an electron microscopy and showed elevated transendothellial electrical resistance and reduced inulin permeability. To our knowledge, this is the first report for the establishment of a porcine astrocyte/endothelial cell co-culture BBB model, which avoids interspecies and age differences between the two cell types, usually encountered in the other reported co-culture BBB models. Considering the availability of the porcine brain tissue and the close physiological and anatomical relation between the human and pig brain, the porcine astrocyte/endothelial cell co-culture system can serve as a reliable and easily reproducible model for different in vitro BBB studies.

  10. Iron oxide nanoparticles induce human microvascular endothelial cell permeability through reactive oxygen species production and microtubule remodeling

    Directory of Open Access Journals (Sweden)

    Shi Xianglin

    2009-01-01

    Full Text Available Abstract Background Engineered iron nanoparticles are being explored for the development of biomedical applications and many other industry purposes. However, to date little is known concerning the precise mechanisms of translocation of iron nanoparticles into targeted tissues and organs from blood circulation, as well as the underlying implications of potential harmful health effects in human. Results The confocal microscopy imaging analysis demonstrates that exposure to engineered iron nanoparticles induces an increase in cell permeability in human microvascular endothelial cells. Our studies further reveal iron nanoparticles enhance the permeability through the production of reactive oxygen species (ROS and the stabilization of microtubules. We also showed Akt/GSK-3β signaling pathways are involved in iron nanoparticle-induced cell permeability. The inhibition of ROS demonstrate ROS play a major role in regulating Akt/GSK-3β – mediated cell permeability upon iron nanoparticle exposure. These results provide new insights into the bioreactivity of engineered iron nanoparticles which can inform potential applications in medical imaging or drug delivery. Conclusion Our results indicate that exposure to iron nanoparticles induces an increase in endothelial cell permeability through ROS oxidative stress-modulated microtubule remodeling. The findings from this study provide new understandings on the effects of nanoparticles on vascular transport of macromolecules and drugs.

  11. Cell proliferation along vascular islands during microvascular network growth

    Directory of Open Access Journals (Sweden)

    Kelly-Goss Molly R

    2012-06-01

    Full Text Available Abstract Background Observations in our laboratory provide evidence of vascular islands, defined as disconnected endothelial cell segments, in the adult microcirculation. The objective of this study was to determine if vascular islands are involved in angiogenesis during microvascular network growth. Results Mesenteric tissues, which allow visualization of entire microvascular networks at a single cell level, were harvested from unstimulated adult male Wistar rats and Wistar rats 3 and 10 days post angiogenesis stimulation by mast cell degranulation with compound 48/80. Tissues were immunolabeled for PECAM and BRDU. Identification of vessel lumens via injection of FITC-dextran confirmed that endothelial cell segments were disconnected from nearby patent networks. Stimulated networks displayed increases in vascular area, length density, and capillary sprouting. On day 3, the percentage of islands with at least one BRDU-positive cell increased compared to the unstimulated level and was equal to the percentage of capillary sprouts with at least one BRDU-positive cell. At day 10, the number of vascular islands per vascular area dramatically decreased compared to unstimulated and day 3 levels. Conclusions These results show that vascular islands have the ability to proliferate and suggest that they are able to incorporate into the microcirculation during the initial stages of microvascular network growth.

  12. Method for isolation and molecular characterization of extracellular microvesicles released from brain endothelial cells

    Directory of Open Access Journals (Sweden)

    Haqqani Arsalan S

    2013-01-01

    Full Text Available Abstract Background In addition to possessing intracellular vesicles, eukaryotic cells also produce extracellular microvesicles, ranging from 50 to 1000 nm in diameter that are released or shed into the microenvironment under physiological and pathological conditions. These membranous extracellular organelles include both exosomes (originating from internal vesicles of endosomes and ectosomes (originating from direct budding/shedding of plasma membranes. Extracellular microvesicles contain cell-specific collections of proteins, glycoproteins, lipids, nucleic acids and other molecules. These vesicles play important roles in intercellular communication by acting as carrier for essential cell-specific information to target cells. Endothelial cells in the brain form the blood–brain barrier, a specialized interface between the blood and the brain that tightly controls traffic of nutrients and macromolecules between two compartments and interacts closely with other cells forming the neurovascular unit. Therefore, brain endothelial cell extracellular microvesicles could potentially play important roles in ‘externalizing’ brain-specific biomarkers into the blood stream during pathological conditions, in transcytosis of blood-borne molecules into the brain, and in cell-cell communication within the neurovascular unit. Methods To study cell-specific molecular make-up and functions of brain endothelial cell exosomes, methods for isolation of extracellular microvesicles using mass spectrometry-compatible protocols and the characterization of their signature profiles using mass spectrometry -based proteomics were developed. Results A total of 1179 proteins were identified in the isolated extracellular microvesicles from brain endothelial cells. The microvesicles were validated by identification of almost 60 known markers, including Alix, TSG101 and the tetraspanin proteins CD81 and CD9. The surface proteins on isolated microvesicles could potentially

  13. In vitro models of the blood–brain barrier: An overview of commonly used brain endothelial cell culture models and guidelines for their use

    Science.gov (United States)

    Helms, Hans C; Abbott, N Joan; Burek, Malgorzata; Cecchelli, Romeo; Couraud, Pierre-Olivier; Deli, Maria A; Förster, Carola; Galla, Hans J; Romero, Ignacio A; Shusta, Eric V; Stebbins, Matthew J; Vandenhaute, Elodie; Weksler, Babette

    2016-01-01

    The endothelial cells lining the brain capillaries separate the blood from the brain parenchyma. The endothelial monolayer of the brain capillaries serves both as a crucial interface for exchange of nutrients, gases, and metabolites between blood and brain, and as a barrier for neurotoxic components of plasma and xenobiotics. This “blood-brain barrier” function is a major hindrance for drug uptake into the brain parenchyma. Cell culture models, based on either primary cells or immortalized brain endothelial cell lines, have been developed, in order to facilitate in vitro studies of drug transport to the brain and studies of endothelial cell biology and pathophysiology. In this review, we aim to give an overview of established in vitro blood–brain barrier models with a focus on their validation regarding a set of well-established blood–brain barrier characteristics. As an ideal cell culture model of the blood–brain barrier is yet to be developed, we also aim to give an overview of the advantages and drawbacks of the different models described. PMID:26868179

  14. Bcl-2 silencing attenuates hypoxia-induced apoptosis resistance in pulmonary microvascular endothelial cells.

    Science.gov (United States)

    Cao, Yongmei; Jiang, Zhen; Zeng, Zhen; Liu, Yujing; Gu, Yuchun; Ji, Yingying; Zhao, Yupeng; Li, Yingchuan

    2016-01-01

    Pulmonary arterial hypertension (PAH) is a life-threatening disorder that ultimately causes heart failure. While the underlying causes of this condition are not well understood, previous studies suggest that the anti-apoptotic nature of pulmonary microvascular endothelial cells (PMVECs) in hypoxic environments contributes to PAH pathogenesis. In this study, we focus on the contribution of Bcl-2 and hypoxia response element (HRE) to apoptosis-resistant endothelial cells and investigate the mechanism. PMVECs obtained from either normal rats or apoptosis-resistant PMVECs obtained from PAH rats were transduced with recombinant lentiviral vectors carrying either Bcl-2-shRNA or HRE combined Bcl-2-shRNA, and then cultured these cells for 24 h under hypoxic (5% O2) or normoxic (21% O2) conditions. In normal PMVECs, Bcl-2-shRNA or HRE combined with Bcl-2-shRNA transduction successfully decreased Bcl-2 expression, while increasing apoptosis as well as caspase-3 and P53 expression in a normoxic environment. In a hypoxic environment, the effects of Bcl-2-shRNA treatment on cell apoptosis, and on Bcl-2, caspase-3, P53 expression were significantly suppressed. Conversely, HRE activation combined with Bcl-2-shRNA transduction markedly enhanced cell apoptosis and upregulated caspase-3 and P53 expression, while decreasing Bcl-2 expression. Furthermore, in apoptosis-resistant PMVECs, HRE-mediated Bcl-2 silencing effectively enhanced cell apoptosis and caspase-3 activity. The apoptosis rate was significantly depressed when Lv-HRE-Bcl-2-shRNA was combined with Lv-P53-shRNA or Lv-caspase3-shRNA transduction in a hypoxic environment. These results suggest that HRE-mediated Bcl-2 inhibition can effectively attenuate hypoxia-induced apoptosis resistance in PMVECs by downregulating Bcl-2 expression and upregulating caspase-3 and P53 expression. This study therefore reveals critical insight into potential therapeutic targets for treating PAH.

  15. Binding of human endothelium to Ulex europaeus I-coated Dynabeads: application to the isolation of microvascular endothelium.

    Science.gov (United States)

    Jackson, C J; Garbett, P K; Nissen, B; Schrieber, L

    1990-06-01

    A major problem encountered when isolating human microvascular endothelium is the presence of contaminating cells such as fibroblasts that rapidly over-grow the endothelial cells. We describe here a simple, rapid technique for purifying endothelial cells derived from the microvasculature of neonatal foreskin and osteoarthritic and rheumatoid arthritic synovium. This technique is based on the selective binding of the lectin Ulex europaeus I (UEA I) to the endothelial cell surface via fucose residues. Initially UEA I was covalently bound to tosyl-activated super-paramagnetic polystyrene beads (Dynabeads) by incubation for 24 h at room temperature. Cells were isolated by extracting microvascular segments from enzyme-treated (trypsin and Pronase) cubes of tissue. The mixed population of cells obtained were purified by incubating them at 4 degrees C for 10 min with the UEA I-coated Dynabeads. Endothelium bound to the beads whilst contaminating cells were removed by five washes with HBSS using a magnetic particle concentrator. The endothelial cells thus obtained grew to confluence as a cobblestone-like monolayer and expressed von Willebrand factor antigen. The cells were released from the Dynabeads by the competitive binding of fucose (10 min at 4 degrees C). This new method is simple and reproducible and allows pure human microvascular endothelial cells to be cultured within 2 h of obtaining a specimen.

  16. Sphingosine 1-phosphate receptor 5 mediates the immune quiescence of the human brain endothelial barrier

    Directory of Open Access Journals (Sweden)

    van Doorn Ruben

    2012-06-01

    Full Text Available Abstract Background The sphingosine 1-phosphate (S1P receptor modulator FTY720P (Gilenya® potently reduces relapse rate and lesion activity in the neuroinflammatory disorder multiple sclerosis. Although most of its efficacy has been shown to be related to immunosuppression through the induction of lymphopenia, it has been suggested that a number of its beneficial effects are related to altered endothelial and blood–brain barrier (BBB functionality. However, to date it remains unknown whether brain endothelial S1P receptors are involved in the maintenance of the function of the BBB thereby mediating immune quiescence of the brain. Here we demonstrate that the brain endothelial receptor S1P5 largely contributes to the maintenance of brain endothelial barrier function. Methods We analyzed the expression of S1P5 in human post-mortem tissues using immunohistochemistry. The function of S1P5 at the BBB was assessed in cultured human brain endothelial cells (ECs using agonists and lentivirus-mediated knockdown of S1P5. Subsequent analyses of different aspects of the brain EC barrier included the formation of a tight barrier, the expression of BBB proteins and markers of inflammation and monocyte transmigration. Results We show that activation of S1P5 on cultured human brain ECs by a selective agonist elicits enhanced barrier integrity and reduced transendothelial migration of monocytes in vitro. These results were corroborated by genetically silencing S1P5 in brain ECs. Interestingly, functional studies with these cells revealed that S1P5 strongly contributes to brain EC barrier function and underlies the expression of specific BBB endothelial characteristics such as tight junctions and permeability. In addition, S1P5 maintains the immunoquiescent state of brain ECs with low expression levels of leukocyte adhesion molecules and inflammatory chemokines and cytokines through lowering the activation of the transcription factor NFκB. Conclusion Our

  17. Implications Enzymatic Degradation of the Endothelial Glycocalyx on the Microvascular Hemodynamics and the Arteriolar Red Cell Free Layer of the Rat Cremaster Muscle

    Directory of Open Access Journals (Sweden)

    Ozlem Yalcin

    2018-03-01

    Full Text Available The endothelial glycocalyx is a complex network of glycoproteins, proteoglycans, and glycosaminoglycans; it lines the vascular endothelial cells facing the lumen of blood vessels forming the endothelial glycocalyx layer (EGL. This study aims to investigate the microvascular hemodynamics implications of the EGL by quantifying changes in blood flow hydrodynamics post-enzymatic degradation of the glycocalyx layer. High-speed intravital microscopy videos of small arteries (around 35 μm of the rat cremaster muscle were recorded at various time points after enzymatic degradation of the EGL. The thickness of the cell free layer (CFL, blood flow velocity profiles, and volumetric flow rates were quantified. Hydrodynamic effects of the presence of the EGL were observed in the differences between the thickness of CFL in microvessels with an intact EGL and glass tubes of similar diameters. Maximal changes in the thickness of CFL were observed 40 min post-enzymatic degradation of the EGL. Analysis of the frequency distribution of the thickness of CFL allows for estimation of the thickness of the endothelial surface layer (ESL, the plasma layer, and the glycocalyx. Peak flow, maximum velocity, and mean velocity were found to statistically increase by 24, 27, and 25%, respectively, after enzymatic degradation of the glycocalyx. The change in peak-to-peak maximum velocity and mean velocity were found to statistically increase by 39 and 32%, respectively, after 40 min post-enzymatic degradation of the EGL. The bluntness of blood flow velocity profiles was found to be reduced post-degradation of the EGL, as the exclusion volume occupied by the EGL increased the effective volume impermeable to RBCs in microvessels. This study presents the effects of the EGL on microvascular hemodynamics. Enzymatic degradation of the EGL resulted in a decrease in the thickness of CFL, an increase in blood velocity, blood flow, and decrease of the bluntness of the blood flow

  18. Implications Enzymatic Degradation of the Endothelial Glycocalyx on the Microvascular Hemodynamics and the Arteriolar Red Cell Free Layer of the Rat Cremaster Muscle.

    Science.gov (United States)

    Yalcin, Ozlem; Jani, Vivek P; Johnson, Paul C; Cabrales, Pedro

    2018-01-01

    The endothelial glycocalyx is a complex network of glycoproteins, proteoglycans, and glycosaminoglycans; it lines the vascular endothelial cells facing the lumen of blood vessels forming the endothelial glycocalyx layer (EGL). This study aims to investigate the microvascular hemodynamics implications of the EGL by quantifying changes in blood flow hydrodynamics post-enzymatic degradation of the glycocalyx layer. High-speed intravital microscopy videos of small arteries (around 35 μm) of the rat cremaster muscle were recorded at various time points after enzymatic degradation of the EGL. The thickness of the cell free layer (CFL), blood flow velocity profiles, and volumetric flow rates were quantified. Hydrodynamic effects of the presence of the EGL were observed in the differences between the thickness of CFL in microvessels with an intact EGL and glass tubes of similar diameters. Maximal changes in the thickness of CFL were observed 40 min post-enzymatic degradation of the EGL. Analysis of the frequency distribution of the thickness of CFL allows for estimation of the thickness of the endothelial surface layer (ESL), the plasma layer, and the glycocalyx. Peak flow, maximum velocity, and mean velocity were found to statistically increase by 24, 27, and 25%, respectively, after enzymatic degradation of the glycocalyx. The change in peak-to-peak maximum velocity and mean velocity were found to statistically increase by 39 and 32%, respectively, after 40 min post-enzymatic degradation of the EGL. The bluntness of blood flow velocity profiles was found to be reduced post-degradation of the EGL, as the exclusion volume occupied by the EGL increased the effective volume impermeable to RBCs in microvessels. This study presents the effects of the EGL on microvascular hemodynamics. Enzymatic degradation of the EGL resulted in a decrease in the thickness of CFL, an increase in blood velocity, blood flow, and decrease of the bluntness of the blood flow velocity profile in

  19. Endothelial ErbB4 deficit induces alterations in exploratory behavior and brain energy metabolism in mice.

    Science.gov (United States)

    Wu, Gang; Liu, Xiu-Xiu; Lu, Nan-Nan; Liu, Qi-Bing; Tian, Yun; Ye, Wei-Feng; Jiang, Guo-Jun; Tao, Rong-Rong; Han, Feng; Lu, Ying-Mei

    2017-06-01

    The receptor tyrosine kinase ErbB4 is present throughout the primate brain and has a distinct functional profile. In this study, we investigate the potential role of endothelial ErbB4 receptor signaling in the brain. Here, we show that the endothelial cell-specific deletion of ErbB4 induces decreased exploratory behavior in adult mice. However, the water maze task for spatial memory and the memory reconsolidation test reveal no changes; additionally, we observe no impairment in CaMKII phosphorylation in Cdh5Cre;ErbB4 f/f mice, which indicates that the endothelial ErbB4 deficit leads to decreased exploratory activity rather than direct memory deficits. Furthermore, decreased brain metabolism, which was measured using micro-positron emission tomography, is observed in the Cdh5Cre;ErbB4 f/f mice. Consistently, the immunoblot data demonstrate the downregulation of brain Glut1, phospho-ULK1 (Ser555), and TIGAR in the endothelial ErbB4 conditional knockout mice. Collectively, our findings suggest that endothelial ErbB4 plays a critical role in regulating brain function, at least in part, through maintaining normal brain energy homeostasis. Targeting ErbB4 or the modulation of endothelial ErbB4 signaling may represent a rational pharmacological approach to treat neurological disorders. © 2017 John Wiley & Sons Ltd.

  20. An improved in vitro blood-brain barrier model: rat brain endothelial cells co-cultured with astrocytes.

    Science.gov (United States)

    Abbott, N Joan; Dolman, Diana E M; Drndarski, Svetlana; Fredriksson, Sarah M

    2012-01-01

    In vitro blood-brain barrier (BBB) models using primary cultured brain endothelial cells are important for establishing cellular and molecular mechanisms of BBB function. Co-culturing with BBB-associated cells especially astrocytes to mimic more closely the in vivo condition leads to upregulation of the BBB phenotype in the brain endothelial cells. Rat brain endothelial cells (RBECs) are a valuable tool allowing ready comparison with in vivo studies in rodents; however, it has been difficult to obtain pure brain endothelial cells, and few models achieve a transendothelial electrical resistance (TEER, measure of tight junction efficacy) of >200 Ω cm(2), i.e. the models are still relatively leaky. Here, we describe methods for preparing high purity RBECs and neonatal rat astrocytes, and a co-culture method that generates a robust, stable BBB model that can achieve TEER >600 Ω cm(2). The method is based on >20 years experience with RBEC culture, together with recent improvements to kill contaminating cells and encourage BBB differentiation.Astrocytes are isolated by mechanical dissection and cell straining and are frozen for later co-culture. RBECs are isolated from 3-month-old rat cortices. The brains are cleaned of meninges and white matter and enzymatically and mechanically dissociated. Thereafter, the tissue homogenate is centrifuged in bovine serum albumin to separate vessel fragments from other cells that stick to the myelin plug. The vessel fragments undergo a second enzyme digestion to separate pericytes from vessels and break down vessels into shorter segments, after which a Percoll gradient is used to separate capillaries from venules, arterioles, and single cells. To kill remaining contaminating cells such as pericytes, the capillary fragments are plated in puromycin-containing medium and RBECs grown to 50-60% confluence. They are then passaged onto filters for co-culture with astrocytes grown in the bottom of the wells. The whole procedure takes ∼2

  1. Accelerated differentiation of human induced pluripotent stem cells to blood-brain barrier endothelial cells.

    Science.gov (United States)

    Hollmann, Emma K; Bailey, Amanda K; Potharazu, Archit V; Neely, M Diana; Bowman, Aaron B; Lippmann, Ethan S

    2017-04-13

    Due to their ability to limitlessly proliferate and specialize into almost any cell type, human induced pluripotent stem cells (iPSCs) offer an unprecedented opportunity to generate human brain microvascular endothelial cells (BMECs), which compose the blood-brain barrier (BBB), for research purposes. Unfortunately, the time, expense, and expertise required to differentiate iPSCs to purified BMECs precludes their widespread use. Here, we report the use of a defined medium that accelerates the differentiation of iPSCs to BMECs while achieving comparable performance to BMECs produced by established methods. Induced pluripotent stem cells were seeded at defined densities and differentiated to BMECs using defined medium termed E6. Resultant purified BMEC phenotypes were assessed through trans-endothelial electrical resistance (TEER), fluorescein permeability, and P-glycoprotein and MRP family efflux transporter activity. Expression of endothelial markers and their signature tight junction proteins were confirmed using immunocytochemistry. The influence of co-culture with astrocytes and pericytes on purified BMECs was assessed via TEER measurements. The robustness of the differentiation method was confirmed across independent iPSC lines. The use of E6 medium, coupled with updated culture methods, reduced the differentiation time of iPSCs to BMECs from thirteen to 8 days. E6-derived BMECs expressed GLUT-1, claudin-5, occludin, PECAM-1, and VE-cadherin and consistently achieved TEER values exceeding 2500 Ω × cm 2 across multiple iPSC lines, with a maximum TEER value of 4678 ± 49 Ω × cm 2 and fluorescein permeability below 1.95 × 10 -7 cm/s. E6-derived BMECs maintained TEER above 1000 Ω × cm 2 for a minimum of 8 days and showed no statistical difference in efflux transporter activity compared to BMECs differentiated by conventional means. The method was also found to support long-term stability of BMECs harboring biallelic PARK2 mutations associated

  2. Edaravone Protects against Methylglyoxal-Induced Barrier Damage in Human Brain Endothelial Cells

    Science.gov (United States)

    Tóth, Andrea E.; Walter, Fruzsina R.; Bocsik, Alexandra; Sántha, Petra; Veszelka, Szilvia; Nagy, Lajos; Puskás, László G.; Couraud, Pierre-Olivier; Takata, Fuyuko; Dohgu, Shinya; Kataoka, Yasufumi; Deli, Mária A.

    2014-01-01

    Background Elevated level of reactive carbonyl species, such as methylglyoxal, triggers carbonyl stress and activates a series of inflammatory responses leading to accelerated vascular damage. Edaravone is the active substance of a Japanese medicine, which aids neurological recovery following acute brain ischemia and subsequent cerebral infarction. Our aim was to test whether edaravone can exert a protective effect on the barrier properties of human brain endothelial cells (hCMEC/D3 cell line) treated with methylglyoxal. Methodology Cell viability was monitored in real-time by impedance-based cell electronic sensing. The barrier function of the monolayer was characterized by measurement of resistance and flux of permeability markers, and visualized by immunohistochemistry for claudin-5 and β-catenin. Cell morphology was also examined by holographic phase imaging. Principal Findings Methylglyoxal exerted a time- and dose-dependent toxicity on cultured human brain endothelial cells: a concentration of 600 µM resulted in about 50% toxicity, significantly reduced the integrity and increased the permeability of the barrier. The cell morphology also changed dramatically: the area of cells decreased, their optical height significantly increased. Edaravone (3 mM) provided a complete protection against the toxic effect of methylglyoxal. Co-administration of edaravone restored cell viability, barrier integrity and functions of brain endothelial cells. Similar protection was obtained by the well-known antiglycating molecule, aminoguanidine, our reference compound. Conclusion These results indicate for the first time that edaravone is protective in carbonyl stress induced barrier damage. Our data may contribute to the development of compounds to treat brain endothelial dysfunction in carbonyl stress related diseases. PMID:25033388

  3. Edaravone protects against methylglyoxal-induced barrier damage in human brain endothelial cells.

    Directory of Open Access Journals (Sweden)

    Andrea E Tóth

    Full Text Available Elevated level of reactive carbonyl species, such as methylglyoxal, triggers carbonyl stress and activates a series of inflammatory responses leading to accelerated vascular damage. Edaravone is the active substance of a Japanese medicine, which aids neurological recovery following acute brain ischemia and subsequent cerebral infarction. Our aim was to test whether edaravone can exert a protective effect on the barrier properties of human brain endothelial cells (hCMEC/D3 cell line treated with methylglyoxal.Cell viability was monitored in real-time by impedance-based cell electronic sensing. The barrier function of the monolayer was characterized by measurement of resistance and flux of permeability markers, and visualized by immunohistochemistry for claudin-5 and β-catenin. Cell morphology was also examined by holographic phase imaging.Methylglyoxal exerted a time- and dose-dependent toxicity on cultured human brain endothelial cells: a concentration of 600 µM resulted in about 50% toxicity, significantly reduced the integrity and increased the permeability of the barrier. The cell morphology also changed dramatically: the area of cells decreased, their optical height significantly increased. Edaravone (3 mM provided a complete protection against the toxic effect of methylglyoxal. Co-administration of edaravone restored cell viability, barrier integrity and functions of brain endothelial cells. Similar protection was obtained by the well-known antiglycating molecule, aminoguanidine, our reference compound.These results indicate for the first time that edaravone is protective in carbonyl stress induced barrier damage. Our data may contribute to the development of compounds to treat brain endothelial dysfunction in carbonyl stress related diseases.

  4. Radiation Effects on the Cytoskeleton of Endothelial Cells and Endothelial Monolayer Permeability

    International Nuclear Information System (INIS)

    Gabrys, Dorota; Greco, Olga; Patel, Gaurang; Prise, Kevin M.; Tozer, Gillian M.; Kanthou, Chryso

    2007-01-01

    Purpose: To investigate the effects of radiation on the endothelial cytoskeleton and endothelial monolayer permeability and to evaluate associated signaling pathways, which could reveal potential mechanisms of known vascular effects of radiation. Methods and Materials: Cultured endothelial cells were X-ray irradiated, and actin filaments, microtubules, intermediate filaments, and vascular endothelial (VE)-cadherin junctions were examined by immunofluorescence. Permeability was determined by the passage of fluorescent dextran through cell monolayers. Signal transduction pathways were analyzed using RhoA, Rho kinase, and stress-activated protein kinase-p38 (SAPK2/p38) inhibitors by guanosine triphosphate-RhoA activation assay and transfection with RhoAT19N. The levels of junction protein expression and phosphorylation of myosin light chain and SAPK2/p38 were assessed by Western blotting. The radiation effects on cell death were verified by clonogenic assays. Results: Radiation induced rapid and persistent actin stress fiber formation and redistribution of VE-cadherin junctions in microvascular, but not umbilical vein endothelial cells, and microtubules and intermediate filaments remained unaffected. Radiation also caused a rapid and persistent increase in microvascular permeability. RhoA-guanosine triphosphatase and Rho kinase were activated by radiation and caused phosphorylation of downstream myosin light chain and the observed cytoskeletal and permeability changes. SAPK2/p38 was activated by radiation but did not influence either the cytoskeleton or permeability. Conclusion: This study is the first to show rapid activation of the RhoA/Rho kinase by radiation in endothelial cells and has demonstrated a link between this pathway and cytoskeletal remodeling and permeability. The results also suggest that the RhoA pathway might be a useful target for modulating the permeability and other effects of radiation for therapeutic gain

  5. Circulating IGF-1 deficiency exacerbates hypertension-induced microvascular rarefaction in the mouse hippocampus and retrosplenial cortex: implications for cerebromicrovascular and brain aging.

    Science.gov (United States)

    Tarantini, Stefano; Tucsek, Zsuzsanna; Valcarcel-Ares, M Noa; Toth, Peter; Gautam, Tripti; Giles, Cory B; Ballabh, Praveen; Wei, Jeanne Y; Wren, Jonathan D; Ashpole, Nicole M; Sonntag, William E; Ungvari, Zoltan; Csiszar, Anna

    2016-08-01

    Strong epidemiological and experimental evidence indicate that both age and hypertension lead to significant functional and structural impairment of the cerebral microcirculation, predisposing to the development of vascular cognitive impairment (VCI) and Alzheimer's disease. Preclinical studies establish a causal link between cognitive decline and microvascular rarefaction in the hippocampus, an area of brain important for learning and memory. Age-related decline in circulating IGF-1 levels results in functional impairment of the cerebral microvessels; however, the mechanistic role of IGF-1 deficiency in impaired hippocampal microvascularization remains elusive. The present study was designed to characterize the additive/synergistic effects of IGF-1 deficiency and hypertension on microvascular density and expression of genes involved in angiogenesis and microvascular regression in the hippocampus. To achieve that goal, we induced hypertension in control and IGF-1 deficient mice (Igf1 f/f  + TBG-Cre-AAV8) by chronic infusion of angiotensin II. We found that circulating IGF-1 deficiency is associated with decreased microvascular density and exacerbates hypertension-induced microvascular rarefaction both in the hippocampus and the neocortex. The anti-angiogenic hippocampal gene expression signature observed in hypertensive IGF-1 deficient mice in the present study provides important clues for subsequent studies to elucidate mechanisms by which hypertension may contribute to the pathogenesis and clinical manifestation of VCI. In conclusion, adult-onset, isolated endocrine IGF-1 deficiency exerts deleterious effects on the cerebral microcirculation, leading to a significant decline in cortical and hippocampal capillarity and exacerbating hypertension-induced cerebromicrovascular rarefaction. The morphological impairment of the cerebral microvasculature induced by IGF-1 deficiency and hypertension reported here, in combination with neurovascular uncoupling, increased

  6. In vitro models of the blood–brain barrier: An overview of commonly used brain endothelial cell culture models and guidelines for their use

    OpenAIRE

    Helms, Hans C; Abbott, N Joan; Burek, Malgorzata; Cecchelli, Romeo; Couraud, Pierre-Olivier; Deli, Maria A; Förster, Carola; Galla, Hans J; Romero, Ignacio A; Shusta, Eric V; Stebbins, Matthew J; Vandenhaute, Elodie; Weksler, Babette; Brodin, Birger

    2016-01-01

    The endothelial cells lining the brain capillaries separate the blood from the brain parenchyma. The endothelial monolayer of the brain capillaries serves both as a crucial interface for exchange of nutrients, gases, and metabolites between blood and brain, and as a barrier for neurotoxic components of plasma and xenobiotics. This “blood-brain barrier” function is a major hindrance for drug uptake into the brain parenchyma. Cell culture models, based on either primary cells or immortalized br...

  7. Pathways for insulin access to the brain: the role of the microvascular endothelial cell

    OpenAIRE

    Meijer, Rick I.; Gray, Sarah M.; Aylor, Kevin W.; Barrett, Eugene J.

    2016-01-01

    New understanding of the directional flow of subarachnoid cerebrospinal fluid (CSF) through the Virchow-Robin space (VRS) to brain parenchyma, coupled with the demonstration here of rapid, insulin receptor-dependent trapping of plasma insulin by the brain microvasculature, underscores the direct role of insulin's blood-brain barrier transit to insulin delivery to the brain.

  8. Resveratrol protects vascular endothelial cells from high glucose-induced apoptosis through inhibition of NADPH oxidase activation-driven oxidative stress.

    Science.gov (United States)

    Chen, Feng; Qian, Li-Hua; Deng, Bo; Liu, Zhi-Min; Zhao, Ying; Le, Ying-Ying

    2013-09-01

    Hyperglycemia-induced oxidative stress has been implicated in diabetic vascular complications in which NADPH oxidase is a major source of reactive oxygen species (ROS) generation. Resveratrol is a naturally occurring polyphenol, which has vasoprotective effects in diabetic animal models and inhibits high glucose (HG)-induced oxidative stress in endothelial cells. We aimed to examine whether HG-induced NADPH oxidase activation and ROS production contribute to glucotoxicity to endothelial cells and the effect of resveratrol on glucotoxicity. Using a murine brain microvascular endothelial cell line bEnd3, we found that NADPH oxidase inhibitor (apocynin) and resveratrol both inhibited HG-induced endothelial cell apoptosis. HG-induced elevation of NADPH oxidase activity and production of ROS were inhibited by apocynin, suggesting that HG induces endothelial cell apoptosis through NADPH oxidase-mediated ROS production. Mechanistic studies revealed that HG upregulated NADPH oxidase subunit Nox1 but not Nox2, Nox4, and p22(phox) expression through NF-κB activation, which resulted in elevation of NADPH oxidase activity and consequent ROS production. Resveratrol prevented HG-induced endothelial cell apoptosis through inhibiting HG-induced NF-κB activation, NADPH oxidase activity elevation, and ROS production. HG induces endothelial cell apoptosis through NF-κB/NADPH oxidase/ROS pathway, which was inhibited by resveratrol. Our findings provide new potential therapeutic targets against brain vascular complications of diabetes. © 2013 John Wiley & Sons Ltd.

  9. Targeting the dominant mechanism of coronary microvascular dysfunction with intracoronary physiology tests

    NARCIS (Netherlands)

    Mejia-Renteria, H.; Hoeven, N. van der; Hoef, T.P. van de; Heemelaar, J.; Ryan, N.; Lerman, A.; Royen, N. van; Escaned, J.

    2017-01-01

    The coronary microcirculation plays a key role in modulating blood supply to the myocardium. Several factors like myocardial oxygen demands, endothelial and neurogenic conditions determine its function. Although there is available evidence supporting microvascular dysfunction as an important cause

  10. Dysregulation of coronary microvascular reactivity in asymptomatic patients with type 2 diabetes mellitus

    Energy Technology Data Exchange (ETDEWEB)

    Momose, Mitsuru; Neverve, Jodi; Nekolla, Stephan G.; Schwaiger, Markus; Bengel, Frank M. [Nuklearmedizinische Klinik und Poliklinik der Technischen Universitaet Muenchen, Klinikum rechts der Isar, Ismaninger Strasse 22, 81675 Munich (Germany); Abletshauser, Claudia [Department of Medicine, Novartis Pharma GmbH, Nuernberg (Germany); Schnell, Oliver; Standl, Eberhard [Institut fuer Diabetesforschung, Munich (Germany)

    2002-12-01

    In diabetic patients, a number of studies have suggested an impairment of vascular reactivity in response to vasodilatory stimuli. The pattern of dysregulation at the coronary microcirculatory level, however, has not been clearly defined. Thus, it was the aim of this study to characterise coronary microvascular function non-invasively in a homogeneous group of asymptomatic type 2 diabetic patients. In 46 patients with type 2 diabetes, myocardial blood flow (MBF) was quantified at baseline, in response to cold pressor test (CPT) and during adenosine-mediated vasodilation using positron emission tomography and nitrogen-13 ammonia. None of the patients had been treated with insulin, and none had symptoms of cardiac disease. Decreased MBF during CPT, indicating microvascular dysregulation, was observed in 16 patients (CPT-), while 30 patients demonstrated increased MBF during CPT (CPT+). Response to CPT was mildly, but significantly correlated with response to adenosine (r=0.44, P=0.0035). There was no difference in HbA1c, serum lipid levels or serum endothelial markers between the groups. Microvascular dysregulation in the CPT- group was associated with elevated baseline MBF (P<0.0001), reduced baseline vascular resistance (P=0.0026) and an abnormal increase in resistance during CPT (P=0.0002). In conclusion, coronary microvascular dysregulation is present in approximately one-third of asymptomatic, non-insulin-treated type 2 diabetic patients. Elevated baseline blood flow and reduced microvascular resistance at rest are characteristics of this dysregulation. These data suggest a state of activation of endothelial-dependent vasodilation at baseline which appears to limit the flow response to stress conditions. (orig.)

  11. Inflammation-induced microvascular insulin resistance is an early event in diet-induced obesity

    Science.gov (United States)

    Zhao, Lina; Fu, Zhuo; Wu, Jing; Aylor, Kevin W.; Barrett, Eugene J.; Cao, Wenhong

    2015-01-01

    Endothelial dysfunction and vascular insulin resistance usually coexist and chronic inflammation engenders both. In the present study, we investigate the temporal relationship between vascular insulin resistance and metabolic insulin resistance. We assessed insulin responses in all arterial segments, including aorta, distal saphenous artery and the microvasculature, as well as the metabolic insulin responses in muscle in rats fed on a high-fat diet (HFD) for various durations ranging from 3 days to 4 weeks with or without sodium salicylate treatment. Compared with controls, HFD feeding significantly blunted insulin-mediated Akt (protein kinase B) and eNOS [endothelial nitric oxide (NO) synthase] phosphorylation in aorta in 1 week, blunted vasodilatory response in small resistance vessel in 4 weeks and microvascular recruitment in as early as 3 days. Insulin-stimulated whole body glucose disposal did not begin to progressively decrease until after 1 week. Salicylate treatment fully inhibited vascular inflammation, prevented microvascular insulin resistance and significantly improved muscle metabolic responses to insulin. We conclude that microvascular insulin resistance is an early event in diet-induced obesity and insulin resistance and inflammation plays an essential role in this process. Our data suggest microvascular insulin resistance contributes to the development of metabolic insulin resistance in muscle and muscle microvasculature is a potential therapeutic target in the prevention and treatment of diabetes and its related complications. PMID:26265791

  12. A novel effective method for the assessment of microvascular function in male patients with coronary artery disease: a pilot study using laser speckle contrast imaging

    Energy Technology Data Exchange (ETDEWEB)

    Borges, J.P. [Laboratório de Atividade Física e Promoção è Saúde, Departamento de Desporto Coletivo, Instituto de Educação Física e Desportos, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, RJ (Brazil); Lopes, G.O. [Laboratório de Atividade Física e Promoção è Saúde, Departamento de Desporto Coletivo, Instituto de Educação Física e Desportos, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, RJ (Brazil); Instituto Nacional de Cardiologia, Rio de Janeiro, RJ (Brazil); Verri, V.; Coelho, M.P.; Nascimento, P.M.C.; Kopiler, D.A. [Instituto Nacional de Cardiologia, Rio de Janeiro, RJ (Brazil); Tibirica, E. [Instituto Nacional de Cardiologia, Rio de Janeiro, RJ (Brazil); Laboratório de Investigação Cardiovascular, Departamento Osório de Almeida, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, RJ (Brazil)

    2016-09-01

    Evaluation of microvascular endothelial function is essential for investigating the pathophysiology and treatment of cardiovascular and metabolic diseases. Although laser speckle contrast imaging technology is well accepted as a noninvasive methodology for assessing microvascular endothelial function, it has never been used to compare male patients with coronary artery disease with male age-matched healthy controls. Thus, the aim of this study was to determine whether laser speckle contrast imaging could be used to detect differences in the systemic microvascular functions of patients with established cardiovascular disease (n=61) and healthy age-matched subjects (n=24). Cutaneous blood flow was assessed in the skin of the forearm using laser speckle contrast imaging coupled with the transdermal iontophoretic delivery of acetylcholine and post-occlusive reactive hyperemia. The maximum increase in skin blood flow induced by acetylcholine was significantly reduced in the cardiovascular disease patients compared with the control subjects (74 vs 116%; P<0.01). With regard to post-occlusive reactive hyperemia-induced vasodilation, the patients also presented reduced responses compared to the controls (0.42±0.15 vs 0.50±0.13 APU/mmHg; P=0.04). In conclusion, laser speckle contrast imaging can identify endothelial and microvascular dysfunctions in male individuals with cardiovascular disease. Thus, this technology appears to be an efficient non-invasive technique for evaluating systemic microvascular and endothelial functions, which could be valuable as a peripheral marker of atherothrombotic diseases in men.

  13. A novel effective method for the assessment of microvascular function in male patients with coronary artery disease: a pilot study using laser speckle contrast imaging

    International Nuclear Information System (INIS)

    Borges, J.P.; Lopes, G.O.; Verri, V.; Coelho, M.P.; Nascimento, P.M.C.; Kopiler, D.A.; Tibirica, E.

    2016-01-01

    Evaluation of microvascular endothelial function is essential for investigating the pathophysiology and treatment of cardiovascular and metabolic diseases. Although laser speckle contrast imaging technology is well accepted as a noninvasive methodology for assessing microvascular endothelial function, it has never been used to compare male patients with coronary artery disease with male age-matched healthy controls. Thus, the aim of this study was to determine whether laser speckle contrast imaging could be used to detect differences in the systemic microvascular functions of patients with established cardiovascular disease (n=61) and healthy age-matched subjects (n=24). Cutaneous blood flow was assessed in the skin of the forearm using laser speckle contrast imaging coupled with the transdermal iontophoretic delivery of acetylcholine and post-occlusive reactive hyperemia. The maximum increase in skin blood flow induced by acetylcholine was significantly reduced in the cardiovascular disease patients compared with the control subjects (74 vs 116%; P<0.01). With regard to post-occlusive reactive hyperemia-induced vasodilation, the patients also presented reduced responses compared to the controls (0.42±0.15 vs 0.50±0.13 APU/mmHg; P=0.04). In conclusion, laser speckle contrast imaging can identify endothelial and microvascular dysfunctions in male individuals with cardiovascular disease. Thus, this technology appears to be an efficient non-invasive technique for evaluating systemic microvascular and endothelial functions, which could be valuable as a peripheral marker of atherothrombotic diseases in men

  14. Longitudinal assessment of endothelial function in the microvasculature of mice in-vivo.

    Science.gov (United States)

    Belch, Jill J F; Akbar, Naveed; Alapati, Venkateswara; Petrie, John; Arthur, Simon; Khan, Faisel

    2013-01-01

    Endothelial dysfunction is associated with early development of cardiovascular disease, making longitudinal measurements desirable. We devised a protocol using laser Doppler imaging (LDI) and iontophoresis of acetylcholine (ACh) and sodium nitroprusside (SNP) to assess the skin microcirculation longitudinally in mice every 4 weeks for 24 weeks in two groups of C57BL/6 mice, chow versus high-cholesterol diet(known to induce endothelial dysfunction). LDI measurements were compared with vascular function (isometric tension) measured using wire myography in the tail artery in response to ACh and SNP. Microvascular responses to ACh were significantly reduced in cholesterol-fed versus chow-fed mice from week 4 onwards (Phydrochloride (L-NAME) showed a significant reduction in ACh response compared with vehicle-treated animals (P<0.05) at baseline and at 12 weeks. In cholesterol-fed mice, ACh responses were 226 ± 21 and 180 ± 21 AU (P=0.03) before and after L-NAME, respectively. A reduction in ex-vivo ACh response was detected in the tail artery in cholesterol-fed mice, and a significant correlation found between peak microvascular ACh response and maximum ACh response in the tail artery (r=0.699, P=0.017). No changes were found in SNP responses in the microvasculature or tail artery. Using this protocol, we have shown longitudinal decreases in microvascular endothelial function to cholesterol feeding. L-NAME studies confirm that the reduced vasodilatation to ACh in cholesterol-fed mice was mediated partly through reduced NO bioavailability. Wire myography of tail arteries confirmed that in-vivo measurements of microvascular function reflect ex-vivo vascular function in other beds. Longitudinal assessments of skin microvascular function in mice could provide a useful translatable model for assessing early endothelial dysfunction. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Benfotiamine prevents macro- and microvascular endothelial dysfunction and oxidative stress following a meal rich in advanced glycation end products in individuals with type 2 diabetes.

    Science.gov (United States)

    Stirban, Alin; Negrean, Monica; Stratmann, Bernd; Gawlowski, Thomas; Horstmann, Tina; Götting, Christian; Kleesiek, Knut; Mueller-Roesel, Michaela; Koschinsky, Theodor; Uribarri, Jaime; Vlassara, Helen; Tschoepe, Diethelm

    2006-09-01

    Diabetes is characterized by marked postprandial endothelial dysfunction induced by hyperglycemia, hypertriglyceridemia, advanced glycation end products (AGEs), and dicarbonyls (e.g., methylglyoxal [MG]). In vitro hyperglycemia-induced MG formation and endothelial dysfunction could be blocked by benfotiamine, but in vivo effects of benfotiamine on postprandial endothelial dysfunction and MG synthesis have not been investigated in humans until now. Thirteen people with type 2 diabetes were given a heat-processed test meal with a high AGE content (HAGE; 15.100 AGE kU, 580 kcal, 54 g protein, 17 g lipids, and 48 g carbohydrates) before and after a 3-day therapy with benfotiamine (1,050 mg/day). Macrovascular flow-mediated dilatation (FMD) and microvascular reactive hyperemia, along with serum markers of endothelial disfunction (E-selectin, vascular cell adhesion molecule-1, and intracellular adhesion molecule-1), oxidative stress, AGE, and MG were measured during both test meal days after an overnight fast and then at 2, 4, and 6 h postprandially. The HAGE induced a maximum reactive hyperemia decrease of -60.0% after 2 h and a maximum FMD impairment of -35.1% after 4 h, without affecting endothelium-independent vasodilatation. The effects of HAGE on both FMD and reactive hyperemia were completely prevented by benfotiamine. Serum markers of endothelial dysfunction and oxidative stress, as well as AGE, increased after HAGE. These effects were significantly reduced by benfotiamine. Our study confirms micro- and macrovascular endothelial dysfunction accompanied by increased oxidative stress following a real-life, heat-processed, AGE-rich meal in individuals with type 2 diabetes and suggests benfotiamine as a potential treatment.

  16. Epigalloccatechin-3-gallate Inhibits Ocular Neovascularization and Vascular Permeability in Human Retinal Pigment Epithelial and Human Retinal Microvascular Endothelial Cells via Suppression of MMP-9 and VEGF Activation

    Directory of Open Access Journals (Sweden)

    Hak Sung Lee

    2014-08-01

    Full Text Available Epigalloccatechin-3-gallate (EGCG is the main polyphenol component of green tea (leaves of Camellia sinensis. EGCG is known for its antioxidant, anti-inflammatory, antiviral, and anti-carcinogenic properties. Here, we identify EGCG as a new inhibitor of ocular angiogenesis and its vascular permeability. Matrix metalloproteinases (MMPs and vascular endothelial growth factor (VEGF play a key role in the processes of extracellular matrix (ECM remodeling and microvascular permeability during angiogenesis. We investigated the inhibitory effects of EGCG on ocular neovascularization and vascular permeability using the retina oriented cells and animal models induced by VEGF and alkaline burn. EGCG treatment significantly decreased mRNA and protein expression levels of MMP-9 in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA and tumor necrosis factor alpha (TNF-α in human retinal pigment epithelial cells (HRPECs. EGCG also effectively protected ARPE-19 cells from cell death and attenuated mRNA expressions of key angiogenic factors (MMP-9, VEGF, VEGF Receptor-2 by inhibiting generation of reactive oxygen species (ROS. EGCG significantly inhibited proliferation, vascular permeability, and tube formation in VEGF-induced human retinal microvascular endothelial cells (HRMECs. Furthermore, EGCG significantly reduced vascular leakage and permeability by blood-retinal barrier breakdown in VEGF-induced animal models. In addition, EGCG effectively limited upregulation of MMP-9 and platelet endothelial cell adhesion molecule (PECAM/CD31 on corneal neovascularization (CNV induced by alkaline burn. Our data suggest that MMP-9 and VEGF are key therapeutic targets of EGCG for treatment and prevention of ocular angiogenic diseases such as age-related macular degeneration, diabetic retinopathy, and corneal neovascularization.

  17. Plasmalemmal V-H+-ATPases regulate intracellular pH in human lung microvascular endothelial cells

    International Nuclear Information System (INIS)

    Rojas, Jose D.; Sennoune, Souad R.; Maiti, Debasish; Martinez, Gloria M.; Bakunts, Karina; Wesson, Donald E.; Martinez-Zaguilan, Raul

    2004-01-01

    The lung endothelium layer is exposed to continuous CO 2 transit which exposes the endothelium to a substantial acid load that could be detrimental to cell function. The Na + /H + exchanger and HCO 3 - -dependent H + -transporting mechanisms regulate intracellular pH (pH cyt ) in most cells. Cells that cope with high acid loads might require additional primary energy-dependent mechanisms. V-H + -ATPases localized at the plasma membranes (pmV-ATPases) have emerged as a novel pH regulatory system. We hypothesized that human lung microvascular endothelial (HLMVE) cells use pmV-ATPases, in addition to Na + /H + exchanger and HCO 3 - -based H + -transporting mechanisms, to maintain pH cyt homeostasis. Immunocytochemical studies revealed V-H + -ATPase at the plasma membrane, in addition to the predicted distribution in vacuolar compartments. Acid-loaded HLMVE cells exhibited proton fluxes in the absence of Na + and HCO 3 - that were similar to those observed in the presence of either Na + , or Na + and HCO 3 - . The Na + - and HCO 3 - -independent pH cyt recovery was inhibited by bafilomycin A 1 , a V-H + -ATPase inhibitor. These studies show a Na + - and HCO 3 - -independent pH cyt regulatory mechanism in HLMVE cells that is mediated by pmV-ATPases

  18. Endothelial β-Catenin Signaling Is Required for Maintaining Adult Blood-Brain Barrier Integrity and Central Nervous System Homeostasis.

    Science.gov (United States)

    Tran, Khiem A; Zhang, Xianming; Predescu, Dan; Huang, Xiaojia; Machado, Roberto F; Göthert, Joachim R; Malik, Asrar B; Valyi-Nagy, Tibor; Zhao, You-Yang

    2016-01-12

    The blood-brain barrier (BBB) formed by brain endothelial cells interconnected by tight junctions is essential for the homeostasis of the central nervous system. Although studies have shown the importance of various signaling molecules in BBB formation during development, little is known about the molecular basis regulating the integrity of the adult BBB. Using a mouse model with tamoxifen-inducible endothelial cell-restricted disruption of ctnnb1 (iCKO), we show here that endothelial β-catenin signaling is essential for maintaining BBB integrity and central nervous system homeostasis in adult mice. The iCKO mice developed severe seizures accompanied by neuronal injury, multiple brain petechial hemorrhages, and central nervous system inflammation, and all had postictal death. Disruption of endothelial β-catenin induced BBB breakdown and downregulation of the specific tight junction proteins claudin-1 and -3 in adult brain endothelial cells. The clinical relevance of the data is indicated by the observation of decreased expression of claudin-1 and nuclear β-catenin in brain endothelial cells of hemorrhagic lesions of hemorrhagic stroke patients. These results demonstrate the prerequisite role of endothelial β-catenin in maintaining the integrity of adult BBB. The results suggest that BBB dysfunction secondary to defective β-catenin transcription activity is a key pathogenic factor in hemorrhagic stroke, seizure activity, and central nervous system inflammation. © 2015 American Heart Association, Inc.

  19. Contacting co-culture of human retinal microvascular endothelial cells alters barrier function of human embryonic stem cell derived retinal pigment epithelial cells.

    Science.gov (United States)

    Skottman, H; Muranen, J; Lähdekorpi, H; Pajula, E; Mäkelä, K; Koivusalo, L; Koistinen, A; Uusitalo, H; Kaarniranta, K; Juuti-Uusitalo, K

    2017-10-01

    Here we evaluated the effects of human retinal microvascular endothelial cells (hREC) on mature human embryonic stem cell (hESC) derived retinal pigment epithelial (RPE) cells. The hESC-RPE cells (Regea08/017, Regea08/023 or Regea11/013) and hREC (ACBRI 181) were co-cultured on opposite sides of transparent membranes for up to six weeks. Thereafter barrier function, small molecule permeability, localization of RPE and endothelial cell marker proteins, cellular fine structure, and growth factor secretion of were evaluated. After co-culture, the RPE specific CRALBP and endothelial cell specific von Willebrand factor were appropriately localized. In addition, the general morphology, pigmentation, and fine structure of hESC-RPE cells were unaffected. Co-culture increased the barrier function of hESC-RPE cells, detected both with TEER measurements and cumulative permeability of FD4 - although the differences varied among the cell lines. Co-culturing significantly altered VEGF and PEDF secretion, but again the differences were cell line specific. The results of this study showed that co-culture with hREC affects hESC-RPE functionality. In addition, co-culture revealed drastic cell line specific differences, most notably in growth factor secretion. This model has the potential to be used as an in vitro outer blood-retinal barrier model for drug permeability testing. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  20. Inhaled nitric oxide pretreatment but not posttreatment attenuates ischemia-reperfusion-induced pulmonary microvascular leak.

    Science.gov (United States)

    Chetham, P M; Sefton, W D; Bridges, J P; Stevens, T; McMurtry, I F

    1997-04-01

    Ischemia-reperfusion (I/R) pulmonary edema probably reflects a leukocyte-dependent, oxidant-mediated mechanism. Nitric oxide (NO) attenuates leukocyte-endothelial cell interactions and I/R-induced microvascular leak. Cyclic adenosine monophosphate (cAMP) agonists reverse and prevent I/R-induced microvascular leak, but reversal by inhaled NO (INO) has not been tested. In addition, the role of soluble guanylyl cyclase (sGC) activation in the NO protection effect is unknown. Rat lungs perfused with salt solution were grouped as either I/R, I/R with INO (10 or 50 ppm) on reperfusion, or time control. Capillary filtration coefficients (Kfc) were estimated 25 min before ischemia (baseline) and after 30 and 75 min of reperfusion. Perfusate cell counts and lung homogenate myeloperoxidase activity were determined in selected groups. Additional groups were treated with either INO (50 ppm) or isoproterenol (ISO-10 microM) after 30 min of reperfusion. Guanylyl cyclase was inhibited with 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ-15 microM), and Kfc was estimated at baseline and after 30 min of reperfusion. (1) Inhaled NO attenuated I/R-induced increases in Kfc. (2) Cell counts were similar at baseline. After 75 min of reperfusion, lung neutrophil retention (myeloperoxidase activity) and decreased perfusate neutrophil counts were similar in all groups. (3) In contrast to ISO, INO did not reverse microvascular leak. (4) 8-bromoguanosine 3',5'-cyclic monophosphate (8-br-cGMP) prevented I/R-induced microvascular leak in ODQ-treated lungs, but INO was no longer effective. Inhaled NO attenuates I/R-induced pulmonary microvascular leak, which requires sGC activation and may involve a mechanism independent of inhibition of leukocyte-endothelial cell interactions. In addition, INO is ineffective in reversing I/R-induced microvascular leak.

  1. Expression and deposition of basement membrane proteins by brain capillary endothelial cells in a primary murine model of the blood-brain barrier

    DEFF Research Database (Denmark)

    Thomsen, Maj Schneider; Birkelund, Svend; Larsen, Annette Burkhart

    2016-01-01

    The blood-brain barrier (BBB) represents the interface between the blood and the brain parenchyma and consists of endothelial cells which are tightly sealed together by tight junction proteins. The endothelial cells are in addition supported by pericytes, which are embedded in the vascular basement...... of the present study was to create four different in vitro constructs of the murine BBB to characterise if the expression and secretion of basement membrane proteins by the murine brain capillary endothelial cells (mBCECs) was affected by co-culturing with pericytes, mixed glial cells, or both. Primary m......BCECs and pericytes were isolated from brains of adult mice. Mixed glial cells were prepared from cerebral cortices of newborn mice. The mBCECs were grown as mono-culture, or co-cultured with pericytes, mixed glial cells, or both. To study the expression of basement membrane proteins RT-qPCR, mass spectrometry...

  2. Treatment of Angina and Microvascular Coronary Dysfunction

    Science.gov (United States)

    Samim, Arang; Nugent, Lynn; Mehta, Puja K.; Shufelt, Chrisandra; Merz, C. Noel Bairey

    2014-01-01

    Opinion statement Microvascular coronary dysfunction (MCD) is an increasingly recognized cause of cardiac ischemia and angina, more commonly diagnosed in women. Patients with MCD present with the triad of persistent chest pain, ischemic changes on stress testing, and no obstructive coronary artery disease (CAD) on cardiac catheterization. Data from National Heart, Lung and Blood Institute (NHLBI)-sponsored Women’s Ischemia Syndrome Evaluation (WISE) study has shown that the diagnosis of MCD is not benign, with a 2.5% annual risk of adverse cardiac events including myocardial infarction, stroke, congestive heart failure, or death. The gold standard diagnostic test for MCD is an invasive coronary reactivity test (CRT), which uses acetylcholine, adenosine, and nitroglycerin to test the endothelial dependent and independent, microvascular and macrovascular coronary function. The CRT allows for diagnostic and treatment options as well as further risk stratifying patients for future cardiovascular events. Treatment of angina and MCD should be aimed at ischemia disease management to reduce risk of adverse cardiac events, ameliorating symptoms to improve quality of life, and to decrease the morbidity from unnecessary and repeated cardiac catheterization in patients with open coronary arteries. A comprehensive treatment approach aimed at risk factor managment, including lifestyle counseling regarding smoking cessation, nutrition and physical activity should be initiated. Current pharmacotherapy for MCD can include the treatment of microvascular endothelial dysfunction (statins, angiotensin-converting enzyme inhibitor, low dose aspirin), as well as treatment for angina and myocardial ischemia (beta blockers, calcium channel blockers, nitrates, ranolazine). Additional symptom management techniques can include tri-cyclic medication, enhanced external counterpulsation, autogenic training, and spinal cord stimulation. While our current therapies are effective in the treatment

  3. Evaluation of the potential toxicity of unmodified and modified cyclodextrins on murine blood-brain barrier endothelial cells.

    Science.gov (United States)

    Shityakov, Sergey; Salmas, Ramin Ekhteiari; Salvador, Ellaine; Roewer, Norbert; Broscheit, Jens; Förster, Carola

    2016-04-01

    In this study, we investigated the cytotoxic effects of unmodified α-cyclodextrin (α-CD) and modified cyclodextrins, including trimethyl-β-cyclodextrin (TRIMEB) and hydroxypropyl-β-cyclodextrin (HPβCD), on immortalized murine microvascular endothelial (cEND) cells of the blood-brain barrier (BBB). A CellTiter-Glo viability test, performed on the cEND cells showed significant differences among the different cyclodextrins. After 24 hr of incubation, TRIMEB was the most cytotoxic, and HPβCD was non-toxic. α-CD and TRIMEB exhibited greater cytotoxicity in the Dulbecco's modified Eagle's medium than in heat-inactivated human serum indicating protective properties of the human serum. The predicted dynamic toxicity profiles (Td) for α-CD and TRIMEB indicated higher cytotoxicity for these cyclodextrins compared to the reference compound (dimethylsulfoxide). Molecular dynamics simulation of cholesterol binding to the CDs suggested that not just cholesterol but phospholipids extraction might be involved in the cytotoxicity. Overall, the results demonstrate that HPβCD has the potential to be used as a candidate for drug delivery vector development and signify a correlation between the in vitro cytotoxic effect and cholesterol binding of cyclodextrins.

  4. Early impairment of coronary microvascular perfusion capacity in rats on a high fat diet

    NARCIS (Netherlands)

    van Haare, Judith; Kooi, M. Eline; Vink, Hans; Post, Mark J.; van Teeffelen, Jurgen W. G. E.; Slenter, Jos; Munts, Chantal; Cobelens, Hanneke; Strijkers, Gustav J.; Koehn, Dennis; van Bilsen, Marc

    2015-01-01

    It remains to be established if, and to what extent, the coronary microcirculation becomes compromised during the development of obesity and insulin resistance. Recent studies suggest that changes in endothelial glycocalyx properties contribute to microvascular dysfunction under (pre-)diabetic

  5. Experimental inflammation following dural application of complete Freund's adjuvant or inflammatory soup does not alter brain and trigeminal microvascular passage

    DEFF Research Database (Denmark)

    Lundblad, Cornelia; Haanes, Kristian A; Grände, Gustaf

    2015-01-01

    , following dural application of complete Freund's adjuvant (CFA) or inflammatory soup (IS) on brain and trigeminal microvascular passage. METHODS: In order to address this issue, we induced local inflammation in male Sprague-Dawley-rats dura mater by the addition of CFA or IS directly on the dural surface...

  6. Gene delivery of therapeutic polypeptides to brain capillary endothelial cells for protein secretion

    DEFF Research Database (Denmark)

    Larsen, Annette Burkhart; Thomsen, Louiza Bohn; Moos, Torben

    . Results: mRNA expression of proteins with neuroprotective potential in RBEC were enabled. Their expression patters were compared with those of RBE4 and HeLa cells using RT-qPCR analyzes. The evidence for protein synthesis and secretion was obtained by detection of FLAG-tagged to the C-terminal of any......Background: The potential for treatment of chronic disorders affecting the CNS is complicated by the inability of several drugs to cross the blood-brain barrier (BBB). None-viral gene therapy applied to brain capillary endothelial cells (BCECs) denotes a novel approach to overcome the restraints...... in this passage, as turning BCECs into recombinant protein factories by transfection could result in protein secretion into the brain. Aim: The aim of the present study was to investigate the possibility of transfection to primary rat brain capillary endothelial cells (RBEC) for recombinant protein synthesis...

  7. Smuggling Drugs into the Brain: An Overview of Ligands Targeting Transcytosis for Drug Delivery across the Blood-Brain Barrier.

    Science.gov (United States)

    Georgieva, Julia V; Hoekstra, Dick; Zuhorn, Inge S

    2014-11-17

    The blood-brain barrier acts as a physical barrier that prevents free entry of blood-derived substances, including those intended for therapeutic applications. The development of molecular Trojan horses is a promising drug targeting technology that allows for non-invasive delivery of therapeutics into the brain. This concept relies on the application of natural or genetically engineered proteins or small peptides, capable of specifically ferrying a drug-payload that is either directly coupled or encapsulated in an appropriate nanocarrier, across the blood-brain barrier via receptor-mediated transcytosis. Specifically, in this process the nanocarrier-drug system ("Trojan horse complex") is transported transcellularly across the brain endothelium, from the blood to the brain interface, essentially trailed by a native receptor. Naturally, only certain properties would favor a receptor to serve as a transporter for nanocarriers, coated with appropriate ligands. Here we briefly discuss brain microvascular endothelial receptors that have been explored until now, highlighting molecular features that govern the efficiency of nanocarrier-mediated drug delivery into the brain.

  8. Low intensity shear stress increases endothelial ELR+ CXC chemokine production via a focal adhesion kinase-p38{beta} MAPK-NF-{kappa}B pathway.

    Science.gov (United States)

    Shaik, Sadiq S; Soltau, Thomas D; Chaturvedi, Gaurav; Totapally, Balagangadhar; Hagood, James S; Andrews, William W; Athar, Mohammad; Voitenok, Nikolai N; Killingsworth, Cheryl R; Patel, Rakesh P; Fallon, Michael B; Maheshwari, Akhil

    2009-02-27

    CXC chemokines with a glutamate-leucine-arginine (ELR) tripeptide motif (ELR(+) CXC chemokines) play an important role in leukocyte trafficking into the tissues. For reasons that are not well elucidated, circulating leukocytes are recruited into the tissues mainly in small vessels such as capillaries and venules. Because ELR(+) CXC chemokines are important mediators of endothelial-leukocyte interaction, we compared chemokine expression by microvascular and aortic endothelium to investigate whether differences in chemokine expression by various endothelial types could, at least partially, explain the microvascular localization of endothelial-leukocyte interaction. Both in vitro and in vivo models indicate that ELR(+) CXC chemokine expression is higher in microvascular endothelium than in aortic endothelial cells. These differences can be explained on the basis of the preferential activation of endothelial chemokine production by low intensity shear stress. Low shear activated endothelial ELR(+) CXC chemokine production via cell surface heparan sulfates, beta(3)-integrins, focal adhesion kinase, the mitogen-activated protein kinase p38beta, mitogen- and stress-associated protein kinase-1, and the transcription factor.

  9. Targeting immunoliposomes to transferrin receptors on brain capillary endothelial cells as a mean for cargo transport across the blood-brain barrier

    DEFF Research Database (Denmark)

    Johnsen, Kasper Bendix; Larsen, Annette Burkhart; Bruun, Jonas

    2016-01-01

    Brain capillary endothelial cells (BCECs) express transferrin receptors as opposed to endothelial cells of any organ in the remaining body, suggesting that targeting to the transferrin receptors as a reasonable strategy for delivering drugs to the CNS. However, as the intracellular trafficking...

  10. Better microvascular function on long-term treatment with lisinopril than with nifedipine in renal transplant recipients.

    Science.gov (United States)

    Asberg, A; Midtvedt, K; Vassbotn, T; Hartmann, A

    2001-07-01

    The prevalence of hypertension in renal transplant recipients is high but the pathophysiology is poorly defined. Impaired endothelial function may be a factor of major importance. The present study addresses the effects of long-term treatment with either lisinopril or slow-release nifedipine on microvascular function and plasma endothelin in renal transplant recipients on cyclosporin A (CsA). Seventy-five hypertensive renal transplant recipients were double-blind randomized to receive slow-release nifedipine (NIF, n=40) or lisinopril (LIS, n=35). Ten normotensive, age-matched recipients served as controls. All patients received CsA-based immunosuppressive therapy including prednisolone and azathioprine. Microvascular function was assessed in the forearm skin vasculature, using laser Doppler flowmetry in combination with post-occlusive reactive hyperaemia and endothelial-dependent function during local acetylcholine (ACh) stimulation. The analysis of microvascular function (AUC(rh)) showed that nifedipine-treated patients had significantly lower responses compared with lisinopril-treated patients (20+/-17 and 43+/-20 AU x min respectively, P=0.0016). Endothelial function was borderline significantly lower in the NIF group compared with the LIS group (640+/-345 and 817+/-404 AU x min respectively, P=0.056). The responses in the LIS group were comparable with those in non-hypertensive controls (AUC(rh) was 37+/-16 and AUC(ACh) was 994+/-566 AU x min). Plasma endothelin-1 concentrations were significantly higher in the NIF group compared with the LIS group (0.44+/-0.19 vs. 0.34+/-0.10 fmol/ml respectively, P=0.048), and were 0.29+/-0.09 fmol/ml in the control patients. AUC(ACh) was associated with plasma endothelin-1 (P=0.0053), while AUC(rh) was not (P=0.080). The study indicates that long-term treatment with lisinopril, when compared with nifedipine, yields a more beneficial effect on microvascular function in hypertensive renal transplant recipients on CsA. The

  11. Novel Mechanism of Attenuation of LPS-Induced NF-κB Activation by the Heat Shock Protein 90 Inhibitor, 17-N-allylamino-17-demethoxygeldanamycin, in Human Lung Microvascular Endothelial Cells

    Science.gov (United States)

    Thangjam, Gagan S.; Dimitropoulou, Chistiana; Joshi, Atul D.; Barabutis, Nektarios; Shaw, Mary C.; Kovalenkov, Yevgeniy; Wallace, Chistopher M.; Fulton, David J.; Patel, Vijay

    2014-01-01

    Heat shock protein (hsp) 90 inhibition attenuates NF-κB activation and blocks inflammation. However, the precise mechanism of NF-κB regulation by hsp90 in the endothelium is not clear. We investigated the mechanisms of hsp90 inhibition by 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) on NF-κB activation by LPS in primary human lung microvascular endothelial cells. Transcriptional activation of NF-κB was measured by luciferase reporter assay, gene expression by real-time RT-PCR, DNA binding of transcription factors by chromatin immunoprecipitation assay, protein–protein interaction by coimmunoprecipitation/immunoblotting, histone deacetylase (HDAC)/histone acetyltransferase enzyme activity by fluorometry, and nucleosome eviction by partial microccocal DNase digestion. In human lung microvascular endothelial cells, 17-AAG–induced degradation of IKBα was accomplished regardless of the phosphorylation/ubiquitination state of the protein. Hence, 17-AAG did not block LPS-induced NF-κB nuclear translocation and DNA binding activity. Instead, 17-AAG blocked the recruitment of the coactivator, cAMP response element binding protein binding protein, and prevented the assembly of a transcriptionally competent RNA polymerase II complex at the κB elements of the IKBα (an NF-κB–responsive gene) promoter. The effect of LPS on IKBα mRNA expression was associated with rapid deacetylation of histone-H3(Lys9) and a dramatic down-regulation of core histone H3 binding. Even though treatment with an HDAC inhibitor produced the same effect as hsp90 inhibition, the effect of 17-AAG was independent of HDAC. We conclude that hsp90 inhibition attenuates NF-κB transcriptional activation by preventing coactivator recruitment and nucleosome eviction from the target promoter in human lung endothelial cells. PMID:24303801

  12. In vitro evidence for the brain glutamate efflux hypothesis: brain endothelial cells cocultured with astrocytes display a polarized brain-to-blood transport of glutamate.

    Science.gov (United States)

    Helms, Hans Christian; Madelung, Rasmus; Waagepetersen, Helle Sønderby; Nielsen, Carsten Uhd; Brodin, Birger

    2012-05-01

    The concentration of the excitotoxic amino acid, L-glutamate, in brain interstitial fluid is tightly regulated by uptake transporters and metabolism in astrocytes and neurons. The aim of this study was to investigate the possible role of the blood-brain barrier endothelium in brain L-glutamate homeostasis. Transendothelial transport- and accumulation studies of (3) H-L-glutamate, (3) H-L-aspartate, and (3) H-D-aspartate in an electrically tight bovine endothelial/rat astrocyte blood-brain barrier coculture model were performed. After 6 days in culture, the endothelium displayed transendothelial resistance values of 1014 ± 70 Ω cm(2) , and (14) C-D-mannitol permeability values of 0.88 ± 0.13 × 10(-6) cm s(-1) . Unidirectional flux studies showed that L-aspartate and L-glutamate, but not D-aspartate, displayed polarized transport in the brain-to-blood direction, however, all three amino acids accumulated in the cocultures when applied from the abluminal side. The transcellular transport kinetics were characterized with a K(m) of 69 ± 15 μM and a J(max) of 44 ± 3.1 pmol min(-1) cm(-2) for L-aspartate and a K(m) of 138 ± 49 μM and J(max) of 28 ± 3.1 pmol min(-1) cm(-2) for L-glutamate. The EAAT inhibitor, DL-threo-ß-Benzyloxyaspartate, inhibited transendothelial brain-to-blood fluxes of L-glutamate and L-aspartate. Expression of EAAT-1 (Slc1a3), -2 (Slc1a2), and -3 (Slc1a1) mRNA in the endothelial cells was confirmed by conventional PCR and localization of EAAT-1 and -3 in endothelial cells was shown with immunofluorescence. Overall, the findings suggest that the blood-brain barrier itself may participate in regulating brain L-glutamate concentrations. Copyright © 2012 Wiley Periodicals, Inc.

  13. Uptake mechanism of ApoE-modified nanoparticles on brain capillary endothelial cells as a blood-brain barrier model.

    Science.gov (United States)

    Wagner, Sylvia; Zensi, Anja; Wien, Sascha L; Tschickardt, Sabrina E; Maier, Wladislaw; Vogel, Tikva; Worek, Franz; Pietrzik, Claus U; Kreuter, Jörg; von Briesen, Hagen

    2012-01-01

    The blood-brain barrier (BBB) represents an insurmountable obstacle for most drugs thus obstructing an effective treatment of many brain diseases. One solution for overcoming this barrier is a transport by binding of these drugs to surface-modified nanoparticles. Especially apolipoprotein E (ApoE) appears to play a major role in the nanoparticle-mediated drug transport across the BBB. However, at present the underlying mechanism is incompletely understood. In this study, the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells was investigated to differentiate between active and passive uptake mechanism by flow cytometry and confocal laser scanning microscopy. Furthermore, different in vitro co-incubation experiments were performed with competing ligands of the respective receptor. This study confirms an active endocytotic uptake mechanism and shows the involvement of low density lipoprotein receptor family members, notably the low density lipoprotein receptor related protein, on the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells. This knowledge of the uptake mechanism of ApoE-modified nanoparticles enables future developments to rationally create very specific and effective carriers to overcome the blood-brain barrier.

  14. Uptake mechanism of ApoE-modified nanoparticles on brain capillary endothelial cells as a blood-brain barrier model.

    Directory of Open Access Journals (Sweden)

    Sylvia Wagner

    Full Text Available BACKGROUND: The blood-brain barrier (BBB represents an insurmountable obstacle for most drugs thus obstructing an effective treatment of many brain diseases. One solution for overcoming this barrier is a transport by binding of these drugs to surface-modified nanoparticles. Especially apolipoprotein E (ApoE appears to play a major role in the nanoparticle-mediated drug transport across the BBB. However, at present the underlying mechanism is incompletely understood. METHODOLOGY/PRINCIPAL FINDINGS: In this study, the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells was investigated to differentiate between active and passive uptake mechanism by flow cytometry and confocal laser scanning microscopy. Furthermore, different in vitro co-incubation experiments were performed with competing ligands of the respective receptor. CONCLUSIONS/SIGNIFICANCE: This study confirms an active endocytotic uptake mechanism and shows the involvement of low density lipoprotein receptor family members, notably the low density lipoprotein receptor related protein, on the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells. This knowledge of the uptake mechanism of ApoE-modified nanoparticles enables future developments to rationally create very specific and effective carriers to overcome the blood-brain barrier.

  15. Microvascular and Macrovascular Abnormalities and Cognitive and Physical Function in Older Adults: Cardiovascular Health Study.

    Science.gov (United States)

    Kim, Dae Hyun; Grodstein, Francine; Newman, Anne B; Chaves, Paulo H M; Odden, Michelle C; Klein, Ronald; Sarnak, Mark J; Lipsitz, Lewis A

    2015-09-01

    To evaluate and compare the associations between microvascular and macrovascular abnormalities and cognitive and physical function Cross-sectional analysis of the Cardiovascular Health Study (1998-1999). Community. Individuals with available data on three or more of five microvascular abnormalities (brain, retina, kidney) and three or more of six macrovascular abnormalities (brain, carotid artery, heart, peripheral artery) (N = 2,452; mean age 79.5). Standardized composite scores derived from three cognitive tests (Modified Mini-Mental State Examination, Digit-Symbol Substitution Test, Trail-Making Test (TMT)) and three physical tests (gait speed, grip strength, 5-time sit to stand) Participants with high microvascular and macrovascular burden had worse cognitive (mean score difference = -0.30, 95% confidence interval (CI) = -0.37 to -0.24) and physical (mean score difference = -0.32, 95% CI = -0.38 to -0.26) function than those with low microvascular and macrovascular burden. Individuals with high microvascular burden alone had similarly lower scores than those with high macrovascular burden alone (cognitive function: -0.16, 95% CI = -0.24 to -0.08 vs -0.13, 95% CI = -0.20 to -0.06; physical function: -0.15, 95% CI = -0.22 to -0.08 vs -0.12, 95% CI = -0.18 to -0.06). Psychomotor speed and working memory, assessed using the TMT, were only impaired in the presence of high microvascular burden. Of the 11 vascular abnormalities considered, white matter hyperintensity, cystatin C-based glomerular filtration rate, large brain infarct, and ankle-arm index were independently associated with cognitive and physical function. Microvascular and macrovascular abnormalities assessed using noninvasive tests of the brain, kidney, and peripheral artery were independently associated with poor cognitive and physical function in older adults. Future research should evaluate the usefulness of these tests in prognostication. © 2015, Copyright the Authors Journal compilation © 2015

  16. Normal saline influences coagulation and endothelial function after traumatic brain injury and hemorrhagic shock in pigs

    DEFF Research Database (Denmark)

    Dekker, Simone E; Sillesen, Martin; Bambakidis, Ted

    2014-01-01

    ), colloids (Hextend [HEX]), and fresh frozen plasma (FFP) resuscitation are associated with differential effects on coagulation and endothelial systems. METHODS: We subjected 15 Yorkshire swine to TBI and HS (40% blood volume), and kept in HS for 2 hours before resuscitation with NS, HEX, or FFP. Markers......BACKGROUND: Traumatic brain injury (TBI) and hemorrhagic shock (HS) are the leading causes of trauma-related deaths. These insults disrupt coagulation and endothelial systems. This study investigated whether previously reported differences in lesion size and brain swelling during normal saline (NS...... of endothelial activation (E-selectin, Intercellular adhesion molecule [ICAM]-1), coagulation activation (prothrombin fragment 1 + 2), and natural anticoagulation (activated protein C [aPC]) were determined in serum and brain whole cell lysates. RESULTS: Serum levels of aPC were greater in the NS group (203 ± 30...

  17. Contribution of thrombin-reactive brain pericytes to blood-brain barrier dysfunction in an in vivo mouse model of obesity-associated diabetes and an in vitro rat model.

    Directory of Open Access Journals (Sweden)

    Takashi Machida

    Full Text Available Diabetic complications are characterized by the dysfunction of pericytes located around microvascular endothelial cells. The blood-brain barrier (BBB exhibits hyperpermeability with progression of diabetes. Therefore, brain pericytes at the BBB may be involved in diabetic complications of the central nervous system (CNS. We hypothesized that brain pericytes respond to increased brain thrombin levels in diabetes, leading to BBB dysfunction and diabetic CNS complications. Mice were fed a high-fat diet (HFD for 2 or 8 weeks to induce obesity. Transport of i.v.-administered sodium fluorescein and 125I-thrombin across the BBB were measured. We evaluated brain endothelial permeability and expression of tight junction proteins in the presence of thrombin-treated brain pericytes using a BBB model of co-cultured rat brain endothelial cells and pericytes. Mice fed a HFD for 8 weeks showed both increased weight gain and impaired glucose tolerance. In parallel, the brain influx rate of sodium fluorescein was significantly greater than that in mice fed a normal diet. HFD feeding inhibited the decline in brain thrombin levels occurring during 6 weeks of feeding. In the HFD fed mice, plasma thrombin levels were significantly increased, by up to 22%. 125I-thrombin was transported across the BBB in normal mice after i.v. injection, with uptake further enhanced by co-injection of unlabeled thrombin. Thrombin-treated brain pericytes increased brain endothelial permeability and caused decreased expression of zona occludens-1 (ZO-1 and occludin and morphological disorganization of ZO-1. Thrombin also increased mRNA expression of interleukin-1β and 6 and tumor necrosis factor-α in brain pericytes. Thrombin can be transported from circulating blood through the BBB, maintaining constant levels in the brain, where it can stimulate pericytes to induce BBB dysfunction. Thus, the brain pericyte-thrombin interaction may play a key role in causing BBB dysfunction in

  18. A Triple Culture Model of the Blood-Brain Barrier Using Porcine Brain Endothelial cells, Astrocytes and Pericytes.

    Science.gov (United States)

    Thomsen, Louiza Bohn; Burkhart, Annette; Moos, Torben

    2015-01-01

    In vitro blood-brain barrier (BBB) models based on primary brain endothelial cells (BECs) cultured as monoculture or in co-culture with primary astrocytes and pericytes are useful for studying many properties of the BBB. The BECs retain their expression of tight junction proteins and efflux transporters leading to high trans-endothelial electric resistance (TEER) and low passive paracellular permeability. The BECs, astrocytes and pericytes are often isolated from small rodents. Larger species as cows and pigs however, reveal a higher yield, are readily available and have a closer resemblance to humans, which make them favorable high-throughput sources for cellular isolation. The aim of the present study has been to determine if the preferable combination of purely porcine cells isolated from the 6 months old domestic pigs, i.e. porcine brain endothelial cells (PBECs) in co-culture with porcine astrocytes and pericytes, would compare with PBECs co-cultured with astrocytes and pericytes isolated from newborn rats with respect to TEER value and low passive permeability. The astrocytes and pericytes were grown both as contact and non-contact co-cultures as well as in triple culture to examine their effects on the PBECs for barrier formation as revealed by TEER, passive permeability, and expression patterns of tight junction proteins, efflux transporters and the transferrin receptor. This syngenic porcine in vitro BBB model is comparable to triple cultures using PBECs, rat astrocytes and rat pericytes with respect to TEER formation, low passive permeability, and expression of hallmark proteins signifying the brain endothelium (tight junction proteins claudin 5 and occludin, the efflux transporters P-glycoprotein (PgP) and breast cancer related protein (BCRP), and the transferrin receptor).

  19. A Triple Culture Model of the Blood-Brain Barrier Using Porcine Brain Endothelial cells, Astrocytes and Pericytes.

    Directory of Open Access Journals (Sweden)

    Louiza Bohn Thomsen

    Full Text Available In vitro blood-brain barrier (BBB models based on primary brain endothelial cells (BECs cultured as monoculture or in co-culture with primary astrocytes and pericytes are useful for studying many properties of the BBB. The BECs retain their expression of tight junction proteins and efflux transporters leading to high trans-endothelial electric resistance (TEER and low passive paracellular permeability. The BECs, astrocytes and pericytes are often isolated from small rodents. Larger species as cows and pigs however, reveal a higher yield, are readily available and have a closer resemblance to humans, which make them favorable high-throughput sources for cellular isolation. The aim of the present study has been to determine if the preferable combination of purely porcine cells isolated from the 6 months old domestic pigs, i.e. porcine brain endothelial cells (PBECs in co-culture with porcine astrocytes and pericytes, would compare with PBECs co-cultured with astrocytes and pericytes isolated from newborn rats with respect to TEER value and low passive permeability. The astrocytes and pericytes were grown both as contact and non-contact co-cultures as well as in triple culture to examine their effects on the PBECs for barrier formation as revealed by TEER, passive permeability, and expression patterns of tight junction proteins, efflux transporters and the transferrin receptor. This syngenic porcine in vitro BBB model is comparable to triple cultures using PBECs, rat astrocytes and rat pericytes with respect to TEER formation, low passive permeability, and expression of hallmark proteins signifying the brain endothelium (tight junction proteins claudin 5 and occludin, the efflux transporters P-glycoprotein (PgP and breast cancer related protein (BCRP, and the transferrin receptor.

  20. Sphingosine kinase inhibition alleviates endothelial permeability induced by thrombin and activated neutrophils.

    Science.gov (United States)

    Itagaki, Kiyoshi; Zhang, Qin; Hauser, Carl J

    2010-04-01

    Inflammation and microvascular thrombosis are interrelated causes of acute lung injury in the systemic inflammatory response syndrome. Neutrophils (polymorphonuclear neutrophil [PMN]) and endothelial cells (EC) activated by systemic inflammatory response syndrome interact to increase pulmonary vascular permeability, but the interactions between PMN and EC are difficult to study. Recently, we reported that sphingosine 1-phosphate is a second messenger eliciting store-operated calcium entry (SOCE) in response to inflammatory agonists in both PMN and EC. Store-operated calcium entry is therefore a target mechanism for the therapeutic modulation of inflammatory PMN-EC interactions. Here, we isolated, modeled, and studied the effects of pharmacologic SOCE inhibition using real-time systems to monitor EC permeability after exposure to activated PMN. We created systems to continuously assess permeability of human pulmonary artery endothelial cells and human microvascular endothelial cells from lung. Endothelial cells show increased permeability after challenge by activated PMN. Such permeability increases can be attenuated by exposure of the cocultures to sphingosine kinase (SK) inhibitors (SKI-2, N,N-dimethylsphingosine [DMS]) or Ca2+ entry inhibitors (Gd3+, MRS-1845). Human microvascular endothelial cells from lung pretreated with SKI-2 or DMS showed decreased permeability when later exposed to activated PMN. Likewise, when PMNs were activated with thapsigargin (TG) in the presence of SKI-2, DMS, Gd, or MRS-1845, their ability to cause EC permeability subsequently was reduced. SKI-2 also inhibited the activation of human pulmonary artery ECs by thrombin. These studies will provide a firm mechanistic foundation for understanding how systemic SOCE inhibition may be used to prevent acute lung injury in vivo.

  1. MicroRNA Signature of Human Microvascular Endothelium Infected with Rickettsia rickettsii

    Directory of Open Access Journals (Sweden)

    Abha Sahni

    2017-07-01

    Full Text Available MicroRNAs (miRNAs mediate gene silencing by destabilization and/or translational repression of target mRNA. Infection of human microvascular endothelial cells as primary targets of Rickettsia rickettsii, the etiologic agent of Rocky Mountain spotted fever, triggers host responses appertaining to alterations in cellular gene expression. Microarray-based profiling of endothelial cells infected with R. rickettsii for 3 or 24 h revealed differential expression of 33 miRNAs, of which miRNAs129-5p, 200a-3p, 297, 200b-3p, and 595 were identified as the top five up-regulated miRNAs (5 to 20-fold, p ≤ 0.01 and miRNAs 301b-3p, 548a-3p, and 377-3p were down-regulated (2 to 3-fold, p ≤ 0.01. Changes in the expression of selected miRNAs were confirmed by q-RT-PCR in both in vitro and in vivo models of infection. As potential targets, expression of genes encoding NOTCH1, SMAD2, SMAD3, RIN2, SOD1, and SOD2 was either positively or negatively regulated. Using a miRNA-specific mimic or inhibitor, NOTCH1 was determined to be a target of miRNA 200a-3p in R. rickettsii-infected human dermal microvascular endothelial cells (HMECs. Predictive interactome mapping suggested the potential for miRNA-mediated modulation of regulatory gene networks underlying important host cell signaling pathways. This first demonstration of altered endothelial miRNA expression provides new insights into regulatory elements governing mechanisms of host responses and pathogenesis during human rickettsial infections.

  2. N-n-butyl haloperidol iodide ameliorates hypoxia/reoxygenation injury through modulating the LKB1/AMPK/ROS pathway in cardiac microvascular endothelial cells.

    Science.gov (United States)

    Lu, Binger; Wang, Bin; Zhong, Shuping; Zhang, Yanmei; Gao, Fenfei; Chen, Yicun; Zheng, Fuchun; Shi, Ganggang

    2016-06-07

    Endothelial cells are highly sensitive to hypoxia and contribute to myocardial ischemia/reperfusion injury. We have reported that N-n-butyl haloperidol iodide (F2) can attenuate hypoxia/reoxygenation (H/R) injury in cardiac microvascular endothelial cells (CMECs). However, the molecular mechanisms remain unclear. Neonatal rat CMECs were isolated and subjected to H/R. Pretreatment of F2 leads to a reduction in H/R injury, as evidenced by increased cell viability, decreased lactate dehydrogenase (LDH) leakage and apoptosis, together with enhanced AMP-activated protein kinase (AMPK) and liver kinase B1 (LKB1) phosphorylation in H/R ECs. Blockade of AMPK with compound C reversed F2-induced inhibition of H/R injury, as evidenced by decreased cell viability, increased LDH release and apoptosis. Moreover, compound C also blocked the ability of F2 to reduce H/R-induced reactive oxygen species (ROS) generation. Supplementation with the ROS scavenger N-acetyl-L-cysteine (NAC) reduced ROS levels, increased cell survival rate, and decreased both LDH release and apoptosis after H/R. In conclusion, our data indicate that F2 may mitigate H/R injury by stimulating LKB1/AMPK signaling pathway and subsequent suppression of ROS production in CMECs.

  3. Adenosine formation in contracting primary rat skeletal muscle cells and endothelial cells in culture

    DEFF Research Database (Denmark)

    Hellsten, Ylva; Frandsen, Ulrik

    1997-01-01

    1. The present study examined the capacity for adenosine formation, uptake and metabolism in contracting primary rat muscle cells and in microvascular endothelial cells in culture. 2. Strong and moderate electrical simulation of skeletal muscle cells led to a significantly greater increase....... 3. Addition of microvascular endothelial cells to the cultured skeletal muscle cells enhanced the contraction-induced accumulation of extracellular adenosine (P Skeletal muscle cells were...... in the extracellular adenosine concentration (421 +/- 91 and 235 +/- 30 nmol (g protein)-1, respectively; P muscle cells (161 +/- 20 nmol (g protein)-1). The ATP concentration was lower (18%; P contracted, but not in the moderately contracted muscle cells...

  4. Early invasion of brain parenchyma by African trypanosomes.

    Directory of Open Access Journals (Sweden)

    Ute Frevert

    Full Text Available Human African trypanosomiasis or sleeping sickness is a vector-borne parasitic disease that has a major impact on human health and welfare in sub-Saharan countries. Based mostly on data from animal models, it is currently thought that trypanosome entry into the brain occurs by initial infection of the choroid plexus and the circumventricular organs followed days to weeks later by entry into the brain parenchyma. However, Trypanosoma brucei bloodstream forms rapidly cross human brain microvascular endothelial cells in vitro and appear to be able to enter the murine brain without inflicting cerebral injury. Using a murine model and intravital brain imaging, we show that bloodstream forms of T. b. brucei and T. b. rhodesiense enter the brain parenchyma within hours, before a significant level of microvascular inflammation is detectable. Extravascular bloodstream forms were viable as indicated by motility and cell division, and remained detectable for at least 3 days post infection suggesting the potential for parasite survival in the brain parenchyma. Vascular inflammation, as reflected by leukocyte recruitment and emigration from cortical microvessels, became apparent only with increasing parasitemia at later stages of the infection, but was not associated with neurological signs. Extravascular trypanosomes were predominantly associated with postcapillary venules suggesting that early brain infection occurs by parasite passage across the neuroimmunological blood brain barrier. Thus, trypanosomes can invade the murine brain parenchyma during the early stages of the disease before meningoencephalitis is fully established. Whether individual trypanosomes can act alone or require the interaction from a quorum of parasites remains to be shown. The significance of these findings for disease development is now testable.

  5. Impairment of brain endothelial glucose transporter by methamphetamine causes blood-brain barrier dysfunction

    Directory of Open Access Journals (Sweden)

    Murrin L Charles

    2011-03-01

    Full Text Available Abstract Background Methamphetamine (METH, an addictive psycho-stimulant drug with euphoric effect is known to cause neurotoxicity due to oxidative stress, dopamine accumulation and glial cell activation. Here we hypothesized that METH-induced interference of glucose uptake and transport at the endothelium can disrupt the energy requirement of the blood-brain barrier (BBB function and integrity. We undertake this study because there is no report of METH effects on glucose uptake and transport across the blood-brain barrier (BBB to date. Results In this study, we demonstrate that METH-induced disruption of glucose uptake by endothelium lead to BBB dysfunction. Our data indicate that a low concentration of METH (20 μM increased the expression of glucose transporter protein-1 (GLUT1 in primary human brain endothelial cell (hBEC, main component of BBB without affecting the glucose uptake. A high concentration of 200 μM of METH decreased both the glucose uptake and GLUT1 protein levels in hBEC culture. Transcription process appeared to regulate the changes in METH-induced GLUT1 expression. METH-induced decrease in GLUT1 protein level was associated with reduction in BBB tight junction protein occludin and zonula occludens-1. Functional assessment of the trans-endothelial electrical resistance of the cell monolayers and permeability of dye tracers in animal model validated the pharmacokinetics and molecular findings that inhibition of glucose uptake by GLUT1 inhibitor cytochalasin B (CB aggravated the METH-induced disruption of the BBB integrity. Application of acetyl-L-carnitine suppressed the effects of METH on glucose uptake and BBB function. Conclusion Our findings suggest that impairment of GLUT1 at the brain endothelium by METH may contribute to energy-associated disruption of tight junction assembly and loss of BBB integrity.

  6. Distribution of a 69-kD laminin-binding protein in aortic and microvascular endothelial cells: modulation during cell attachment, spreading, and migration

    DEFF Research Database (Denmark)

    Yannariello-Brown, J; Wewer, U; Liotta, L

    1988-01-01

    cells identified this protein in BAEC lysates. In frozen sections, these polyclonal antibodies and monoclonal antibodies raised against human tumor 69-kD stained the endothelium of bovine aorta and the medial smooth muscle cells, but not surrounding connective tissue or elastin fibers. When...... nonpermeabilized BAEC were stained in an in vitro migration assay, there appeared to be apical patches of 69 kD staining in stationary cells. However, when released from contact inhibition, 69 kD was localized to ruffling membranes on cells at the migrating front. Permeabilized BAEC stained for 69 kD diffusely...... in permeabilized cultured microvascular endothelial cells in a continuous staining pattern at 6 h postplating which redistributed to punctate patches along the length of the filaments at confluence (96 h). In addition, 69 kD co-distribution with laminin could also be demonstrated in cultured subconfluent cells...

  7. Exosomal signaling during hypoxia mediates microvascular endothelial cell migration and vasculogenesis.

    Directory of Open Access Journals (Sweden)

    Carlos Salomon

    Full Text Available Vasculogenesis and angiogenesis are critical processes in fetal circulation and placental vasculature development. Placental mesenchymal stem cells (pMSC are known to release paracrine factors (some of which are contained within exosomes that promote angiogenesis and cell migration. The aims of this study were: to determine the effects of oxygen tension on the release of exosomes from pMSC; and to establish the effects of pMSC-derived exosomes on the migration and angiogenic tube formation of placental microvascular endothelial cells (hPMEC. pMSC were isolated from placental villi (8-12 weeks of gestation, n = 6 and cultured under an atmosphere of 1%, 3% or 8% O2. Cell-conditioned media were collected and exosomes (exo-pMSC isolated by differential and buoyant density centrifugation. The dose effect (5-20 µg exosomal protein/ml of pMSC-derived exosomes on hPMEC migration and tube formation were established using a real-time, live-cell imaging system (Incucyte™. The exosome pellet was resuspended in PBS and protein content was established by mass spectrometry (MS. Protein function and canonical pathways were identified using the PANTHER program and Ingenuity Pathway Analysis, respectively. Exo-pMSC were identified, by electron microscopy, as spherical vesicles, with a typical cup-shape and diameters around of 100 nm and positive for exosome markers: CD63, CD9 and CD81. Under hypoxic conditions (1% and 3% O2 exo-pMSC released increased by 3.3 and 6.7 folds, respectively, when compared to the controls (8% O2; p<0.01. Exo-pMSC increased hPMEC migration by 1.6 fold compared to the control (p<0.05 and increased hPMEC tube formation by 7.2 fold (p<0.05. MS analysis identified 390 different proteins involved in cytoskeleton organization, development, immunomodulatory, and cell-to-cell communication. The data obtained support the hypothesis that pMSC-derived exosomes may contribute to placental vascular adaptation to low oxygen tension under both

  8. Smuggling Drugs into the Brain: An Overview of Ligands Targeting Transcytosis for Drug Delivery across the Blood–Brain Barrier

    Directory of Open Access Journals (Sweden)

    Julia V. Georgieva

    2014-11-01

    Full Text Available The blood–brain barrier acts as a physical barrier that prevents free entry of blood-derived substances, including those intended for therapeutic applications. The development of molecular Trojan horses is a promising drug targeting technology that allows for non-invasive delivery of therapeutics into the brain. This concept relies on the application of natural or genetically engineered proteins or small peptides, capable of specifically ferrying a drug-payload that is either directly coupled or encapsulated in an appropriate nanocarrier, across the blood–brain barrier via receptor-mediated transcytosis. Specifically, in this process the nanocarrier–drug system (“Trojan horse complex” is transported transcellularly across the brain endothelium, from the blood to the brain interface, essentially trailed by a native receptor. Naturally, only certain properties would favor a receptor to serve as a transporter for nanocarriers, coated with appropriate ligands. Here we briefly discuss brain microvascular endothelial receptors that have been explored until now, highlighting molecular features that govern the efficiency of nanocarrier-mediated drug delivery into the brain.

  9. Microvascular dysfunction in the immediate aftermath of chronic total coronary occlusion recanalization.

    Science.gov (United States)

    Ladwiniec, Andrew; Cunnington, Michael S; Rossington, Jennifer; Thackray, Simon; Alamgir, Farquad; Hoye, Angela

    2016-05-01

    The aim of this study was to compare microvascular resistance under both baseline and hyperemic conditions immediately after percutaneous coronary intervention (PCI) of a chronic total occlusion (CTO) with an unobstructed reference vessel in the same patient Microvascular dysfunction has been reported to be prevalent immediately after CTO PCI. However, previous studies have not made comparison with a reference vessel. Patients with a CTO may have global microvascular and/or endothelial dysfunction, making comparison with established normal values misleading. After successful CTO PCI in 21 consecutive patients, coronary pressure and flow velocity were measured at baseline and hyperemia in distal segments of the CTO/target vessel and an unobstructed reference vessel. Hemodynamics including hyperemic microvascular resistance (HMR), basal microvascular resistance (BMR), and instantaneous minimal microvascular resistance at baseline and hyperemia were calculated and compared between reference and target/CTO vessels. After CTO PCI, BMR was reduced in the target/CTO vessel compared with the reference vessel: 3.58 mm Hg/cm/s vs 4.94 mm Hg/cm/s, difference -1.36 mm Hg/cm/s (-2.33 to -0.39, p = 0.008). We did not detect a difference in HMR: 1.82 mm Hg/cm/s vs 2.01 mm Hg/cm/s, difference -0.20 (-0.78 to 0.39, p = 0.49). Instantaneous minimal microvascular resistance correlated strongly with the length of stented segment at baseline (r = 0.63, p = 0.005) and hyperemia (r = 0.68, p = 0.002). BMR is reduced in a recanalized CTO in the immediate aftermath of PCI compared to an unobstructed reference vessel; however, HMR appears to be preserved. A longer stented segment is associated with increased microvascular resistance. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  10. Generation of primary cultures of bovine brain endothelial cells and setup of cocultures with rat astrocytes

    DEFF Research Database (Denmark)

    Helms, Hans C; Brodin, Birger

    2014-01-01

    -brain barrier. The present protocol describes the setup of an in vitro coculture model based on primary cultures of endothelial cells from bovine brain microvessels and primary cultures of rat astrocytes. The model displays a high electrical tightness and expresses blood-brain barrier marker proteins....

  11. Stimulated mast cells promote maturation of myocardial microvascular endothelial cell neovessels by modulating the angiopoietin-Tie-2 signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Z.H. [Division of Cardiology, Shanghai Sixth People' s Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China, Division of Cardiology, Shanghai Sixth People’s Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai (China); Yancheng People' s First Hospital, Division of Cardiology, Yancheng, Jiangsu, China, Division of Cardiology, Yancheng People’s First Hospital, Yancheng, Jiangsu (China); Zhu, W.; Tao, J.P.; Zhang, Q.Y.; Wei, M. [Division of Cardiology, Shanghai Sixth People' s Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China, Division of Cardiology, Shanghai Sixth People’s Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai (China)

    2013-10-22

    Angiopoietin (Ang)-1 and Ang-2 interact in angiogenesis to activate the Tie-2 receptor, which may be involved in new vessel maturation and regression. Mast cells (MCs) are also involved in formation of new blood vessels and angiogenesis. The present study was designed to test whether MCs can mediate angiogenesis in myocardial microvascular endothelial cells (MMVECs). Using a rat MMVEC and MC co-culture system, we observed that Ang-1 protein levels were very low even though its mRNA levels were increased by MCs. Interestingly, MCs were able to enhance migration, proliferation, and capillary-like tube formation, which were associated with suppressed Ang-2 protein expression, but not Tie-2 expression levels. These MCs induced effects that could be reversed by either tryptase inhibitor [N-tosyl-L-lysine chloromethyl ketone (TLCK)] or chymase inhibitor (N-tosyl-L-phenylalanyl chloromethyl ketone), with TLCK showing greater effects. In conclusion, our data indicated that MCs can interrupt neovessel maturation via suppression of the Ang-2/Tie-2 signaling pathway.

  12. Endothelium-targeted overexpression of heat shock protein 27 ameliorates blood–brain barrier disruption after ischemic brain injury

    Science.gov (United States)

    Jiang, Xiaoyan; Zhang, Lili; Pu, Hongjian; Hu, Xiaoming; Zhang, Wenting; Cai, Wei; Gao, Yanqin; Leak, Rehana K.; Keep, Richard F.; Bennett, Michael V. L.; Chen, Jun

    2017-01-01

    The damage borne by the endothelial cells (ECs) forming the blood–brain barrier (BBB) during ischemic stroke and other neurological conditions disrupts the structure and function of the neurovascular unit and contributes to poor patient outcomes. We recently reported that structural aberrations in brain microvascular ECs—namely, uncontrolled actin polymerization and subsequent disassembly of junctional proteins, are a possible cause of the early onset BBB breach that arises within 30–60 min of reperfusion after transient focal ischemia. Here, we investigated the role of heat shock protein 27 (HSP27) as a direct inhibitor of actin polymerization and protectant against BBB disruption after ischemia/reperfusion (I/R). Using in vivo and in vitro models, we found that targeted overexpression of HSP27 specifically within ECs—but not within neurons—ameliorated BBB impairment 1–24 h after I/R. Mechanistically, HSP27 suppressed I/R-induced aberrant actin polymerization, stress fiber formation, and junctional protein translocation in brain microvascular ECs, independent of its protective actions against cell death. By preserving BBB integrity after I/R, EC-targeted HSP27 overexpression attenuated the infiltration of potentially destructive neutrophils and macrophages into brain parenchyma, thereby improving long-term stroke outcome. Notably, early poststroke administration of HSP27 attached to a cell-penetrating transduction domain (TAT-HSP27) rapidly elevated HSP27 levels in brain microvessels and ameliorated I/R-induced BBB disruption and subsequent neurological deficits. Thus, the present study demonstrates that HSP27 can function at the EC level to preserve BBB integrity after I/R brain injury. HSP27 may be a therapeutic agent for ischemic stroke and other neurological conditions involving BBB breakdown. PMID:28137866

  13. Blood-brain barrier alterations provide evidence of subacute diaschisis in an ischemic stroke rat model.

    Directory of Open Access Journals (Sweden)

    Svitlana Garbuzova-Davis

    Full Text Available Comprehensive stroke studies reveal diaschisis, a loss of function due to pathological deficits in brain areas remote from initial ischemic lesion. However, blood-brain barrier (BBB competence in subacute diaschisis is uncertain. The present study investigated subacute diaschisis in a focal ischemic stroke rat model. Specific focuses were BBB integrity and related pathogenic processes in contralateral brain areas.In ipsilateral hemisphere 7 days after transient middle cerebral artery occlusion (tMCAO, significant BBB alterations characterized by large Evans Blue (EB parenchymal extravasation, autophagosome accumulation, increased reactive astrocytes and activated microglia, demyelinization, and neuronal damage were detected in the striatum, motor and somatosensory cortices. Vascular damage identified by ultrastuctural and immunohistochemical analyses also occurred in the contralateral hemisphere. In contralateral striatum and motor cortex, major ultrastructural BBB changes included: swollen and vacuolated endothelial cells containing numerous autophagosomes, pericyte degeneration, and perivascular edema. Additionally, prominent EB extravasation, increased endothelial autophagosome formation, rampant astrogliosis, activated microglia, widespread neuronal pyknosis and decreased myelin were observed in contralateral striatum, and motor and somatosensory cortices.These results demonstrate focal ischemic stroke-induced pathological disturbances in ipsilateral, as well as in contralateral brain areas, which were shown to be closely associated with BBB breakdown in remote brain microvessels and endothelial autophagosome accumulation. This microvascular damage in subacute phase likely revealed ischemic diaschisis and should be considered in development of treatment strategies for stroke.

  14. Effect of perilipin-5 on apoptosis of cardiac microvascular endothelial cells induced by high fat and high glucose in mice

    Directory of Open Access Journals (Sweden)

    Jin DU

    2017-12-01

    Full Text Available Objective To investigate the effects and mechanisms of perilipin-5 (Plin5 on the apoptosis of mouse cardiac microvascular endothelial cells induced by high fat and high glucose. Methods The mouse cardiac microvascular endothelial cells (MCMECs cultured with high glucose medium were respectively given 0, 100, 300 and 500μmol/L palmitic acid for 24 hours. In order to explore the effects and mechanisms of Plin5 on MCMECs injuries induced by high fat and high glucose, MCMECs exposed to 300μmol/L palmitic acid for 24 hours were divided into control group, Scra siRNA group and Plin5 siRNA group. The control group was only treated with transfection reagent, the Scra siRNA group was given treatment of transfection reagent and garbled RNA, the Plin5 siRNA group was given treatment of transfection reagent and Plin5 specific siRNA. In order to further confirm the specific mechanism of Plin5 in high fat/glucose inducing MCMECs injury, MCMECs in Plin5 siRNA group were divided into vehicle group and N-acetyl cysteine (NAC group, and given the same intervention of high fat. The apoptotic rate was detected by flow cytometry, qRT-PCR and Western blotting were respectively used to detect the mRNA and protein expression of Plin5, and the intracellular reactive oxygen species (ROS level was tested by DHE staining and ELISA kit. Results The apoptotic rate of MCMECs was increased in a fat concentration-dependent manner (P<0.05. Compared with 0μmol/L palmitic acid group, the intracellular ROS content and the expression of Plin5 increased significantly in 300μmol/L palmitic acid group (P<0.05. Compared with the control group and the Scra siRNA group, the intracellular ROS content and apoptotic rate increased significantly in Plin5 siRNA group under the action of 300μmol/L palmitic acid (P<0.05. Compared with the vehicle group, the intracellular ROS content and apoptotic rate decreased remarkably in NAC group (P<0.05. Conclusion With inhibition of oxidative stress

  15. Tick-borne encephalitis virus infects human brain microvascular endothelial cells without compromising blood-brain barrier integrity

    Czech Academy of Sciences Publication Activity Database

    Palus, Martin; Vancová, Marie; Širmarová, J.; Elsterová, Jana; Perner, Jan; Růžek, Daniel

    2017-01-01

    Roč. 507, JUL (2017), s. 110-122 ISSN 0042-6822 R&D Projects: GA MZd(CZ) NV16-34238A; GA MŠk(CZ) LM2015062; GA TA ČR(CZ) TE01020118 Institutional support: RVO:60077344 Keywords : tick-borne encephalitis * tick-borne encephalitis virus * blood- brain barrier * neuroinfection Subject RIV: EE - Microbiology, Virology OBOR OECD: Virology Impact factor: 3.353, year: 2016

  16. Globular adiponectin ameliorates metabolic insulin resistance via AMPK-mediated restoration of microvascular insulin responses

    Science.gov (United States)

    Zhao, Lina; Fu, Zhuo; Wu, Jing; Aylor, Kevin W; Barrett, Eugene J; Cao, Wenhong; Liu, Zhenqi

    2015-01-01

    Abstract Hypoadiponectinaemia is closely associated with endothelial dysfunction and insulin resistance, and microvasculature plays a critical role in the regulation of insulin action in muscle. Here we tested whether adiponectin replenishment could improve metabolic insulin sensitivity in male rats fed a high-fat diet (HFD) via the modulation of microvascular insulin responses. Male Sprague–Dawley rats were fed either a HFD or low-fat diet (LFD) for 4 weeks. Small resistance artery myograph changes in tension, muscle microvascular recruitment and metabolic response to insulin were determined. Compared with rats fed a LFD, HFD feeding abolished the vasodilatory actions of globular adiponectin (gAd) and insulin on pre-constricted distal saphenous arteries. Pretreatment with gAd improved insulin responses in arterioles isolated from HFD rats, which was blocked by AMP-activated protein kinase (AMPK) inhibition. Similarly, HFD abolished microvascular responses to either gAd or insulin and decreased insulin-stimulated glucose disposal by ∼60%. However, supplementing gAd fully rescued insulin’s microvascular action and significantly improved the metabolic responses to insulin in HFD male rats and these actions were abolished by inhibition of either AMPK or nitric oxide production. We conclude that HFD induces vascular adiponectin and insulin resistance but gAd administration can restore vascular insulin responses and improve insulin’s metabolic action via an AMPK- and nitric oxide-dependent mechanism in male rats. Key points Adiponectin is an adipokine with anti-inflammatory and anti-diabetic properties. Hypoadiponectinaemia is closely associated with endothelial dysfunction and insulin resistance in obesity and diabetes. Insulin resistance is present in muscle microvasculature and this may contribute to decreased insulin delivery to, and action in, muscle. In this study we examined whether adiponectin ameliorates metabolic insulin resistance by affecting muscle

  17. A SAGE based approach to human glomerular endothelium : defining the transcriptome, finding a novel molecule and highlighting endothelial diversity

    NARCIS (Netherlands)

    Sengoelge, Guerkan; Winnicki, Wolfgang; Kupczok, Anne; von Haeseler, Arndt; Schuster, Michael; Pfaller, Walter; Jennings, Paul; Weltermann, Ansgar; Blake, Sophia; Sunder-Plassmann, Gere

    2014-01-01

    BACKGROUND: Large scale transcript analysis of human glomerular microvascular endothelial cells (HGMEC) has never been accomplished. We designed this study to define the transcriptome of HGMEC and facilitate a better characterization of these endothelial cells with unique features. Serial analysis

  18. Expression of Shiga toxin 2e glycosphingolipid receptors of primary porcine brain endothelial cells and toxin-mediated breakdown of the blood-brain barrier.

    Science.gov (United States)

    Meisen, Iris; Rosenbrück, Regina; Galla, Hans-Joachim; Hüwel, Sabine; Kouzel, Ivan U; Mormann, Michael; Karch, Helge; Müthing, Johannes

    2013-06-01

    Shiga toxin (Stx) 2e, released by certain Stx-producing Escherichia coli, is presently the best characterized virulence factor responsible for pig edema disease, which is characterized by hemorrhagic lesions, neurological disorders and often fatal outcomes. Although Stx2e-mediated brain vascular injury is the key event in development of neurologic signs, the glycosphingolipid (GSL) receptors of Stx2e and toxin-mediated impairment of pig brain endothelial cells have not been investigated so far. Here, we report on the detailed structural characterization of Stx2e receptors globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), which make up the major neutral GSLs in primary porcine brain capillary endothelial cells (PBCECs). Various Gb3Cer and Gb4Cer lipoforms harboring sphingenine (d18:1) or sphinganine (d18:0) and mostly a long-chain fatty acid (C20-C24) were detected. A notable batch-to-batch heterogeneity of primary endothelial cells was observed regarding the extent of ceramide hydroxylation of Gb3Cer or Gb4Cer species. Gb3Cer, Gb4Cer and sphingomyelin preferentially distribute to detergent-resistant membrane fractions and can be considered lipid raft markers in PBCECs. Moreover, we employed an in vitro model of the blood-brain barrier (BBB), which exhibited strong cytotoxic effects of Stx2e on the endothelial monolayer and a rapid collapse of the BBB. These data strongly suggest the involvement of Stx2e in cerebral vascular damage with resultant neurological disturbance characteristic of edema disease.

  19. Silver nanoparticles induce tight junction disruption and astrocyte neurotoxicity in a rat blood–brain barrier primary triple coculture model

    Directory of Open Access Journals (Sweden)

    Xu L

    2015-09-01

    Full Text Available Liming Xu,1,2,* Mo Dan,1,* Anliang Shao,1 Xiang Cheng,1,3 Cuiping Zhang,4 Robert A Yokel,5 Taro Takemura,6 Nobutaka Hanagata,6 Masami Niwa,7,8 Daisuke Watanabe7,81National Institutes for Food and Drug Control, No 2, Temple of Heaven, Beijing, 2School of Information and Engineering, Wenzhou Medical University, Wenzhou, 3School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu, 4Beijing Neurosurgical Institute, Capital Medical University, Beijing, People’s Republic of China; 5College of Pharmacy, University of Kentucky, Lexington, KY, USA; 6Nanotechnology Innovation Station for Nanoscale Science and Technology, National Institute for Materials Science, Tsukuba, Ibaraki, 7Department of Pharmacology, Nagasaki University, 8BBB Laboratory, PharmaCo-Cell Company, Ltd., Nagasaki, Japan*These authors contributed equally to this workBackground: Silver nanoparticles (Ag-NPs can enter the brain and induce neurotoxicity. However, the toxicity of Ag-NPs on the blood–brain barrier (BBB and the underlying mechanism(s of action on the BBB and the brain are not well understood.Method: To investigate Ag-NP suspension (Ag-NPS-induced toxicity, a triple coculture BBB model of rat brain microvascular endothelial cells, pericytes, and astrocytes was established. The BBB permeability and tight junction protein expression in response to Ag-NPS, NP-released Ag ions, and polystyrene-NP exposure were investigated. Ultrastructural changes of the microvascular endothelial cells, pericytes, and astrocytes were observed using transmission electron microscopy (TEM. Global gene expression of astrocytes was measured using a DNA microarray.Results: A triple coculture BBB model of primary rat brain microvascular endothelial cells, pericytes, and astrocytes was established, with the transendothelial electrical resistance values >200 Ω·cm2. After Ag-NPS exposure for 24 hours, the BBB permeability was significantly increased and expression of the

  20. The SGLT2 Inhibitor Dapagliflozin Significantly Improves the Peripheral Microvascular Endothelial Function in Patients with Uncontrolled Type 2 Diabetes Mellitus.

    Science.gov (United States)

    Sugiyama, Seigo; Jinnouchi, Hideaki; Kurinami, Noboru; Hieshima, Kunio; Yoshida, Akira; Jinnouchi, Katsunori; Nishimura, Hiroyuki; Suzuki, Tomoko; Miyamoto, Fumio; Kajiwara, Keizo; Jinnouchi, Tomio

    2018-03-30

    Objective Sodium-glucose cotransporter-2 (SGLT2) inhibitors reduce cardiovascular events and decrease the body fat mass in patients with type 2 diabetes mellitus (T2DM). We examined whether or not the SGLT2-inhibitor dapagliflozin can improve the endothelial function associated with a reduction in abdominal fat mass. Methods We prospectively recruited patients with uncontrolled (hemoglobin A1c [HbA1c] >7.0%) T2DM who were not being treated by SGLT2 inhibitors. Patients were treated with add-on dapagliflozin (5 mg/day) or non-SGLT2 inhibitor medicines for 6 months to improve their HbA1c. We measured the peripheral microvascular endothelial function as assessed by reactive hyperemia peripheral arterial tonometry (RH-PAT) and calculated the natural logarithmic transformed value of the RH-PAT index (LnRHI). We then investigated changes in the LnRHI and abdominal fat area using computed tomography (CT). Results The subjects were 54 patients with uncontrolled T2DM (72.2% men) with a mean HbA1c of 8.1%. The HbA1c was significantly decreased in both groups, with no significant difference between the groups. Dapagliflozin treatment, but not non-SGLT2 inhibitor treatment, significantly increased the LnRHI. The changes in the LnRHI were significantly greater in the dapagliflozin group than in the non-SGLT2 inhibitor group. Dapagliflozin treatment, but not non-SGLT2 inhibitor treatment, significantly decreased the abdominal visceral fat area, subcutaneous fat area (SFA), and total fat area (TFA) as assessed by CT and significantly increased the plasma adiponectin levels. The percentage changes in the LnRHI were significantly correlated with changes in the SFA, TFA, systolic blood pressure, and adiponectin. Conclusion Add-on treatment with dapagliflozin significantly improves the glycemic control and endothelial function associated with a reduction in the abdominal fat mass in patients with uncontrolled T2DM.

  1. Effects of the PPARγ agonist troglitazone on endothelial cells in vivo and in vitro: Differences between human and mouse

    International Nuclear Information System (INIS)

    Kakiuchi-Kiyota, Satoko; Vetro, Joseph A.; Suzuki, Shugo; Varney, Michelle L.; Han, Huai-Yun; Nascimento, Merielen; Pennington, Karen L.; Arnold, Lora L.; Singh, Rakesh K.; Cohen, Samuel M.

    2009-01-01

    Peroxisome proliferator-activated receptor gamma (PPARγ) agonists and PPARγ/α dual agonists have been or are being developed for clinical use in the treatment of type 2 diabetes mellitus and hyperlipidemias. A common tumor finding in rodent carcinogenicity studies for these agonists is hemangioma/hemangiosarcoma in mice but not in rats. We hypothesized that increased endothelial cell proliferation may be involved in the mechanism of PPAR agonist-induced vascular tumors in mice, and we investigated the effects on endothelial cells utilizing troglitazone, the first clinically used PPARγ agonist, in vivo and in vitro. Troglitazone (400 and 800 mg/kg/day) induced hemangiosarcomas in mice in a 2-year bioassay. We showed that troglitazone increased endothelial cell proliferation in brown and white adipose tissue and liver in mice at sarcomagenic doses after 4 weeks of treatment. Troglitazone was cytotoxic both to human dermal microvascular endothelial cells (HMEC1) and mouse mammary fat pad microvascular endothelial cells (MFP MVEC) at high concentrations. However, MFP MVEC were more resistant to the cytotoxic effects of troglitazone based on the much lower LC 50 in HMEC1 (17.4 μM) compared to MFP MVEC (92.2 μM). Troglitazone increased the proliferation and survival of MFP MVEC but not HMEC1 in growth factor reduced conditions. Our data demonstrate that troglitazone may induce hemangiosarcomas in mice, at least in part, through enhancement of survival and proliferation of microvascular endothelial cells. Such an effect does not occur with human cells, suggesting that human may react differently to exposure to PPAR agonists compared with mice.

  2. Fabrication of a reticular poly(lactide-co-glycolide) cylindrical scaffold for the in vitro development of microvascular networks

    Science.gov (United States)

    Tung, Yen-Ting; Chang, Cheng-Chung; Ju, Jyh-Cherng; Wang, Gou-Jen

    2017-12-01

    The microvascular network is a simple but critical system that is responsible for a range of important biological mechanisms in the bodies of all animals. The ability to generate a functional microvessel not only makes it possible to engineer vital tissue of considerable size but also serves as a platform for biomedical studies. However, most of the current methods for generating microvessel networks in vitro use rectangular channels which cannot represent real vessels in vivo and have dead zones at their corners, hence hindering the circulation of culture medium. We propose a scaffold-wrapping method which enables fabrication of a customized microvascular network in vitro in a more biomimetic way. By integrating microelectromechanical techniques with thermal reflow, we designed and fabricated a microscale hemi-cylindrical photoresist template. A replica mold of polydimethylsiloxane, produced by casting, was then used to generate cylindrical scaffolds with biodegradable poly(lactide-co-glycolide) (PLGA). Human umbilical vein endothelial cells were seeded on both sides of the PLGA scaffold and cultured using a traditional approach. The expression of endothelial cell marker CD31 and intercellular junction vascular endothelial cadherin on the cultured cell demonstrated the potential of generating a microvascular network with a degradable cylindrical scaffold. Our method allows cells to be cultured on a scaffold using a conventional culture approach and monitors cell conditions continuously. We hope our cell-covered scaffold can serve as a framework for building large tissues or can be used as the core of a vascular chip for in vitro circulation studies.

  3. Defibrotide prevents the activation of macrovascular and microvascular endothelia caused by soluble factors released to blood by autologous hematopoietic stem cell transplantation.

    Science.gov (United States)

    Palomo, Marta; Diaz-Ricart, Maribel; Rovira, Montserrat; Escolar, Ginés; Carreras, Enric

    2011-04-01

    Endothelial activation and damage occur in association with autologous hematopoietic stem cell transplantation (HSCT). Several of the early complications associated with HSCT seem to have a microvascular location. Through the present study, we have characterized the activation and damage of endothelial cells of both macro (HUVEC) and microvascular (HMEC) origin, occurring early after autologous HSCT, and the potential protective effect of defibrotide (DF). Sera samples from patients were collected before conditioning (Pre), at the time of transplantation (day 0), and at days 7, 14, and 21 after autologous HSCT. Changes in the expression of endothelial cell receptors at the surface, presence and reactivity of extracellular adhesive proteins, and the signaling pathways involved were analyzed. The expression of ICAM-1 at the cell surface increased progressively in both HUVEC and HMEC. However, a more prothrombotic profile was denoted for HMEC, in particular at the time of transplantation (day 0), reflecting the deleterious effect of the conditioning treatment on the endothelium, especially at a microvascular location. Interestingly, this observation correlated with a higher increase in the expression of both tissue factor and von Willebrand factor on the extracellular matrix, together with activation of intracellular p38 MAPK and Akt. Previous exposure and continuous incubation of cells with DF prevented the signs of activation and damage induced by the autologous sera. These observations corroborate that conditioning treatment in autologous HSCT induces a proinflammatory and a prothrombotic phenotype, especially at a microvascular location, and indicate that DF has protective antiinflammatory and antithrombotic effects in this setting. Copyright © 2011 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

  4. Comparison of a Rat Primary Cell-Based Blood-Brain Barrier Model With Epithelial and Brain Endothelial Cell Lines: Gene Expression and Drug Transport

    Directory of Open Access Journals (Sweden)

    Szilvia Veszelka

    2018-05-01

    Full Text Available Cell culture-based blood-brain barrier (BBB models are useful tools for screening of CNS drug candidates. Cell sources for BBB models include primary brain endothelial cells or immortalized brain endothelial cell lines. Despite their well-known differences, epithelial cell lines are also used as surrogate models for testing neuropharmaceuticals. The aim of the present study was to compare the expression of selected BBB related genes including tight junction proteins, solute carriers (SLC, ABC transporters, metabolic enzymes and to describe the paracellular properties of nine different culture models. To establish a primary BBB model rat brain capillary endothelial cells were co-cultured with rat pericytes and astrocytes (EPA. As other BBB and surrogate models four brain endothelial cells lines, rat GP8 and RBE4 cells, and human hCMEC/D3 cells with or without lithium treatment (D3 and D3L, and four epithelial cell lines, native human intestinal Caco-2 and high P-glycoprotein expressing vinblastine-selected VB-Caco-2 cells, native MDCK and MDR1 transfected MDCK canine kidney cells were used. To test transporter functionality, the permeability of 12 molecules, glucopyranose, valproate, baclofen, gabapentin, probenecid, salicylate, rosuvastatin, pravastatin, atorvastatin, tacrine, donepezil, was also measured in the EPA and epithelial models. Among the junctional protein genes, the expression level of occludin was high in all models except the GP8 and RBE4 cells, and each model expressed a unique claudin pattern. Major BBB efflux (P-glycoprotein or ABCB1 and influx transporters (GLUT-1, LAT-1 were present in all models at mRNA levels. The transcript of BCRP (ABCG2 was not expressed in MDCK, GP8 and RBE4 cells. The absence of gene expression of important BBB efflux and influx transporters BCRP, MRP6, -9, MCT6, -8, PHT2, OATPs in one or both types of epithelial models suggests that Caco-2 or MDCK models are not suitable to test drug candidates which

  5. Regulation of human feto-placental endothelial barrier integrity by vascular endothelial growth factors: competitive interplay between VEGF-A165a, VEGF-A165b, PIGF and VE-cadherin.

    Science.gov (United States)

    Pang, Vincent; Bates, David O; Leach, Lopa

    2017-12-01

    The human placenta nourishes and protects the developing foetus whilst influencing maternal physiology for fetal advantage. It expresses several members of the vascular endothelial growth factor (VEGF) family including the pro-angiogenic/pro-permeability VEGF-A 165 a isoform, the anti-angiogenic VEGF-A 165 b, placental growth factor (PIGF) and their receptors, VEGFR1 and VEGFR2. Alterations in the ratio of these factors during gestation and in complicated pregnancies have been reported; however, the impact of this on feto-placental endothelial barrier integrity is unknown. The present study investigated the interplay of these factors on junctional occupancy of VE-cadherin and macromolecular leakage in human endothelial monolayers and the perfused placental microvascular bed. Whilst VEGF-A 165 a (50 ng/ml) increased endothelial monolayer albumin permeability ( P 0.05) or PlGF ( P >0.05) did not. Moreover, VEGF-A 165 b (100 ng/ml; P 0.05) inhibited VEGF-A 165 a-induced permeability when added singly. PlGF abolished the VEGF-A 165 b-induced reduction in VEGF-A 165 a-mediated permeability ( P >0.05); PlGF was found to compete with VEGF-A 165 b for binding to Flt-1 at equimolar affinity. Junctional occupancy of VE-cadherin matched alterations in permeability. In the perfused microvascular bed, VEGF-A 165 b did not induce microvascular leakage but inhibited and reversed VEGF-A 165 a-induced loss of junctional VE-cadherin and tracer leakage. These results indicate that the anti-angiogenic VEGF-A 165 b isoform does not increase permeability in human placental microvessels or HUVEC primary cells and can interrupt VEGF-A 165 a-induced permeability. Moreover, the interplay of these isoforms with PIGF (and s-flt1) suggests that the ratio of these three factors may be important in determining the placental and endothelial barrier in normal and complicated pregnancies. © 2017 The Author(s).

  6. Activation of the endoplasmic reticulum stress response by the amyloid-beta 1-40 peptide in brain endothelial cells.

    Science.gov (United States)

    Fonseca, Ana Catarina R G; Ferreiro, Elisabete; Oliveira, Catarina R; Cardoso, Sandra M; Pereira, Cláudia F

    2013-12-01

    Neurovascular dysfunction arising from endothelial cell damage is an early pathogenic event that contributes to the neurodegenerative process occurring in Alzheimer's disease (AD). Since the mechanisms underlying endothelial dysfunction are not fully elucidated, this study was aimed to explore the hypothesis that brain endothelial cell death is induced upon the sustained activation of the endoplasmic reticulum (ER) stress response by amyloid-beta (Aβ) peptide, which deposits in the cerebral vessels in many AD patients and transgenic mice. Incubation of rat brain endothelial cells (RBE4 cell line) with Aβ1-40 increased the levels of several markers of ER stress-induced unfolded protein response (UPR), in a time-dependent manner, and affected the Ca(2+) homeostasis due to the release of Ca(2+) from this intracellular store. Finally, Aβ1-40 was shown to activate both mitochondria-dependent and -independent apoptotic cell death pathways. Enhanced release of cytochrome c from mitochondria and activation of the downstream caspase-9 were observed in cells treated with Aβ1-40 concomitantly with caspase-12 activation. Furthermore, Aβ1-40 activated the apoptosis effectors' caspase-3 and promoted the translocation of apoptosis-inducing factor (AIF) to the nucleus demonstrating the involvement of caspase-dependent and -independent mechanisms during Aβ-induced endothelial cell death. In conclusion, our data demonstrate that ER stress plays a significant role in Aβ1-40-induced apoptotic cell death in brain endothelial cells suggesting that ER stress-targeted therapeutic strategies might be useful in AD to counteract vascular defects and ultimately neurodegeneration. © 2013.

  7. Microvascular Cranial Nerve Palsy

    Science.gov (United States)

    ... Español Eye Health / Eye Health A-Z Microvascular Cranial Nerve Palsy Sections What Is Microvascular Cranial Nerve Palsy? ... Microvascular Cranial Nerve Palsy Treatment What Is Microvascular Cranial Nerve Palsy? Leer en Español: ¿Qué es una parálisis ...

  8. Altered decorin leads to disrupted endothelial cell function: a possible mechanism in the pathogenesis of fetal growth restriction?

    Science.gov (United States)

    Chui, A; Murthi, P; Gunatillake, T; Brennecke, S P; Ignjatovic, V; Monagle, P T; Whitelock, J M; Said, J M

    2014-08-01

    Fetal growth restriction (FGR) is a key cause of adverse pregnancy outcome where maternal and fetal factors are identified as contributing to this condition. Idiopathic FGR is associated with altered vascular endothelial cell functions. Decorin (DCN) has important roles in the regulation of endothelial cell functions in vascular environments. DCN expression is reduced in FGR. The objectives were to determine the functional consequences of reduced DCN in a human microvascular endothelial cell line model (HMVEC), and to determine downstream targets of DCN and their expression in primary placental microvascular endothelial cells (PLECs) from control and FGR-affected placentae. Short-interference RNA was used to reduce DCN expression in HMVECs and the effect on proliferation, angiogenesis and thrombin generation was determined. A Growth Factor PCR Array was used to identify downstream targets of DCN. The expression of target genes in control and FGR PLECs was performed. DCN reduction decreased proliferation and angiogenesis but increased thrombin generation with no effect on apoptosis. The array identified three targets of DCN: FGF17, IL18 and MSTN. Validation of target genes confirmed decreased expression of VEGFA, MMP9, EGFR1, IGFR1 and PLGF in HMVECs and PLECs from control and FGR pregnancies. Reduction of DCN in vascular endothelial cells leads to disrupted cell functions. The targets of DCN include genes that play important roles in angiogenesis and cellular growth. Therefore, differential expression of these may contribute to the pathogenesis of FGR and disease states in other microvascular circulations. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Modeling of pulsatile flow-dependent nitric oxide regulation in a realistic microvascular network.

    Science.gov (United States)

    Wang, Ruofan; Pan, Qing; Kuebler, Wolfgang M; Li, John K-J; Pries, Axel R; Ning, Gangmin

    2017-09-01

    Hemodynamic pulsatility has been reported to regulate microcirculatory function. To quantitatively assess the impact of flow pulsatility on the microvasculature, a mathematical model was first developed to simulate the regulation of NO production by pulsatile flow in the microcirculation. Shear stress and pressure pulsatility were selected as regulators of endothelial NO production and NO-dependent vessel dilation as feedback to control microvascular hemodynamics. The model was then applied to a real microvascular network of the rat mesentery consisting of 546 microvessels. As compared to steady flow conditions, pulsatile flow increased the average NO concentration in arterioles from 256.8±93.1nM to 274.8±101.1nM (Pflow as compared to steady flow conditions. Network perfusion and flow heterogeneity were improved under pulsatile flow conditions, and vasodilation within the network was more sensitive to heart rate changes than pulse pressure amplitude. The proposed model simulates the role of flow pulsatility in the regulation of a complex microvascular network in terms of NO concentration and hemodynamics under varied physiological conditions. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Vascular endothelial growth factor and basic fibroblast growth factor expression positively correlates with angiogenesis and peritumoural brain oedema in astrocytoma

    International Nuclear Information System (INIS)

    Jang, F.F.; Wei, W.

    2008-01-01

    Astrocytoma is the most malignant intracranial neoplasm and is characterized by high neovascularization and peritumoural brain oedema. Angiogenesis is a complicated process in oncogenesis regulated by the balance between angiogenic and antiangiogenic factors. The expression of two angiogenic growth factors, vascular endothelial growth factor and basic fibroblast growth factor were investigated using immunohistochemistry for astrocytoma from 82 patients and 11 normal human tissues. The expression of vascular endothelial growth factor and basic fibroblast growth factor positively correlate with the pathological grade of astrocytoma, microvessel density numbers and brain oedema, which may be responsible for the increased tumour neovascularization and peritumoural brain oedema. The results support the idea that inhibiting vascular endothelial growth factor and basic fibroblast growth factor are useful for the treatment of human astrocytoma and to improve patient's clinical outcomes and prognosis. (author)

  11. Microvascular function in pre-eclampsia is influenced by insulin resistance and an imbalance of angiogenic mediators.

    Science.gov (United States)

    Ghosh, Anshuman; Freestone, Nicholas S; Anim-Nyame, Nicholas; Arrigoni, Francesca I F

    2017-04-01

    In preeclampsia, maternal microvascular function is disrupted and angiogenesis is dysfunctional. Insulin resistance that occurs in some pregnancies also pathologically affects microvascular function. We wished to examine the relationship of angiogenic mediators and insulin resistance on microvascular health in pregnancy. We performed a nested, case-control study of 16 women who developed preeclampsia with 17 normal pregnant controls. We hypothesized that the impaired microvascular blood flow in preeclamptic women associated with an increased ratio of the antiangiogenic factors; (s-endoglin [sEng] and soluble fms-like tyrosine kinase-1 [sFlt-1]) and proangiogenic molecule (placental growth factor [PlGF]) could be influenced by insulin resistance. Serum samples taken after 28 weeks of gestation were measured for the angiogenic factors, insulin, and glucose alongside the inflammatory marker; tumor necrosis factor-α and endothelial activation, namely; soluble vascular cell adhesion molecule 1, intercellular adhesion molecule-1, and e-selectin. Maternal microvascular blood flow, measured by strain gauge plethysmography, correlated with ratios of pro- and antiangiogenic mediators independently of preeclampsia. Decreased microvascular function measured in preeclampsia strongly correlated with both the antiangiogenic factor (sFlt-1 + sEng): PlGF ratio and high levels of insulin resistance, and combining insulin resistance with antiangiogenic factor ratios further strengthened this relationship. In pregnancy, microvascular blood flow is strongly associated with perturbations in pro- and antiangiogenic mediators. In preeclampsia, the relationship of maternal microvascular dysfunction with antiangiogenic mediators is strengthened when combined with insulin resistance. © 2017 Kingston University. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

  12. Microvascular Anastomosis Training in Neurosurgery: A Review

    Directory of Open Access Journals (Sweden)

    Vadim A. Byvaltsev

    2018-01-01

    Full Text Available Cerebrovascular diseases are among the most widespread diseases in the world, which largely determine the structure of morbidity and mortality rates. Microvascular anastomosis techniques are important for revascularization surgeries on brachiocephalic and carotid arteries and complex cerebral aneurysms and even during resection of brain tumors that obstruct major cerebral arteries. Training in microvascular surgery became even more difficult with less case exposure and growth of the use of endovascular techniques. In this text we will briefly discuss the history of microvascular surgery, review current literature on simulation models with the emphasis on their merits and shortcomings, and describe the views and opinions on the future of the microvascular training in neurosurgery. In “dry” microsurgical training, various models created from artificial materials that simulate biological tissues are used. The next stage in training more experienced surgeons is to work with nonliving tissue models. Microvascular training using live models is considered to be the most relevant due to presence of the blood flow. Training on laboratory animals has high indicators of face and constructive validity. One of the future directions in the development of microsurgical techniques is the use of robotic systems. Robotic systems may play a role in teaching future generations of microsurgeons. Modern technologies allow access to highly accurate learning environments that are extremely similar to real environment. Additionally, assessment of microsurgical skills should become a fundamental part of the current evaluation of competence within a microneurosurgical training program. Such an assessment tool could be utilized to ensure a constant level of surgical competence within the recertification process. It is important that this evaluation be based on validated models.

  13. Morphofunctional aspects of the blood-brain barrier.

    Science.gov (United States)

    Nico, Beatrice; Ribatti, Domenico

    2012-01-01

    The blood-brain barrier (BBB) selectively controls the homeostasis of the Central Nervous System (CNS) environment by the specific structural and biochemical features of the endothelial cells, pericytes and glial endfeet, which represent the cellular components of the mature BBB. Endothelial tight junctions (TJs) are the most important structural component of the BBB, and molecular alteration in the phosphorylation state of some TJs proteins, like ZO-1 or occludin, are crucial in determining alterations in the control of BBB vascular permeability. Astrocytes endfeet enveloping the vessels wall, are considered important in the induction and maintenance of the BBB, through secretion of soluble factors, which modulate the expression of enzymatic complexes and antigens by endothelial cells and TJs - associated proteins. Moreover, astrocytes control water flux at BBB site by expressing a specific water channel, namely aquaporin-4 (AQP4), involved in the molecular composition of the orthogonal particles arrays (OAPs) on the perivascular glial endfeet and tightly coupled with the maintenance of the BBB integrity. Disruption of the BBB is a consistent event occurring in the development of several CNS diseases, including demyelinating lesions in the course of relapsing multiple sclerosis, stroke, Duchenne muscular dystrophy (DMD), but also mechanical injures, neurological insults, septic encephalopathy, brain tumors, permanent ischemia or transient ischemia followed by reperfusion. In most cases, these pathological conditions are associated with an increase in microvascular permeability, vasogenic edema, swollen atrocyte endfeet, and BBB disruption.

  14. Hyperglycemia and Diabetes Downregulate the Functional Expression of TRPV4 Channels in Retinal Microvascular Endothelium

    Science.gov (United States)

    Monaghan, Kevin; McNaughten, Jennifer; McGahon, Mary K.; Kelly, Catriona; Kyle, Daniel; Yong, Phaik Har

    2015-01-01

    Retinal endothelial cell dysfunction is believed to play a key role in the etiology and pathogenesis of diabetic retinopathy. Numerous studies have shown that TRPV4 channels are critically involved in maintaining normal endothelial cell function. In the current paper, we demonstrate that TRPV4 is functionally expressed in the endothelium of the retinal microcirculation and that both channel expression and activity is downregulated by hyperglycaemia. Quantitative PCR and immunostaining demonstrated molecular expression of TRPV4 in cultured bovine retinal microvascular endothelial cells (RMECs). Functional TRPV4 activity was assessed in cultured RMECs from endothelial Ca2+-responses recorded using fura-2 microfluorimetry and electrophysiological recordings of membrane currents. The TRPV4 agonist 4α-phorbol 12,13-didecanoate (4-αPDD) increased [Ca2+]i in RMECs and this response was largely abolished using siRNA targeted against TRPV4. These Ca2+-signals were completely inhibited by removal of extracellular Ca2+, confirming their dependence on influx of extracellular Ca2+. The 4-αPDD Ca2+-response recorded in the presence of cyclopiazonic acid (CPA), which depletes the intracellular stores preventing any signal amplification through store release, was used as a measure of Ca2+-influx across the cell membrane. This response was blocked by HC067047, a TRPV4 antagonist. Under voltage clamp conditions, the TRPV4 agonist GSK1016790A stimulated a membrane current, which was again inhibited by HC067047. Following incubation with 25mM D-glucose TRPV4 expression was reduced in comparison with RMECs cultured under control conditions, as were 4αPDD-induced Ca2+-responses in the presence of CPA and ion currents evoked by GSK1016790A. Molecular expression of TRPV4 in the retinal vascular endothelium of 3 months’ streptozotocin-induced diabetic rats was also reduced in comparison with that in age-matched controls. We conclude that hyperglycaemia and diabetes reduce the

  15. Rho-Kinase Inhibition Ameliorates Dasatinib-Induced Endothelial Dysfunction and Pulmonary Hypertension

    Directory of Open Access Journals (Sweden)

    Csilla Fazakas

    2018-05-01

    Full Text Available The multi-kinase inhibitor dasatinib is used for treatment of imatinib-resistant chronic myeloid leukemia, but is prone to induce microvascular dysfunction. In lung this can manifest as capillary leakage with pleural effusion, pulmonary edema or even pulmonary arterial hypertension. To understand how dasatinib causes endothelial dysfunction we examined the effects of clinically relevant concentrations of dasatinib on both human pulmonary arterial macro- and microvascular endothelial cells (ECs. The effects of dasatinib was compared to imatinib and nilotinib, two other clinically used BCR/Abl kinase inhibitors that do not inhibit Src. Real three-dimensional morphology and high resolution stiffness mapping revealed softening of both macro- and microvascular ECs upon dasatinib treatment, which was not observed in response to imatinib. In a dose-dependent manner, dasatinib decreased transendothelial electrical resistance/impedance and caused a permeability increase as well as disruption of tight adherens junctions in both cell types. In isolated perfused and ventilated rat lungs, dasatinib increased mean pulmonary arterial pressure, which was accompanied by a gain in lung weight. The Rho-kinase inhibitor Y27632 partly reversed the dasatinib-induced changes in vitro and ex vivo, presumably by acting downstream of Src. Co-administration of the Rho-kinase inhibitor Y27632 completely blunted the increased pulmonary pressure in response to dasatinib. In conclusion, a dasatinib-induced permeability increase in human pulmonary arterial macro- and microvascular ECs might explain many of the adverse effects of dasatinib in patients. Rho-kinase inhibition might be suitable to ameliorate these effects.

  16. Gpr124 is essential for blood-brain barrier integrity in central nervous system disease.

    Science.gov (United States)

    Chang, Junlei; Mancuso, Michael R; Maier, Carolina; Liang, Xibin; Yuki, Kanako; Yang, Lu; Kwong, Jeffrey W; Wang, Jing; Rao, Varsha; Vallon, Mario; Kosinski, Cynthia; Zhang, J J Haijing; Mah, Amanda T; Xu, Lijun; Li, Le; Gholamin, Sharareh; Reyes, Teresa F; Li, Rui; Kuhnert, Frank; Han, Xiaoyuan; Yuan, Jenny; Chiou, Shin-Heng; Brettman, Ari D; Daly, Lauren; Corney, David C; Cheshier, Samuel H; Shortliffe, Linda D; Wu, Xiwei; Snyder, Michael; Chan, Pak; Giffard, Rona G; Chang, Howard Y; Andreasson, Katrin; Kuo, Calvin J

    2017-04-01

    Although blood-brain barrier (BBB) compromise is central to the etiology of diverse central nervous system (CNS) disorders, endothelial receptor proteins that control BBB function are poorly defined. The endothelial G-protein-coupled receptor (GPCR) Gpr124 has been reported to be required for normal forebrain angiogenesis and BBB function in mouse embryos, but the role of this receptor in adult animals is unknown. Here Gpr124 conditional knockout (CKO) in the endothelia of adult mice did not affect homeostatic BBB integrity, but resulted in BBB disruption and microvascular hemorrhage in mouse models of both ischemic stroke and glioblastoma, accompanied by reduced cerebrovascular canonical Wnt-β-catenin signaling. Constitutive activation of Wnt-β-catenin signaling fully corrected the BBB disruption and hemorrhage defects of Gpr124-CKO mice, with rescue of the endothelial gene tight junction, pericyte coverage and extracellular-matrix deficits. We thus identify Gpr124 as an endothelial GPCR specifically required for endothelial Wnt signaling and BBB integrity under pathological conditions in adult mice. This finding implicates Gpr124 as a potential therapeutic target for human CNS disorders characterized by BBB disruption.

  17. Human pericyte-endothelial cell interactions in co-culture models mimicking the diabetic retinal microvascular environment.

    Science.gov (United States)

    Tarallo, Sonia; Beltramo, Elena; Berrone, Elena; Porta, Massimo

    2012-12-01

    Pericytes regulate vascular tone, perfusion pressure and endothelial cell (EC) proliferation in capillaries. Thiamine and benfotiamine counteract high glucose-induced damage in vascular cells. We standardized two human retinal pericyte (HRP)/EC co-culture models to mimic the diabetic retinal microvascular environment. We aimed at evaluating the interactions between co-cultured HRP and EC in terms of proliferation/apoptosis and the possible protective role of thiamine and benfotiamine against high glucose-induced damage. EC and HRP were co-cultured in physiological glucose and stable or intermittent high glucose, with or without thiamine/benfotiamine. No-contact model: EC were plated on a porous membrane suspended into the medium and HRP on the bottom of the same well. Cell-to-cell contact model: EC and HRP were plated on the opposite sides of the same membrane. Proliferation (cell counts and DNA synthesis), apoptosis and tubule formation in Matrigel were assessed. In the no-contact model, stable high glucose reduced proliferation of co-cultured EC/HRP and EC alone and increased co-cultured EC/HRP apoptosis. In the contact model, both stable and intermittent high glucose reduced co-cultured EC/HRP proliferation and increased apoptosis. Stable high glucose had no effects on HRP in separate cultures. Both EC and HRP proliferated better when co-cultured. Thiamine and benfotiamine reversed high glucose-induced damage in all cases. HRP are sensitive to soluble factors released by EC when cultured in high glucose conditions, as suggested by conditioned media assays. In the Matrigel models, addition of thiamine and benfotiamine re-established the high glucose-damaged interactions between EC/HRP and stabilized microtubules.

  18. Uric acid is associated with inflammation, coronary microvascular dysfunction, and adverse outcomes in postmenopausal women

    Science.gov (United States)

    Prasad, Megha; Matteson, Eric L.; Herrmann, Joerg; Gulati, Rajiv; Rihal, Charanjit S.; Lerman, Lilach O.; Lerman, Amir

    2016-01-01

    Uric acid is a risk factor for coronary artery disease (CAD) in postmenopausal women but the association with inflammation and coronary microvascular endothelial dysfunction (CED) is not well-defined. The aim of this study was to determine the relationship of serum uric acid (SUA), inflammatory markers and CED. In this prospective cohort study, serum uric acid, hsCRP levels, and neutrophil count were measured in 229 postmenopausal women who underwent diagnostic catheterization, were found to have no obstructive CAD and underwent coronary microvascular function testing, to measure coronary blood flow (CBF) response to intracoronary acetylcholine. The average age was 58 years (IQR 52, 66) years. Hypertension was present in 48%, type 2 diabetes mellitus in 5.6%, and hyperlipidemia in 61.8%. CED was diagnosed in 59% of postmenopausal women. Mean uric acid level was 4.7 ± 1.3 mg/dL. Postmenopausal women with CED had significantly higher SUA compared to patients without CED (4.9 ± 1.3 vs. 4.4 ± 1.3 mg/dL; p=0.02). There was a significant correlation between SUA and % change in CBF to acetylcholine (p=0.009), and this correlation persisted in multivariable analysis. SUA levels were significantly associated with increased neutrophil count (p=0.02) and hsCRP levels (p=0.006) among patients with CED, but not those without CED. Serum uric acid is associated with coronary microvascular endothelial dysfunction in postmenopausal women and may be related to inflammation. These findings link serum uric acid levels to early coronary atherosclerosis in postmenopausal women. PMID:27993955

  19. Endothelial Activation and Blood-Brain Barrier Disruption in Neurotoxicity after Adoptive Immunotherapy with CD19 CAR-T Cells.

    Science.gov (United States)

    Gust, Juliane; Hay, Kevin A; Hanafi, Laïla-Aïcha; Li, Daniel; Myerson, David; Gonzalez-Cuyar, Luis F; Yeung, Cecilia; Liles, W Conrad; Wurfel, Mark; Lopez, Jose A; Chen, Junmei; Chung, Dominic; Harju-Baker, Susanna; Özpolat, Tahsin; Fink, Kathleen R; Riddell, Stanley R; Maloney, David G; Turtle, Cameron J

    2017-12-01

    Lymphodepletion chemotherapy followed by infusion of CD19-targeted chimeric antigen receptor-modified T (CAR-T) cells can be complicated by neurologic adverse events (AE) in patients with refractory B-cell malignancies. In 133 adults treated with CD19 CAR-T cells, we found that acute lymphoblastic leukemia, high CD19 + cells in bone marrow, high CAR-T cell dose, cytokine release syndrome, and preexisting neurologic comorbidities were associated with increased risk of neurologic AEs. Patients with severe neurotoxicity demonstrated evidence of endothelial activation, including disseminated intravascular coagulation, capillary leak, and increased blood-brain barrier (BBB) permeability. The permeable BBB failed to protect the cerebrospinal fluid from high concentrations of systemic cytokines, including IFNγ, which induced brain vascular pericyte stress and their secretion of endothelium-activating cytokines. Endothelial activation and multifocal vascular disruption were found in the brain of a patient with fatal neurotoxicity. Biomarkers of endothelial activation were higher before treatment in patients who subsequently developed grade ≥4 neurotoxicity. Significance: We provide a detailed clinical, radiologic, and pathologic characterization of neurotoxicity after CD19 CAR-T cells, and identify risk factors for neurotoxicity. We show endothelial dysfunction and increased BBB permeability in neurotoxicity and find that patients with evidence of endothelial activation before lymphodepletion may be at increased risk of neurotoxicity. Cancer Discov; 7(12); 1404-19. ©2017 AACR. See related commentary by Mackall and Miklos, p. 1371 This article is highlighted in the In This Issue feature, p. 1355 . ©2017 American Association for Cancer Research.

  20. Permeability of endothelial and astrocyte cocultures: in vitro blood-brain barrier models for drug delivery studies.

    Science.gov (United States)

    Li, Guanglei; Simon, Melissa J; Cancel, Limary M; Shi, Zhong-Dong; Ji, Xinying; Tarbell, John M; Morrison, Barclay; Fu, Bingmei M

    2010-08-01

    The blood-brain barrier (BBB) is a major obstacle for drug delivery to the brain. To seek for in vitro BBB models that are more accessible than animals for investigating drug transport across the BBB, we compared four in vitro cultured cell models: endothelial monoculture (bEnd3 cell line), coculture of bEnd3 and primary rat astrocytes (coculture), coculture with collagen type I and IV mixture, and coculture with Matrigel. The expression of the BBB tight junction proteins in these in vitro models was assessed using RT-PCR and immunofluorescence. We also quantified the hydraulic conductivity (L (p)), transendothelial electrical resistance (TER) and diffusive solute permeability (P) of these models to three solutes: TAMRA, Dextran 10K and Dextran 70K. Our results show that L (p) and P of the endothelial monoculture and coculture models are not different from each other. Compared with in vivo permeability data from rat pial microvessels, P of the endothelial monoculture and coculture models are not significantly different from in vivo data for Dextran 70K, but they are 2-4 times higher for TAMRA and Dextran 10K. This suggests that the endothelial monoculture and all of the coculture models are fairly good models for studying the transport of relatively large solutes across the BBB.

  1. EGb 761 Protects Cardiac Microvascular Endothelial Cells against Hypoxia/Reoxygenation Injury and Exerts Inhibitory Effect on the ATM Pathway.

    Science.gov (United States)

    Zhang, Chao; Wang, Deng-Feng; Zhang, Zhuang; Han, Dong; Yang, Kan

    2017-03-28

    Ginkgo bilob a extract (EGb 761) has been widely used clinically to reduce myocardial ischemia reperfusion injury (MIRI). Microvascular endothelial cells (MVECs) may be a proper cellular model in vitro for the effect and mechanism study against MIRI. However, the protective effect of EGb 761 on MVECs resisting hypoxia/reoxygenation (H/R) injury is little reported. In this study, H/R-injured MVECs were treated with EGb 761, and then the cell viability, apoptosis, ROS production, SOD activity, caspase-3 activity, and protein level of ATM, γ-H2AX, p53, and Bax were measured. ATM siRNA was transfected to study the changes of protein in the ATM pathway. EGb 761 presented protective effect on H/R-injured MVECs, with decreasing cell death, apoptosis, and ROS, and elevated SOD activity. Next, EGb 761 could inhibit H/R-induced ATM, γ-H2AX, p53, and Bax in a dose-dependent manner. Moreover, ATM siRNA also could inhibit H/R-induced ATM, γ-H2AX, p53, and Bax. Overall, these findings verify that EGb 761 protects cardiac MVECs from H/R injury, and for the first time, illustrate the influence on the ATM pathway and apoptosis by EGb 761 via dampening ROS.

  2. The association of systemic microvascular changes with lung function and lung density: a cross-sectional study.

    Directory of Open Access Journals (Sweden)

    Bianca Harris

    Full Text Available Smoking causes endothelial dysfunction and systemic microvascular disease with resultant end-organ damage in the kidneys, eyes and heart. Little is known about microvascular changes in smoking-related lung disease. We tested if microvascular changes in the retina, kidneys and heart were associated with obstructive spirometry and low lung density on computed tomography. The Multi-Ethnic Study of Atherosclerosis recruited participants age 45-84 years without clinical cardiovascular disease. Measures of microvascular function included retinal arteriolar and venular caliber, urine albumin-to-creatinine ratio and, in a subset, myocardial blood flow on magnetic resonance imaging. Spirometry was measured following ATS/ERS guidelines. Low attenuation areas (LAA were measured on lung fields of cardiac computed tomograms. Regression models adjusted for pulmonary and cardiac risk factors, medications and body size. Among 3,397 participants, retinal venular caliber was inversely associated with forced expiratory volume in one second (FEV(1 (P<0.001 and FEV(1/forced vital capacity (FVC ratio (P = 0.04. Albumin-to-creatinine ratio was inversely associated with FEV(1 (P = 0.002 but not FEV(1/FVC. Myocardial blood flow (n = 126 was associated with lower FEV(1 (P = 0.02, lower FEV(1/FVC (P = 0.001 and greater percentage LAA (P = 0.04. Associations were of greater magnitude among smokers. Low lung function was associated with microvascular changes in the retina, kidneys and heart, and low lung density was associated with impaired myocardial microvascular perfusion. These cross-sectional results suggest that microvascular damage with end-organ dysfunction in all circulations may pertain to the lung, that lung dysfunction may contribute to systemic microvascular disease, or that there may be a shared predisposition.

  3. Evaluation of microvascular endothelial function and capillary density in patients with infective endocarditis using laser speckle contrast imaging and video-capillaroscopy.

    Science.gov (United States)

    Barcelos, Amanda; Tibirica, Eduardo; Lamas, Cristiane

    2018-07-01

    To evaluate the systemic microcirculation of patients with infective endocarditis (IE). This is a comparative study of patients with definite IE by the modified Duke criteria admitted to our center for treatment. A reference group of sex- and age-matched healthy volunteers was included. Microvascular flow was evaluated in the forearm using a laser speckle contrast imaging system, for noninvasive measurement of cutaneous microvascular perfusion, in combination with skin iontophoresis of acetylcholine (ACh) and sodium nitroprusside (SNP) to test microvascular reactivity. Microvascular density was evaluated using skin video-capillaroscopy. We studied 22 patients with IE; 15 were male and seven female. The mean age and standard deviation (SD) were 45.5 ± 17.3 years. Basal skin microvascular conductance was significantly increased in patients with IE, compared with healthy individuals (0.36 ± 0.13 versus 0.21 ± 0.08 APU/mmHg; P < 0.0001). The increase in microvascular conductance induced by ACh in patients was 0.21 ± 0.17 and in the reference group, it was 0.37 ± 0.14 APU/mmHg (P = 0.0012). The increase in microvascular conductance induced by SNP in patients was 0.18 ± 0.14 and it was 0.29 ± 0.15 APU/mmHg (P = 0.0140) in the reference group. The basal mean skin capillary density of patients (135 ± 24 capillaries/mm 2 ) was significantly higher, compared with controls (97 ± 21 capillaries/mm 2 ; P < 0.0001). The main findings in the microcirculation of patients with IE were greater basal vasodilation and a reduction of the endothelium-dependent and -independent microvascular reactivity, as well as greater functional skin capillary density compared to healthy individuals. Copyright © 2018 Elsevier Inc. All rights reserved.

  4. Astrocytic TYMP and VEGFA drive blood-brain barrier opening in inflammatory central nervous system lesions.

    Science.gov (United States)

    Chapouly, Candice; Tadesse Argaw, Azeb; Horng, Sam; Castro, Kamilah; Zhang, Jingya; Asp, Linnea; Loo, Hannah; Laitman, Benjamin M; Mariani, John N; Straus Farber, Rebecca; Zaslavsky, Elena; Nudelman, German; Raine, Cedric S; John, Gareth R

    2015-06-01

    In inflammatory central nervous system conditions such as multiple sclerosis, breakdown of the blood-brain barrier is a key event in lesion pathogenesis, predisposing to oedema, excitotoxicity, and ingress of plasma proteins and inflammatory cells. Recently, we showed that reactive astrocytes drive blood-brain barrier opening, via production of vascular endothelial growth factor A (VEGFA). Here, we now identify thymidine phosphorylase (TYMP; previously known as endothelial cell growth factor 1, ECGF1) as a second key astrocyte-derived permeability factor, which interacts with VEGFA to induce blood-brain barrier disruption. The two are co-induced NFκB1-dependently in human astrocytes by the cytokine interleukin 1 beta (IL1B), and inactivation of Vegfa in vivo potentiates TYMP induction. In human central nervous system microvascular endothelial cells, VEGFA and the TYMP product 2-deoxy-d-ribose cooperatively repress tight junction proteins, driving permeability. Notably, this response represents part of a wider pattern of endothelial plasticity: 2-deoxy-d-ribose and VEGFA produce transcriptional programs encompassing angiogenic and permeability genes, and together regulate a third unique cohort. Functionally, each promotes proliferation and viability, and they cooperatively drive motility and angiogenesis. Importantly, introduction of either into mouse cortex promotes blood-brain barrier breakdown, and together they induce severe barrier disruption. In the multiple sclerosis model experimental autoimmune encephalitis, TYMP and VEGFA co-localize to reactive astrocytes, and correlate with blood-brain barrier permeability. Critically, blockade of either reduces neurologic deficit, blood-brain barrier disruption and pathology, and inhibiting both in combination enhances tissue preservation. Suggesting importance in human disease, TYMP and VEGFA both localize to reactive astrocytes in multiple sclerosis lesion samples. Collectively, these data identify TYMP as an

  5. Energy and glucose pathways in thiamine deficient primary rat brain microvascular endothelial cells.

    Science.gov (United States)

    Ham, D; Karska-Wysocki, B

    2005-12-01

    Thiamine deficiency (TD) results in lactate acidosis, which is associated with neurodegeneration. The aim of this study was to investigate this alteration in primary rat brain endothelia. Spectrophotometric analysis of culture media revealed that only a higher concentration of pyrithiamine, which accelerates the intracellular blocking of thiamine, significantly elevated the lactate level and lactate dehydrogenase activity within 7 days. The medium without pyrithiamine and with a thiamine concentration comparable to pathophysiological plasma levels mildly reduced only the activity of transketolase. This suggests that significant metabolic changes may not occur at the early phase of TD in cerebral capillary cells, while anaerobic glycolysis in capillaries may be mediated during late stage/chronic TD.

  6. Cultured cells of the blood-brain barrier from apolipoprotein B-100 transgenic mice: effects of oxidized low-density lipoprotein treatment.

    Science.gov (United States)

    Lénárt, Nikolett; Walter, Fruzsina R; Bocsik, Alexandra; Sántha, Petra; Tóth, Melinda E; Harazin, András; Tóth, Andrea E; Vizler, Csaba; Török, Zsolt; Pilbat, Ana-Maria; Vígh, László; Puskás, László G; Sántha, Miklós; Deli, Mária A

    2015-07-17

    The apolipoprotein B-100 (ApoB-100) transgenic mouse line is a model of human atherosclerosis. Latest findings suggest the importance of ApoB-100 in the development of neurodegenerative diseases and microvascular/perivascular localization of ApoB-100 protein was demonstrated in the cerebral cortex of ApoB-100 transgenic mice. The aim of the study was to characterize cultured brain endothelial cells, pericytes and glial cells from wild-type and ApoB-100 transgenic mice and to study the effect of oxidized low-density lipoprotein (oxLDL) on these cells. Morphology of cells isolated from brains of wild type and ApoB-100 transgenic mice was characterized by immunohistochemistry and the intensity of immunolabeling was quantified by image analysis. Toxicity of oxLDL treatment was monitored by real-time impedance measurement and lactate dehydrogenase release. Reactive oxygen species and nitric oxide production, barrier permeability in triple co-culture blood-brain barrier model and membrane fluidity were also determined after low-density lipoprotein (LDL) or oxLDL treatment. The presence of ApoB-100 was confirmed in brain endothelial cells, while no morphological change was observed between wild type and transgenic cells. Oxidized but not native LDL exerted dose-dependent toxicity in all three cell types, induced barrier dysfunction and increased reactive oxygen species (ROS) production in both genotypes. A partial protection from oxLDL toxicity was seen in brain endothelial and glial cells from ApoB-100 transgenic mice. Increased membrane rigidity was measured in brain endothelial cells from ApoB-100 transgenic mice and in LDL or oxLDL treated wild type cells. The morphological and functional properties of cultured brain endothelial cells, pericytes and glial cells from ApoB-100 transgenic mice were characterized and compared to wild type cells for the first time. The membrane fluidity changes in ApoB-100 transgenic cells related to brain microvasculature indicate

  7. A SAGE based approach to human glomerular endothelium: defining the transcriptome, finding a novel molecule and highlighting endothelial diversity.

    Science.gov (United States)

    Sengoelge, Guerkan; Winnicki, Wolfgang; Kupczok, Anne; von Haeseler, Arndt; Schuster, Michael; Pfaller, Walter; Jennings, Paul; Weltermann, Ansgar; Blake, Sophia; Sunder-Plassmann, Gere

    2014-08-27

    Large scale transcript analysis of human glomerular microvascular endothelial cells (HGMEC) has never been accomplished. We designed this study to define the transcriptome of HGMEC and facilitate a better characterization of these endothelial cells with unique features. Serial analysis of gene expression (SAGE) was used for its unbiased approach to quantitative acquisition of transcripts. We generated a HGMEC SAGE library consisting of 68,987 transcript tags. Then taking advantage of large public databases and advanced bioinformatics we compared the HGMEC SAGE library with a SAGE library of non-cultured ex vivo human glomeruli (44,334 tags) which contained endothelial cells. The 823 tags common to both which would have the potential to be expressed in vivo were subsequently checked against 822,008 tags from 16 non-glomerular endothelial SAGE libraries. This resulted in 268 transcript tags differentially overexpressed in HGMEC compared to non-glomerular endothelia. These tags were filtered using a set of criteria: never before shown in kidney or any type of endothelial cell, absent in all nephron regions except the glomerulus, more highly expressed than statistically expected in HGMEC. Neurogranin, a direct target of thyroid hormone action which had been thought to be brain specific and never shown in endothelial cells before, fulfilled these criteria. Its expression in glomerular endothelium in vitro and in vivo was then verified by real-time-PCR, sequencing and immunohistochemistry. Our results represent an extensive molecular characterization of HGMEC beyond a mere database, underline the endothelial heterogeneity, and propose neurogranin as a potential link in the kidney-thyroid axis.

  8. Lipid rafts regulate PCB153-induced disruption of occludin and brain endothelial barrier function through protein phosphatase 2A and matrix metalloproteinase-2

    Energy Technology Data Exchange (ETDEWEB)

    Eum, Sung Yong, E-mail: seum@miami.edu; Jaraki, Dima; András, Ibolya E.; Toborek, Michal

    2015-09-15

    Occludin is an essential integral transmembrane protein regulating tight junction (TJ) integrity in brain endothelial cells. Phosphorylation of occludin is associated with its localization to TJ sites and incorporation into intact TJ assembly. The present study is focused on the role of lipid rafts in polychlorinated biphenyl (PCB)-induced disruption of occludin and endothelial barrier function. Exposure of human brain endothelial cells to 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB153) induced dephosphorylation of threonine residues of occludin and displacement of occludin from detergent-resistant membrane (DRM)/lipid raft fractions within 1 h. Moreover, lipid rafts modulated the reduction of occludin level through activation of matrix metalloproteinase 2 (MMP-2) after 24 h PCB153 treatment. Inhibition of protein phosphatase 2A (PP2A) activity by okadaic acid or fostriecin markedly protected against PCB153-induced displacement of occludin and increased permeability of endothelial cells. The implication of lipid rafts and PP2A signaling in these processes was further defined by co-immunoprecipitation of occludin with PP2A and caveolin-1, a marker protein of lipid rafts. Indeed, a significant MMP-2 activity was observed in lipid rafts and was increased by exposure to PCB153. The pretreatment of MMP-2 inhibitors protected against PCB153-induced loss of occludin and disruption of lipid raft structure prevented the increase of endothelial permeability. Overall, these results indicate that lipid raft-associated processes, such as PP2A and MMP-2 activation, participate in PCB153-induced disruption of occludin function in brain endothelial barrier. This study contributes to a better understanding of the mechanisms leading to brain endothelial barrier dysfunction in response to exposure to environmental pollutants, such as ortho-substituted PCBs. - Highlights: • PCB153 disturbed human brain endothelial barrier through disruption of occludin. • Lipid raft-associated PP

  9. Transfection of brain capillary endothelial cells in primary culture with defined blood-brain barrier properties.

    Science.gov (United States)

    Burkhart, Annette; Thomsen, Louiza Bohn; Thomsen, Maj Schneider; Lichota, Jacek; Fazakas, Csilla; Krizbai, István; Moos, Torben

    2015-08-07

    Primary brain capillary endothelial cells (BCECs) are a promising tool to study the blood-brain barrier (BBB) in vitro, as they maintain many important characteristics of the BBB in vivo, especially when co-cultured with pericytes and/or astrocytes. A novel strategy for drug delivery to the brain is to transform BCECs into protein factories by genetic modifications leading to secretion of otherwise BBB impermeable proteins into the central nervous system. However, a huge challenge underlying this strategy is to enable transfection of non-mitotic BCECs, taking a non-viral approach. We therefore aimed to study transfection in primary, non-mitotic BCECs cultured with defined BBB properties without disrupting the cells' integrity. Primary cultures of BCECs, pericytes and astrocytes were generated from rat brains and used in three different in vitro BBB experimental arrangements, which were characterised based on a their expression of tight junction proteins and other BBB specific proteins, high trans-endothelial electrical resistance (TEER), and low passive permeability to radiolabeled mannitol. Recombinant gene expression and protein synthesis were examined in primary BCECs. The BCECs were transfected using a commercially available transfection agent Turbofect™ to express the red fluorescent protein HcRed1-C1. The BCECs were transfected at different time points to monitor transfection in relation to mitotic or non-mitotic cells, as indicated by fluorescence-activated cell sorting analysis after 5-and 6-carboxylfluorescein diacetate succinidyl ester incorporation. The cell cultures exhibited important BBB characteristics judged from their expression of BBB specific proteins, high TEER values, and low passive permeability. Among the three in vitro BBB models, co-culturing with BCECs and astrocytes was well suited for the transfection studies. Transfection was independent of cell division and with equal efficacy between the mitotic and non-mitotic BCECs. Importantly

  10. Endothelial Semaphorin 7A promotes inflammation in seawater aspiration-induced acute lung injury.

    Science.gov (United States)

    Zhang, Minlong; Wang, Li; Dong, Mingqing; Li, Zhichao; Jin, Faguang

    2014-10-28

    Inflammation is involved in the pathogenesis of seawater aspiration-induced acute lung injury (ALI). Although several studies have shown that Semaphorin 7A (SEMA7A) promotes inflammation, there are limited reports regarding immunological function of SEMA7A in seawater aspiration-induced ALI. Therefore, we investigated the role of SEMA7A during seawater aspiration-induced ALI. Male Sprague-Dawley rats were underwent seawater instillation. Then, lung samples were collected at an indicated time for analysis. In addition, rat pulmonary microvascular endothelial cells (RPMVECs) were cultured and then stimulated with 25% seawater for indicated time point. After these treatments, cells samples were collected for analysis. In vivo, seawater instillation induced lung histopathologic changes, pro-inflammation cytokines release and increased expression of SEMA7A. In vitro, seawater stimulation led to pro-inflammation cytokine release, cytoskeleton remodeling and increased monolayer permeability in pulmonary microvascular endothelial cells. In addition, knockdown of hypoxia-inducible factor (HIF)-1α inhibited the seawater induced increase expression of SEMA7A. Meanwhile, knockdown of SEMA7A by specific siRNA inhibited the seawater induced aberrant inflammation, endothelial cytoskeleton remodeling and endothelial permeability. These results suggest that SEMA7A is critical in the development of lung inflammation and pulmonary edema in seawater aspiration-induced ALI, and may be a therapeutic target for this disease.

  11. Role of adhesion molecules and inflammation in Venezuelan equine encephalitis virus infected mouse brain

    Directory of Open Access Journals (Sweden)

    Honnold Shelley P

    2011-04-01

    Full Text Available Abstract Background Neuroinvasion of Venezuelan equine encephalitis virus (VEEV and subsequent initiation of inflammation in the brain plays a crucial role in the outcome of VEEV infection in mice. Adhesion molecules expressed on microvascular endothelial cells in the brain have been implicated in the modulation of the blood brain barrier (BBB and inflammation in brain but their role in VEEV pathogenesis is not very well understood. In this study, we evaluated the expression of extracellular matrix and adhesion molecules genes in the brain of VEEV infected mice. Findings Several cell to cell adhesion molecules and extracellular matrix protein genes such as ICAM-1, VCAM-1, CD44, Cadherins, integrins, MMPs and Timp1 were differentially regulated post-VEEV infection. ICAM-1 knock-out (IKO mice infected with VEEV had markedly reduced inflammation in the brain and demonstrated a delay in the onset of clinical symptoms of disease. A differential regulation of inflammatory genes was observed in the IKO mice brain compared to their WT counterparts. Conclusions These results improve our present understanding of VEEV induced inflammation in mouse brain.

  12. NADPH oxidase and lipid raft-associated redox signaling are required for PCB153-induced upregulation of cell adhesion molecules in human brain endothelial cells

    International Nuclear Information System (INIS)

    Eum, Sung Yong; Andras, Ibolya; Hennig, Bernhard; Toborek, Michal

    2009-01-01

    Exposure to persistent organic pollutants, such as polychlorinated biphenyls (PCBs), can lead to chronic inflammation and the development of vascular diseases. Because cell adhesion molecules (CAMs) of the cerebrovascular endothelium regulate infiltration of inflammatory cells into the brain, we have explored the molecular mechanisms by which ortho-substituted polychlorinated biphenyls (PCBs), such as PCB153, can upregulate CAMs in brain endothelial cells. Exposure to PCB153 increased expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), as well as elevated adhesion of leukocytes to brain endothelial cells. These effects were impeded by inhibitors of EGFR, JAKs, or Src activity. In addition, pharmacological inhibition of NADPH oxidase or disruption of lipid rafts by cholesterol depleting agents blocked PCB153-induced phosphorylation of JAK and Src kinases and upregulation of CAMs. In contrast, silencing of caveolin-1 by siRNA interference did not affect upregulation of ICAM-1 and VCAM-1 in brain endothelial cells stimulated by PCB153. Results of the present study indicate that lipid raft-dependent NADPH oxidase/JAK/EGFR signaling mechanisms regulate the expression of CAMs in brain endothelial cells and adhesion of leukocytes to endothelial monolayers. Due to its role in leukocyte infiltration, induction of CAMs may contribute to PCB-induced cerebrovascular disorders and neurotoxic effects in the CNS.

  13. Tumor and Endothelial Cell Hybrids Participate in Glioblastoma Vasculature

    Directory of Open Access Journals (Sweden)

    Soufiane El Hallani

    2014-01-01

    Full Text Available Background. Recently antiangiogenic therapy with bevacizumab has shown a high but transient efficacy in glioblastoma (GBM. Indeed, GBM is one of the most angiogenic human tumors and endothelial proliferation is a hallmark of the disease. We therefore hypothesized that tumor cells may participate in endothelial proliferation of GBM. Materials and Methods. We used EGFR FISH Probe to detect EGFR amplification and anti-CD31, CD105, VE-cadherin, and vWF to identify endothelial cells. Endothelial and GBM cells were grown separately, labeled with GFP and DsRed lentiviruses, and then cocultured with or without contact. Results. In a subset of GBM tissues, we found that several tumor endothelial cells carry EGFR amplification, characteristic of GBM tumor cells. This observation was reproduced in vitro: when tumor stem cells derived from GBM were grown in the presence of human endothelial cells, a fraction of them acquired endothelial markers (CD31, CD105, VE-cadherin, and vWF. By transduction with GFP and DsRed expressing lentiviral vectors, we demonstrate that this phenomenon is due to cell fusion and not transdifferentiation. Conclusion. A fraction of GBM stem cells thus has the capacity to fuse with endothelial cells and the resulting hybrids may participate in tumor microvascular proliferation and in treatment resistance.

  14. The role of shear stress in Blood-Brain Barrier endothelial physiology

    Directory of Open Access Journals (Sweden)

    Puvenna Vikram

    2011-05-01

    Full Text Available Abstract Background One of the most important and often neglected physiological stimuli contributing to the differentiation of vascular endothelial cells (ECs into a blood-brain barrier (BBB phenotype is shear stress (SS. With the use of a well established humanized dynamic in vitro BBB model and cDNA microarrays, we have profiled the effect of SS in the induction/suppression of ECs genes and related functions. Results Specifically, we found a significant upregulation of tight and adherens junctions proteins and genes. Trans-endothelial electrical resistance (TEER and permeability measurements to know substances have shown that SS promoted the formation of a tight and highly selective BBB. SS also increased the RNA level of multidrug resistance transporters, ion channels, and several p450 enzymes. The RNA level of a number of specialized carrier-mediated transport systems (e.g., glucose, monocarboxylic acid, etc. was also upregulated. RNA levels of modulatory enzymes of the glycolytic pathway (e.g., lactate dehydrogenase were downregulated by SS while those involved in the Krebs cycle (e.g., lactate and other dehydrogenases were upregulated. Measurements of glucose consumption versus lactate production showed that SS negatively modulated the glycolytic bioenergetic pathways of glucose metabolism in favor of the more efficient aerobic respiration. BBB ECs are responsive to inflammatory stimuli. Our data showed that SS increased the RNA levels of integrins and vascular adhesion molecules. SS also inhibited endothelial cell cycle via regulation of BTG family proteins encoding genes. This was paralleled by significant increase in the cytoskeletal protein content while that of membrane, cytosol, and nuclear sub-cellular fractions decreased. Furthermore, analysis of 2D gel electrophoresis (which allows identifying a large number of proteins per sample of EC proteins extracted from membrane sub-cellular endothelial fractions showed that SS increased

  15. Agmatine promotes the migration of murine brain endothelial cells via multiple signaling pathways.

    Science.gov (United States)

    Jung, Hyun-Joo; Jeon, Yong-Heui; Bokara, Kiran Kumar; Koo, Bon-Nyeo; Lee, Won Taek; Park, Kyung Ah; Lee, Jong-Eun

    2013-01-17

    The combination of adhesion and migration of endothelial cells (ECs) is an integral process for evolution, organization, repair and vessel formation in living organisms. Agmatine, a polycationic amine existing in brain, has been investigated to exert neuroprotective effects. Up to date, there are no studies reporting that agmatine modulates murine brain endothelial (bEnd.3) cells migration. In the present study, we intend to investigate the role of agmatine in bEnd.3 cells migration and the molecular mechanism mediating this action. The effect of agmatine on the bEnd.3 cells migration was examined by migration assay, and the mechanism involved for this effect was investigated by western blot analysis and NO contents measurements. Agmatine treatment (50, 100 and 200 μM) significantly accelerated bEnd.3 cells migration in a concentration-dependent manner. Western blotting revealed that agmatine treatment significantly induced vascular endothelial growth factor (VEGF), VEGF receptor 2 (Flk-1/KDR or VEGFR2), phosphatidylinositol 3-kinase (PI3K), Akt/protein kinase B (also known as PKB, PI3K downstream effector protein), endothelial nitric oxide synthase (eNOS) nitric oxide (NO; product by eNOS) and intercellular adhesion molecule 1 (ICAM-1) expressions during bEnd.3 cells migration. The expression of ICAM-1 and migration of bEnd.3 cells, induced by agmatine, were significantly attenuated by treatment of wortmannin, a specific PI3K inhibitor. Taken together, we provide the first evidence that activation of VEGF/VEGFR2 and the consequential PI3K/Akt/eNOS/NO/ICAM-1 signaling pathways are serial events, through which the treatment of agmatine could lead to bEnd.3 cells migration. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. Major involvement of Na(+) -dependent multivitamin transporter (SLC5A6/SMVT) in uptake of biotin and pantothenic acid by human brain capillary endothelial cells.

    Science.gov (United States)

    Uchida, Yasuo; Ito, Katsuaki; Ohtsuki, Sumio; Kubo, Yoshiyuki; Suzuki, Takashi; Terasaki, Tetsuya

    2015-07-01

    The purpose of this study was to clarify the expression of Na(+) -dependent multivitamin transporter (SLC5A6/SMVT) and its contribution to the supply of biotin and pantothenic acid to the human brain via the blood-brain barrier. DNA microarray and immunohistochemical analyses confirmed that SLC5A6 is expressed in microvessels of human brain. The absolute expression levels of SLC5A6 protein in isolated human and monkey brain microvessels were 1.19 and 0.597 fmol/μg protein, respectively, as determined by a quantitative targeted absolute proteomics technique. Using an antibody-free method established by Kubo et al. (2015), we found that SLC5A6 was preferentially localized at the luminal membrane of brain capillary endothelium. Knock-down analysis using SLC5A6 siRNA showed that SLC5A6 accounts for 88.7% and 98.6% of total [(3) H]biotin and [(3) H]pantothenic acid uptakes, respectively, by human cerebral microvascular endothelial cell line hCMEC/D3. SLC5A6-mediated transport in hCMEC/D3 was markedly inhibited not only by biotin and pantothenic acid, but also by prostaglandin E2, lipoic acid, docosahexaenoic acid, indomethacin, ketoprofen, diclofenac, ibuprofen, phenylbutazone, and flurbiprofen. This study is the first to confirm expression of SLC5A6 in human brain microvessels and to provide evidence that SLC5A6 is a major contributor to luminal uptake of biotin and pantothenic acid at the human blood-brain barrier. In humans, it was unclear (not concluded) about what transport system at the blood-brain barrier (BBB) is responsible for the brain uptakes of two vitamins, biotin and pantothenic acid, which are necessary for brain proper function. This study clarified for the first time that the solute carrier 5A6/Na(+) -dependent multivitamin transporter SLC5A6/SMVT is responsible for the supplies of biotin and pantothenic acid into brain across the BBB in humans. DHA, docosahexaenoic acid; NSAID, non-steroidal anti-inflammatory drug; PGE2, prostaglandin E2. © 2015

  17. A model of physical factors in the structural adaptation of microvascular networks in normotension and hypertension

    DEFF Research Database (Denmark)

    Jacobsen, Jens Christian Brings; Gustafsson, Finn; Holstein-Rathlou, N.-H.

    2003-01-01

    Adequate function of the microcirculation is vital to any tissue. To maintain an optimal function, microvascular networks must be able to adapt structurally to changes in the physical environment. Here we present a mathematical network model based on vessel wall mechanics. We assume based...... diameter, until equilibrium is restored. The model explains several of the key features observed experimentally in the microcirculation in normotension and hypertension. Most importantly, it suggests a scenario where overall network structure and network hemodynamics depend on adaptation to local...... hemodynamic stimuli in the individual vessel. Simulated results show emanating microvascular networks with properties similar to those observed in vivo. The model points to an altered endothelial function as a key factor in the development of vascular changes characteristic of hypertension....

  18. Vascular targeting of LIGHT normalizes blood vessels in primary brain cancer and induces intratumoural high endothelial venules.

    Science.gov (United States)

    He, Bo; Jabouille, Arnaud; Steri, Veronica; Johansson-Percival, Anna; Michael, Iacovos P; Kotamraju, Venkata Ramana; Junckerstorff, Reimar; Nowak, Anna K; Hamzah, Juliana; Lee, Gabriel; Bergers, Gabriele; Ganss, Ruth

    2018-06-01

    High-grade brain cancer such as glioblastoma (GBM) remains an incurable disease. A common feature of GBM is the angiogenic vasculature, which can be targeted with selected peptides for payload delivery. We assessed the ability of micelle-tagged, vascular homing peptides RGR, CGKRK and NGR to specifically bind to blood vessels in syngeneic orthotopic GBM models. By using the peptide CGKRK to deliver the tumour necrosis factor (TNF) superfamily member LIGHT (also known as TNF superfamily member 14; TNFSF14) to angiogenic tumour vessels, we have generated a reagent that normalizes the brain cancer vasculature by inducing pericyte contractility and re-establishing endothelial barrier integrity. LIGHT-mediated vascular remodelling also activates endothelia and induces intratumoural high endothelial venules (HEVs), which are specialized blood vessels for lymphocyte infiltration. Combining CGKRK-LIGHT with anti-vascular endothelial growth factor and checkpoint blockade amplified HEV frequency and T-cell accumulation in GBM, which is often sparsely infiltrated by immune effector cells, and reduced tumour burden. Furthermore, CGKRK and RGR peptides strongly bound to blood vessels in freshly resected human GBM, demonstrating shared peptide-binding activities in mouse and human primary brain tumour vessels. Thus, peptide-mediated LIGHT targeting is a highly translatable approach in primary brain cancer to reduce vascular leakiness and enhance immunotherapy. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  19. Sickle erythrocytes inhibit human endothelial cell DNA synthesis

    International Nuclear Information System (INIS)

    Weinstein, R.; Zhou, M.A.; Bartlett-Pandite, A.; Wenc, K.

    1990-01-01

    Patients with sickle cell anemia experience severe vascular occlusive phenomena including acute pain crisis and cerebral infarction. Obstruction occurs at both the microvascular and the arterial level, and the clinical presentation of vascular events is heterogeneous, suggesting a complex etiology. Interaction between sickle erythrocytes and the endothelium may contribute to vascular occlusion due to alteration of endothelial function. To investigate this hypothesis, human vascular endothelial cells were overlaid with sickle or normal erythrocytes and stimulated to synthesize DNA. The erythrocytes were sedimented onto replicate monolayers by centrifugation for 10 minutes at 17 g to insure contact with the endothelial cells. Incorporation of 3H-thymidine into endothelial cell DNA was markedly inhibited during contact with sickle erythrocytes. This inhibitory effect was enhanced more than twofold when autologous sickle plasma was present during endothelial cell labeling. Normal erythrocytes, with or without autologous plasma, had a modest effect on endothelial cell DNA synthesis. When sickle erythrocytes in autologous sickle plasma were applied to endothelial monolayers for 1 minute, 10 minutes, or 1 hour and then removed, subsequent DNA synthesis by the endothelial cells was inhibited by 30% to 40%. Although adherence of sickle erythrocytes to the endothelial monolayers was observed under these experimental conditions, the effect of sickle erythrocytes on endothelial DNA synthesis occurred in the absence of significant adherence. Hence, human endothelial cell DNA synthesis is partially inhibited by contact with sickle erythrocytes. The inhibitory effect of sickle erythrocytes occurs during a brief (1 minute) contact with the endothelial monolayers, and persists for at least 6 hours of 3H-thymidine labeling

  20. Sleep Restriction Impairs Blood–Brain Barrier Function

    Science.gov (United States)

    He, Junyun; Hsuchou, Hung; He, Yi; Kastin, Abba J.; Wang, Yuping

    2014-01-01

    The blood–brain barrier (BBB) is a large regulatory and exchange interface between the brain and peripheral circulation. We propose that changes of the BBB contribute to many pathophysiological processes in the brain of subjects with chronic sleep restriction (CSR). To achieve CSR that mimics a common pattern of human sleep loss, we quantified a new procedure of sleep disruption in mice by a week of consecutive sleep recording. We then tested the hypothesis that CSR compromises microvascular function. CSR not only diminished endothelial and inducible nitric oxide synthase, endothelin1, and glucose transporter expression in cerebral microvessels of the BBB, but it also decreased 2-deoxy-glucose uptake by the brain. The expression of several tight junction proteins also was decreased, whereas the level of cyclooxygenase-2 increased. This coincided with an increase of paracellular permeability of the BBB to the small tracers sodium fluorescein and biotin. CSR for 6 d was sufficient to impair BBB structure and function, although the increase of paracellular permeability returned to baseline after 24 h of recovery sleep. This merits attention not only in neuroscience research but also in public health policy and clinical practice. PMID:25355222

  1. Hypothyroidism Is Associated With Coronary Endothelial Dysfunction in Women

    Science.gov (United States)

    Sara, Jaskanwal D; Zhang, Ming; Gharib, Hossein; Lerman, Lilach O; Lerman, Amir

    2015-01-01

    Background Hypothyroidism is associated with an increased risk of coronary artery disease, beyond that which can be explained by its association with conventional cardiovascular risk factors. Coronary endothelial dysfunction precedes atherosclerosis, has been linked to adverse cardiovascular events, and may account for some of the increased risk in patients with hypothyroidism. The aim of this study was to determine whether there is an association between epicardial and microvascular coronary endothelial dysfunction and hypothyroidism. Methods and Results In 1388 patients (mean age 50.5 [12.3] years, 34% male) presenting with stable chest pain to Mayo Clinic, Rochester, MN for diagnostic coronary angiography, and who were found to have nonobstructive coronary artery disease (hypothyroidism, defined as a documented history of hypothyroidism or a thyroid-stimulating hormone (TSH) >10.0 mU/mL, n=188, and euthyroidism, defined as an absence of a history of hypothyroidism in the clinical record and/or 0.3hypothyroidism had a significantly lower % Δ CBF Ach (48.26 [80.66] versus 64.58 [128.30]) compared to patients with euthyroidism, while the % Δ CAD Ach did not vary significantly between groups. After adjusting for covariates, females with hypothyroidism still had a significantly lower % Δ CBF Ach (estimated difference in % Δ CBF Ach [SE]: −16.79 [8.18]). Conclusions Hypothyroidism in women is associated with microvascular endothelial dysfunction, even after adjusting for confounders, and may explain some of the increased risk of cardiovascular disease in these patients. PMID:26224049

  2. miR-98 and let-7g* protect the blood-brain barrier under neuroinflammatory conditions.

    Science.gov (United States)

    Rom, Slava; Dykstra, Holly; Zuluaga-Ramirez, Viviana; Reichenbach, Nancy L; Persidsky, Yuri

    2015-12-01

    Pathologic conditions in the central nervous system, regardless of the underlying injury mechanism, show a certain level of blood-brain barrier (BBB) impairment. Endothelial dysfunction is the earliest event in the initiation of vascular damage caused by inflammation due to stroke, atherosclerosis, trauma, or brain infections. Recently, microRNAs (miRNAs) have emerged as a class of gene expression regulators. The relationship between neuroinflammation and miRNA expression in brain endothelium remains unexplored. Previously, we showed the BBB-protective and anti-inflammatory effects of glycogen synthase kinase (GSK) 3β inhibition in brain endothelium in in vitro and in vivo models of neuroinflammation. Using microarray screening, we identified miRNAs induced in primary human brain microvascular endothelial cells after exposure to the pro-inflammatory cytokine, tumor necrosis factor-α, with/out GSK3β inhibition. Among the highly modified miRNAs, let-7 and miR-98 were predicted to target the inflammatory molecules, CCL2 and CCL5. Overexpression of let-7 and miR-98 in vitro and in vivo resulted in reduced leukocyte adhesion to and migration across endothelium, diminished expression of pro-inflammatory cytokines, and increased BBB tightness, attenuating barrier 'leakiness' in neuroinflammation conditions. For the first time, we showed that miRNAs could be used as a therapeutic tool to prevent the BBB dysfunction in neuroinflammation.

  3. Functional Differences Between Placental Micro- and Macrovascular Endothelial Colony-Forming Cells

    Science.gov (United States)

    Solomon, Ioana; O’Reilly, Megan; Ionescu, Lavinia; Alphonse, Rajesh S.; Rajabali, Saima; Zhong, Shumei; Vadivel, Arul; Shelley, W. Chris; Yoder, Mervin C.

    2016-01-01

    Alterations in the development of the placental vasculature can lead to pregnancy complications, such as preeclampsia. Currently, the cause of preeclampsia is unknown, and there are no specific prevention or treatment strategies. Further insight into the placental vasculature may aid in identifying causal factors. Endothelial colony-forming cells (ECFCs) are a subset of endothelial progenitor cells capable of self-renewal and de novo vessel formation in vitro. We hypothesized that ECFCs exist in the micro- and macrovasculature of the normal, term human placenta. Human placentas were collected from term pregnancies delivered by cesarean section (n = 16). Placental micro- and macrovasculature was collected from the maternal and fetal side of the placenta, respectively, and ECFCs were isolated and characterized. ECFCs were CD31+, CD105+, CD144+, CD146+, CD14−, and CD45−, took up 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate-labeled acetylated low-density lipoprotein, and bound Ulex europaeus agglutinin 1. In vitro, macrovascular ECFCs had a greater potential to generate high-proliferative colonies and formed more complex capillary-like networks on Matrigel compared with microvascular ECFCs. In contrast, in vivo assessment demonstrated that microvascular ECFCs had a greater potential to form vessels. Macrovascular ECFCs were of fetal origin, whereas microvascular ECFCs were of maternal origin. ECFCs exist in the micro- and macrovasculature of the normal, term human placenta. Although macrovascular ECFCs demonstrated greater vessel and colony-forming potency in vitro, this did not translate in vivo, where microvascular ECFCs exhibited a greater vessel-forming ability. These important findings contribute to the current understanding of normal placental vascular development and may aid in identifying factors involved in preeclampsia and other pregnancy complications. Significance This research confirms that resident endothelial colony

  4. The interaction between circulating complement proteins and cutaneous microvascular endothelial cells in the development of childhood Henoch-Schonlein Purpura.

    Directory of Open Access Journals (Sweden)

    Yao-Hsu Yang

    Full Text Available In addition to IgA, the deposition of complement (C3 in dermal vessels is commonly found in Henoch-Schönlein purpura (HSP. The aim of this study is to elucidate the role of circulating complement proteins in the pathogenesis of childhood HSP.Plasma levels of C3a, C4a, C5a, and Bb in 30 HSP patients and 30 healthy controls were detected by enzyme-linked immunosorbent assay (ELISA. The expression of C3a receptor (C3aR, C5a receptor (CD88, E-selectin, intercellular adhesion molecule 1 (ICAM-1, C3, C5, interleukin (IL-8, monocyte chemotactic protein (MCP-1, and RANTES by human dermal microvascular endothelial cells (HMVEC-d was evaluated either by flow cytometry or by ELISA.At the acute stage, HSP patients had higher plasma levels of C3a (359.5 ± 115.3 vs. 183.3 ± 94.1 ng/ml, p < 0.0001, C5a (181.4 ± 86.1 vs. 33.7 ± 26.3 ng/ml, p < 0.0001, and Bb (3.7 ± 2.6 vs. 1.0 ± 0.6 μg/ml, p < 0.0001, but not C4a than healthy controls. Although HSP patient-derived acute phase plasma did not alter the presentation of C3aR and CD88 on HMVEC-d, it enhanced the production of endothelial C3 and C5. Moreover, C5a was shown in vitro to up-regulate the expression of IL-8, MCP-1, E-selectin, and ICAM-1 by HMVEC-d with a dose-dependent manner.In HSP, the activation of the complement system in part through the alternative pathway may have resulted in increased plasma levels of C3a and C5a, which, especially C5a, may play a role in the disease pathogenesis by activating endothelium of cutaneous small vessels.

  5. Longitudinal assessment of maternal endothelial function and markers of inflammation and placental function throughout pregnancy in lean and obese mothers.

    Science.gov (United States)

    Stewart, Frances M; Freeman, Dilys J; Ramsay, Jane E; Greer, Ian A; Caslake, Muriel; Ferrell, William R

    2007-03-01

    Obesity in pregnancy is increasing and is a risk factor for metabolic pathology such as preeclampsia. In the nonpregnant, obesity is associated with dyslipidemia, vascular dysfunction, and low-grade chronic inflammation. Our aim was to measure microvascular endothelial function in lean and obese pregnant women at intervals throughout their pregnancies and at 4 months after delivery. Plasma markers of endothelial function, inflammation, and placental function and their association with microvascular function were also assessed. Women in the 1st trimester of pregnancy were recruited, 30 with a body mass index (BMI) less than 30 kg/m(2) and 30 with a BMI more than or equal to 30 kg/m(2) matched for age, parity, and smoking status. In vivo endothelial-dependent and -independent microvascular function was measured using laser Doppler imaging in the 1st, 2nd, and 3rd trimesters of pregnancy and at 4 months postnatal. Plasma markers of endothelial activation [soluble intercellular cell adhesion molecule-1 (sVCAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), von Willebrand factor (vWF), and plasminogen activator inhibitor (PAI)-1], inflammation (IL-6, TNFalpha, C-reactive protein, and IL-10), and placental function (PAI-1/PAI-2 ratio) were also assessed at each time point. The pattern of improving endothelial function during pregnancy was the same for lean and obese, but endothelial-dependent vasodilation was significantly lower (P lean women but declined to near 1st trimester levels in the obese (lean/obese difference, 115%; P lean response being greater than obese (P = 0.021), and response declined in both groups in the postpartum period. In multivariate analysis, time of sampling had the most impact on endothelial-independent function [18.5% (adjusted sum of squares expressed as a percentage of total means squared), P lean 0.30 (0.21-0.47), P lean counterparts. There was a higher PAI-1/ PAI-2 ratio in the 1st trimester in obese women, which improved later in

  6. The MTT assays of bovine retinal pericytes and human microvascular endothelial cells on DLC and Si-DLC-coated TCPS.

    Science.gov (United States)

    Okpalugo, T I T; McKenna, E; Magee, A C; McLaughlin, J; Brown, N M D

    2004-11-01

    MTT (Tetrazolium)-assay suggests that diamond-like carbon (DLC) and silicon-doped DLC (Si-DLC) films obtained under appropriate deposition parameters are not toxic to bovine retinal pericytes, and human microvascular endothelial cells (HMEC). The observed frequency distributions of the optical density (OD) values indicative of cell viability are near Gaussian-normal distribution. One-way ANOVA indicates that at 0.05 levels the population means are not significantly different for the coated and control samples. The observed OD values depend on the cell line (cell growth/metabolic rate), possibly cell cycle stage, the deposition parameters-bias voltage, ion energy, pressure, argon precleaning, and the dopant. For colored thin films like DLC with room temperature photoconductivity and photoelectric effects, it is important to account for the OD contribution from the coating itself. MTT assay, not surprisingly, seems not to be highly sensitive to interfacial cellular interaction resulting from the change in the film's nanostructure, because the tetrazolium metabolism is mainly intracellular and not interfacial. The thin films were synthesized by 13.56 MHz RF-PECVD using argon and acetylene as source gases, with tetramethylsilane (TMS) vapor introduced for silicon doping. This study could be relevant to biomedical application of the films in the eye, peri-vascular, vascular compartments, and for cell-tissue engineering. (c) 2004 Wiley Periodicals, Inc.

  7. Pulmonary microvascular hyperpermeability and expression of vascular endothelial growth factor in smoke inhalation- and pneumonia-induced acute lung injury.

    Science.gov (United States)

    Lange, Matthias; Hamahata, Atsumori; Traber, Daniel L; Connelly, Rhykka; Nakano, Yoshimitsu; Traber, Lillian D; Schmalstieg, Frank C; Herndon, David N; Enkhbaatar, Perenlei

    2012-11-01

    Acute lung injury (ALI) and sepsis are major contributors to the morbidity and mortality of critically ill patients. The current study was designed further evaluate the mechanism of pulmonary vascular hyperpermeability in sheep with these injuries. Sheep were randomized to a sham-injured control group (n=6) or ALI/sepsis group (n=7). The sheep in the ALI/sepsis group received inhalation injury followed by instillation of Pseudomonas aeruginosa into the lungs. These groups were monitored for 24 h. Additional sheep (n=16) received the injury and lung tissue was harvested at different time points to measure lung wet/dry weight ratio, vascular endothelial growth factor (VEGF) mRNA and protein expression as well as 3-nitrotyrosine protein expression in lung homogenates. The injury induced severe deterioration in pulmonary gas exchange, increases in lung lymph flow and protein content, and lung water content (P<0.01 each). These alterations were associated with elevated lung and plasma nitrite/nitrate concentrations, increased tracheal blood flow, and enhanced VEGF mRNA and protein expression in lung tissue as well as enhanced 3-nitrotyrosine protein expression (P<0.05 each). This study describes the time course of pulmonary microvascular hyperpermeability in a clinical relevant large animal model and may improve the experimental design of future studies. Copyright © 2012 Elsevier Ltd and ISBI. All rights reserved.

  8. p130Cas scaffolds the signalosome to direct adaptor-effector cross talk during Kaposi's sarcoma-associated herpesvirus trafficking in human microvascular dermal endothelial cells.

    Science.gov (United States)

    Bandyopadhyay, Chirosree; Veettil, Mohanan Valiya; Dutta, Sujoy; Chandran, Bala

    2014-12-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with cell surface receptors, such as heparan sulfate, integrins (α3β1, αVβ3, and αVβ5), and EphrinA2 (EphA2), and activates focal adhesion kinase (FAK), Src, phosphoinositol 3-kinase (PI3-K), c-Cbl, and RhoA GTPase signal molecules early during lipid raft (LR)-dependent productive macropinocytic entry into human dermal microvascular endothelial cells. Our recent studies have identified CIB1 as a signal amplifier facilitating EphA2 phosphorylation and subsequent cytoskeletal cross talk during KSHV macropinocytosis. Although CIB1 lacks an enzymatic activity and traditional adaptor domain or known interacting sequence, it associated with the KSHV entry signal complex and the CIB1-KSHV association was sustained over 30 min postinfection. To identify factors scaffolding the EphA2-CIB1 signal axis, the role of major cellular scaffold protein p130Cas (Crk-associated substrate of Src) was investigated. Inhibitor and small interfering RNA (siRNA) studies demonstrated that KSHV induced p130Cas in an EphA2-, CIB1-, and Src-dependent manner. p130Cas and Crk were associated with KSHV, LRs, EphA2, and CIB1 early during infection. Live-cell microscopy and biochemical studies demonstrated that p130Cas knockdown did not affect KSHV entry but significantly reduced productive nuclear trafficking of viral DNA and routed KSHV to lysosomal degradation. p130Cas aided in scaffolding adaptor Crk to downstream guanine nucleotide exchange factor phospho-C3G possibly to coordinate GTPase signaling during KSHV trafficking. Collectively, these studies demonstrate that p130Cas acts as a bridging molecule between the KSHV-induced entry signal complex and the downstream trafficking signalosome in endothelial cells and suggest that simultaneous targeting of KSHV entry receptors with p130Cas would be an attractive potential avenue for therapeutic intervention in KSHV infection. Eukaryotic cell adaptor molecules, without any intrinsic

  9. Data set characterizing the systemic alterations of microvascular reactivity and capillary density, in patients presenting with infective endocarditis.

    Science.gov (United States)

    Tibirica, Eduardo; Barcelos, Amanda; Lamas, Cristiane

    2018-06-01

    This article represents data associated with a prior publication from our research group, under the title: Evaluation of microvascular endothelial function and capillary density in patients with infective endocarditis using laser speckle contrast imaging and video-capillaroscopy [1]. Patients with definite infective endocarditis, under stable clinical conditions, were prospectively included. The clinical and laboratory features are presented for each of them in raw form. Microvascular reactivity was evaluated using a laser speckle contrast imaging (LSCI) system with a laser wavelength of 785 nm. LSCI was used in combination with the iontophoresis of acetylcholine (ACh) or sodium nitroprusside (SNP) for the noninvasive, continuous measurement of cutaneous microvascular perfusion changes in arbitrary perfusion units (APU). The images were analyzed using the manufacturer's software. One skin site on the ventral surface of the forearm was chosen for the experiment. Microvascular reactivity was also evaluated using post-occlusive reactive hyperemia, whereby arterial occlusion was achieved with supra-systolic pressure (50 mmHg above the systolic arterial pressure) using a sphygmomanometer for three minutes. Following the release of pressure, maximum flux was measured. Data on cutaneous microvascular density were obtained using intravital video-capillaroscopy. The data obtained may be helpful by showing the usefulness of laser-based noninvasive techniques in systemic infectious diseases other than sepsis, in different clinical settings and countries.

  10. Small airway epithelial cells exposure to printer-emitted engineered nanoparticles induces cellular effects on human microvascular endothelial cells in an alveolar-capillary co-culture model.

    Science.gov (United States)

    Sisler, Jennifer D; Pirela, Sandra V; Friend, Sherri; Farcas, Mariana; Schwegler-Berry, Diane; Shvedova, Anna; Castranova, Vincent; Demokritou, Philip; Qian, Yong

    2015-01-01

    The printer is one of the most common office equipment. Recently, it was reported that toner formulations for printing equipment constitute nano-enabled products (NEPs) and contain engineered nanomaterials (ENMs) that become airborne during printing. To date, insufficient research has been performed to understand the potential toxicological properties of printer-emitted particles (PEPs) with several studies using bulk toner particles as test particles. These studies demonstrated the ability of toner particles to cause chronic inflammation and fibrosis in animal models. However, the toxicological implications of inhalation exposures to ENMs emitted from laser printing equipment remain largely unknown. The present study investigates the toxicological effects of PEPs using an in vitro alveolar-capillary co-culture model with Human Small Airway Epithelial Cells (SAEC) and Human Microvascular Endothelial Cells (HMVEC). Our data demonstrate that direct exposure of SAEC to low concentrations of PEPs (0.5 and 1.0 µg/mL) caused morphological changes of actin remodeling and gap formations within the endothelial monolayer. Furthermore, increased production of reactive oxygen species (ROS) and angiogenesis were observed in the HMVEC. Analysis of cytokine and chemokine levels demonstrates that interleukin (IL)-6 and MCP-1 may play a major role in the cellular communication observed between SAEC and HMVEC and the resultant responses in HMVEC. These data indicate that PEPs at low, non-cytotoxic exposure levels are bioactive and affect cellular responses in an alveolar-capillary co-culture model, which raises concerns for potential adverse health effects.

  11. Stimulation of phospholipase C in cultured microvascular endothelial cells from human frontal lobe by histamine, endothelin and purinoceptor agonists.

    Science.gov (United States)

    Purkiss, J. R.; West, D.; Wilkes, L. C.; Scott, C.; Yarrow, P.; Wilkinson, G. F.; Boarder, M. R.

    1994-01-01

    1. Cultures of endothelial cells derived from the microvasculature of human frontal lobe have been investigated for phospholipase C (PLC) responses to histamine, endothelins and purinoceptor agonists. 2. Using cells prelabelled with [3H]-inositol and measuring total [3H]-inositol (poly)phosphates, histamine acting at H1 receptors stimulated a substantial response with an EC50 of about 10 microM. 3. Endothelin-1 also gave a clear stimulation of phosphoinositide-specific phospholipase C. Both concentration-response curves and binding curves showed effective responses and binding in the rank order of endothelin-1 > sarafotoxin S6b > endothelin-3, suggesting an ETA receptor. 4. Assay of total [3H]-inositol (poly)phosphates showed no response to the purinoceptor agonists, 2-methylthioadenosine 5'-trisphosphate (2MeSATP), adenosine 5'-O-(3-thiotrisphosphate) (ATP gamma S) or beta,gamma-methylene ATP. Both ATP and UTP gave a small PLC response. 5. Similarly, when formation of [32P]-phosphatidic acid from cells prelabelled with 32Pi was used as an index of both PLC and phospholipase D, a small response to ATP and UTP was seen but there was no response to the other purinoceptor agonists tested. 6. Study by mass assay of stimulation by ATP of inositol (1,4,5) trisphosphate accumulation revealed a transient response in the first few seconds, a decline to basal, followed by a small sustained response. 7. These results show that human brain endothelial cells in culture are responsive to histamine and endothelins in a manner which may regulate brain capillary permeability. Purines exert a lesser influence. PMID:8032588

  12. Revisiting nanoparticle technology for blood-brain barrier transport: Unfolding at the endothelial gate improves the fate of transferrin receptor-targeted liposomes.

    Science.gov (United States)

    Johnsen, Kasper Bendix; Moos, Torben

    2016-01-28

    An unmet need exists for therapeutic compounds to traverse the brain capillary endothelial cells that denote the blood-brain barrier (BBB) to deliver effective treatment to the diseased brain. The use of nanoparticle technology for targeted delivery to the brain implies that targeted liposomes encapsulating a drug of interest will undergo receptor-mediated uptake and transport through the BBB with a subsequent unfolding of the liposomal content inside the brain, hence revealing drug release to adjacent drug-demanding neurons. As transferrin receptors (TfRs) are present on brain capillary endothelial, but not on endothelial cells elsewhere in the body, the use of TfR-targeted liposomes - colloidal particulates with a phospholipid bilayer membrane - remains the most relevant strategy to obtain efficient drug delivery to the brain. However, many studies have failed to provide sufficient quantitative data to proof passage of the BBB and significant appearance of drugs inside the brain parenchyma. Here, we critically evaluate the current evidence on the use of TfR-targeted liposomes for brain drug delivery based on a thorough investigation of all available studies within this research field. We focus on issues with respect to experimental design and data analysis that may provide an explanation to conflicting reports, and we discuss possible explanations for the current lack of sufficient transcytosis across the BBB for implementation in the design of TfR-targeted liposomes. We finally provide a list of suggestions for strategies to obtain substantial uptake and transport of drug carriers at the BBB with a concomitant transport of therapeutics into the brain. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. The endo-lysosomal system of brain endothelial cells is influenced by astrocytes in vitro

    DEFF Research Database (Denmark)

    Toth, Andrea E; Siupka, Piotr; P Augustine, Thomas J

    2018-01-01

    Receptor- and adsorptive-mediated transport through brain endothelial cells (BEC) of the blood-brain barrier (BBB) involves a complex array of subcellular vesicular structures, the endo-lysosomal system. It consists of several types of vesicles, such as early, recycling, and late endosomes......, retromer-positive structures, and lysosomes. Since this system is important for receptor-mediated transcytosis of drugs across brain capillaries, our aim was to characterise the endo-lysosomal system in BEC with emphasis on their interactions with astrocytes. We used primary porcine BEC in monoculture....... Altogether, our data pin-point unique features of BEC trafficking network, essentially mapping the endo-lysosomal system of in vitro BBB models. Consequently, our findings constitute a valuable basis for planning the optimal route across the BBB when advancing drug delivery to the brain....

  14. The reliability of a single protocol to determine endothelial, microvascular and autonomic functions in adolescents.

    Science.gov (United States)

    Bond, Bert; Williams, Craig A; Barker, Alan R

    2017-11-01

    Impairments in macrovascular, microvascular and autonomic function are present in asymptomatic youths with clustered cardiovascular disease risk factors. This study determines the within-day reliability and between-day reliability of a single protocol to non-invasively assess these outcomes in adolescents. Forty 12- to 15-year-old adolescents (20 boys) visited the laboratory in a fasted state on two occasions, approximately 1 week apart. One hour after a standardized cereal breakfast, macrovascular function was determined via flow-mediated dilation (FMD). Heart rate variability (root mean square of successive R-R intervals; RMSSD) was determined from the ECG-gated ultrasound images acquired during the FMD protocol prior to cuff occlusion. Microvascular function was simultaneously quantified as the peak (PRH) and total (TRH) hyperaemic response to occlusion in the cutaneous circulation of the forearm via laser Doppler imaging. To address within-day reliability, a subset of twenty adolescents (10 boys) repeated these measures 90 min afterwards on one occasion. The within-day typical error and between-day typical error expressed as a coefficient of variation of these outcomes are as follows: ratio-scaled FMD, 5·1% and 10·6%; allometrically scaled FMD, 4·4% and 9·4%; PRH, 11% and 13·3%; TRH, 29·9% and 23·1%; and RMSSD, 17·6% and 17·6%. The within- and between-day test-retest correlation coefficients for these outcomes were all significant (r > 0·54 for all). Macrovascular, microvascular and autonomic functions can be simultaneously and non-invasively determined in adolescents using a single protocol with an appropriate degree of reproducibility. Determining these outcomes may provide greater understanding of the progression of cardiovascular disease and aid early intervention. © 2016 Scandinavian Society of Clinical Physiology and Nuclear Medicine. Published by John Wiley & Sons Ltd.

  15. Brain endothelial cells control fertility through ovarian-steroid-dependent release of semaphorin 3A

    NARCIS (Netherlands)

    Giacobini, Paolo; Parkash, Jyoti; Campagne, Céline; Messina, Andrea; Casoni, Filippo; Vanacker, Charlotte; Langlet, Fanny; Hobo, Barbara; Cagnoni, Gabriella; Gallet, Sarah; Hanchate, Naresh Kumar; Mazur, Danièle; Taniguchi, Masahiko; Mazzone, Massimiliano; Verhaagen, J.; Ciofi, Philippe; Bouret, Sébastien G; Tamagnone, Luca; Prevot, Vincent

    Neuropilin-1 (Nrp1) guides the development of the nervous and vascular systems, but its role in the mature brain remains to be explored. Here we report that the expression of the 65 kDa isoform of Sema3A, the ligand of Nrp1, by adult vascular endothelial cells, is regulated during the ovarian cycle

  16. Anandamide inhibits Theiler's virus induced VCAM-1 in brain endothelial cells and reduces leukocyte transmigration in a model of blood brain barrier by activation of CB1 receptors

    Directory of Open Access Journals (Sweden)

    Loría Frida

    2011-08-01

    Full Text Available Abstract Background VCAM-1 represents one of the most important adhesion molecule involved in the transmigration of blood leukocytes across the blood-brain barrier (BBB that is an essential step in the pathogenesis of MS. Several evidences have suggested the potential therapeutic value of cannabinoids (CBs in the treatment of MS and their experimental models. However, the effects of endocannabinoids on VCAM-1 regulation are poorly understood. In the present study we investigated the effects of anandamide (AEA in the regulation of VCAM-1 expression induced by Theiler's virus (TMEV infection of brain endothelial cells using in vitro and in vivo approaches. Methods i in vitro: VCAM-1 was measured by ELISA in supernatants of brain endothelial cells infected with TMEV and subjected to AEA and/or cannabinoid receptors antagonist treatment. To evaluate the functional effect of VCAM-1 modulation we developed a blood brain barrier model based on a system of astrocytes and brain endothelial cells co-culture. ii in vivo: CB1 receptor deficient mice (Cnr1-/- infected with TMEV were treated with the AEA uptake inhibitor UCM-707 for three days. VCAM-1 expression and microglial reactivity were evaluated by immunohistochemistry. Results Anandamide-induced inhibition of VCAM-1 expression in brain endothelial cell cultures was mediated by activation of CB1 receptors. The study of leukocyte transmigration confirmed the functional relevance of VCAM-1 inhibition by AEA. In vivo approaches also showed that the inhibition of AEA uptake reduced the expression of brain VCAM-1 in response to TMEV infection. Although a decreased expression of VCAM-1 by UCM-707 was observed in both, wild type and CB1 receptor deficient mice (Cnr1-/-, the magnitude of VCAM-1 inhibition was significantly higher in the wild type mice. Interestingly, Cnr1-/- mice showed enhanced microglial reactivity and VCAM-1 expression following TMEV infection, indicating that the lack of CB1 receptor

  17. Molecular and functional characterization of riboflavin specific transport system in rat brain capillary endothelial cells

    Science.gov (United States)

    Patel, Mitesh; Vadlapatla, Ramya Krishna; Pal, Dhananjay; Mitra, Ashim K.

    2012-01-01

    Riboflavin is an important water soluble vitamin (B2) required for metabolic reactions, normal cellular growth, differentiation and function. Mammalian brain cells cannot synthesize riboflavin and must import from systemic circulation. However, the uptake mechanism, cellular translocation and intracellular trafficking of riboflavin in brain capillary endothelial cells are poorly understood. The primary objective of this study is to investigate the existence of riboflavin-specific transport system and delineate the uptake and intracellular regulation of riboflavin in immortalized rat brain capillary endothelial cells (RBE4). The uptake of [3H]-Riboflavin is sodium, temperature and energy dependent but pH independent. [3H]-Riboflavin uptake is saturable with Km and Vmax values of 19 ± 3 µM and 0.235 ± 0.012 picomoles/min/mg protein, respectively. The uptake process is inhibited by unlabelled structural analogs (lumiflavin, lumichrome) but not by structurally unrelated vitamins. Ca++/calmodulin and protein kinase A (PKA) pathways are found to play an important role in the intracellular regulation of [3H]-Riboflavin. Apical and baso-lateral uptake of [3H]-Riboflavin clearly indicate that riboflavin specific transport system is predominantly localized on the apical side of RBE4 cells. A 628 bp band corresponding to riboflavin transporter is revealed in RT-PCR analysis. These findings, for the first time report the existence of a specialized and high affinity transport system for riboflavin in RBE4 cells. Blood-brain barrier (BBB) is a major obstacle limiting drug transport inside the brain as it regulates drug permeation from systemic circulation. This transporter can be utilized for targeted delivery in enhancing brain permeation of highly potent drugs on systemic administration. PMID:22683359

  18. Endothelial progenitor cells in chronic obstructive pulmonary disease and emphysema

    Science.gov (United States)

    Tracy, Russell P.; Parikh, Megha A.; Hoffman, Eric A.; Shimbo, Daichi; Austin, John H. M.; Smith, Benjamin M.; Hueper, Katja; Vogel-Claussen, Jens; Lima, Joao; Gomes, Antoinette; Watson, Karol; Kawut, Steven; Barr, R. Graham

    2017-01-01

    Endothelial injury is implicated in the pathogenesis of COPD and emphysema; however the role of endothelial progenitor cells (EPCs), a marker of endothelial cell repair, and circulating endothelial cells (CECs), a marker of endothelial cell injury, in COPD and its subphenotypes is unresolved. We hypothesized that endothelial progenitor cell populations would be decreased in COPD and emphysema and that circulating endothelial cells would be increased. Associations with other subphenotypes were examined. The Multi-Ethnic Study of Atherosclerosis COPD Study recruited smokers with COPD and controls age 50–79 years without clinical cardiovascular disease. Endothelial progenitor cell populations (CD34+KDR+ and CD34+KDR+CD133+ cells) and circulating endothelial cells (CD45dimCD31+CD146+CD133-) were measured by flow cytometry. COPD was defined by standard spirometric criteria. Emphysema was assessed qualitatively and quantitatively on CT. Full pulmonary function testing and expiratory CTs were measured in a subset. Among 257 participants, both endothelial progenitor cell populations, and particularly CD34+KDR+ endothelial progenitor cells, were reduced in COPD. The CD34+KDR+CD133+ endothelial progenitor cells were associated inversely with emphysema extent. Both endothelial progenitor cell populations were associated inversely with extent of panlobular emphysema and positively with diffusing capacity. Circulating endothelial cells were not significantly altered in COPD but were inversely associated with pulmonary microvascular blood flow on MRI. There was no consistent association of endothelial progenitor cells or circulating endothelial cells with measures of gas trapping. These data provide evidence that endothelial repair is impaired in COPD and suggest that this pathological process is specific to emphysema. PMID:28291826

  19. Downregulation of blood-brain barrier phenotype by proinflammatory cytokines involves NADPH oxidase-dependent ROS generation: consequences for interendothelial adherens and tight junctions.

    Directory of Open Access Journals (Sweden)

    Keith D Rochfort

    Full Text Available Blood-brain barrier (BBB dysfunction is an integral feature of neurological disorders and involves the action of multiple proinflammatory cytokines on the microvascular endothelial cells lining cerebral capillaries. There is still however, considerable ambiguity throughout the scientific literature regarding the mechanistic role(s of cytokines in this context, thereby warranting a comprehensive in vitro investigation into how different cytokines may cause dysregulation of adherens and tight junctions leading to BBB permeabilization.The present study employs human brain microvascular endothelial cells (HBMvECs to compare/contrast the effects of TNF-α and IL-6 on BBB characteristics ranging from the expression of interendothelial junction proteins (VE-cadherin, occludin and claudin-5 to endothelial monolayer permeability. The contribution of cytokine-induced NADPH oxidase activation to altered barrier phenotype was also investigated.In response to treatment with either TNF-α or IL-6 (0-100 ng/ml, 0-24 hrs, our studies consistently demonstrated significant dose- and time-dependent decreases in the expression of all interendothelial junction proteins examined, in parallel with dose- and time-dependent increases in ROS generation and HBMvEC permeability. Increased expression and co-association of gp91 and p47, pivotal NADPH oxidase subunits, was also observed in response to either cytokine. Finally, cytokine-dependent effects on junctional protein expression, ROS generation and endothelial permeability could all be attenuated to a comparable extent using a range of antioxidant strategies, which included ROS depleting agents (superoxide dismutase, catalase, N-acetylcysteine, apocynin and targeted NADPH oxidase blockade (gp91 and p47 siRNA, NSC23766.A timely and wide-ranging investigation comparing the permeabilizing actions of TNF-α and IL-6 in HBMvECs is presented, in which we demonstrate how either cytokine can similarly downregulate the

  20. RCAN1.4 regulates VEGFR-2 internalisation, cell polarity and migration in human microvascular endothelial cells.

    Science.gov (United States)

    Alghanem, Ahmad F; Wilkinson, Emma L; Emmett, Maxine S; Aljasir, Mohammad A; Holmes, Katherine; Rothermel, Beverley A; Simms, Victoria A; Heath, Victoria L; Cross, Michael J

    2017-08-01

    Regulator of calcineurin 1 (RCAN1) is an endogenous inhibitor of the calcineurin pathway in cells. It is expressed as two isoforms in vertebrates: RCAN1.1 is constitutively expressed in most tissues, whereas transcription of RCAN1.4 is induced by several stimuli that activate the calcineurin-NFAT pathway. RCAN1.4 is highly upregulated in response to VEGF in human endothelial cells in contrast to RCAN1.1 and is essential for efficient endothelial cell migration and tubular morphogenesis. Here, we show that RCAN1.4 has a role in the regulation of agonist-stimulated VEGFR-2 internalisation and establishment of endothelial cell polarity. siRNA-mediated gene silencing revealed that RCAN1 plays a vital role in regulating VEGF-mediated cytoskeletal reorganisation and directed cell migration and sprouting angiogenesis. Adenoviral-mediated overexpression of RCAN1.4 resulted in increased endothelial cell migration. Antisense-mediated morpholino silencing of the zebrafish RCAN1.4 orthologue revealed a disrupted vascular development further confirming a role for the RCAN1.4 isoform in regulating vascular endothelial cell physiology. Our data suggest that RCAN1.4 plays a novel role in regulating endothelial cell migration by establishing endothelial cell polarity in response to VEGF.

  1. Mouse lung contains endothelial progenitors with high capacity to form blood and lymphatic vessels

    Directory of Open Access Journals (Sweden)

    Barleon Bernhard

    2010-07-01

    Full Text Available Abstract Background Postnatal endothelial progenitor cells (EPCs have been successfully isolated from whole bone marrow, blood and the walls of conduit vessels. They can, therefore, be classified into circulating and resident progenitor cells. The differentiation capacity of resident lung endothelial progenitor cells from mouse has not been evaluated. Results In an attempt to isolate differentiated mature endothelial cells from mouse lung we found that the lung contains EPCs with a high vasculogenic capacity and capability of de novo vasculogenesis for blood and lymph vessels. Mouse lung microvascular endothelial cells (MLMVECs were isolated by selection of CD31+ cells. Whereas the majority of the CD31+ cells did not divide, some scattered cells started to proliferate giving rise to large colonies (> 3000 cells/colony. These highly dividing cells possess the capacity to integrate into various types of vessels including blood and lymph vessels unveiling the existence of local microvascular endothelial progenitor cells (LMEPCs in adult mouse lung. EPCs could be amplified > passage 30 and still expressed panendothelial markers as well as the progenitor cell antigens, but not antigens for immune cells and hematopoietic stem cells. A high percentage of these cells are also positive for Lyve1, Prox1, podoplanin and VEGFR-3 indicating that a considerabe fraction of the cells are committed to develop lymphatic endothelium. Clonogenic highly proliferating cells from limiting dilution assays were also bipotent. Combined in vitro and in vivo spheroid and matrigel assays revealed that these EPCs exhibit vasculogenic capacity by forming functional blood and lymph vessels. Conclusion The lung contains large numbers of EPCs that display commitment for both types of vessels, suggesting that lung blood and lymphatic endothelial cells are derived from a single progenitor cell.

  2. miR-Let7A Controls the Cell Death and Tight Junction Density of Brain Endothelial Cells under High Glucose Condition.

    Science.gov (United States)

    Song, Juhyun; Yoon, So Ra; Kim, Oh Yoen

    2017-01-01

    Hyperglycemia-induced stress in the brain of patients with diabetes triggers the disruption of blood-brain barrier (BBB), leading to diverse neurological diseases including stroke and dementia. Recently, the role of microRNA becomes an interest in the research for deciphering the mechanism of brain endothelial cell damage under hyperglycemia. Therefore, we investigated whether mircoRNA Let7A (miR-Let7A) controls the damage of brain endothelial (bEnd.3) cells against high glucose condition. Cell viability, cell death marker expressions (p-53, Bax, and cleaved poly ADP-ribose polymerase), the loss of tight junction proteins (ZO-1 and claudin-5), proinflammatory response (interleukin-6, tumor necrosis factor- α ), inducible nitric oxide synthase, and nitrite production were confirmed using MTT, reverse transcription-PCR, quantitative-PCR, Western blotting, immunofluorescence, and Griess reagent assay. miR-Let7A overexpression significantly prevented cell death and loss of tight junction proteins and attenuated proinflammatory response and nitrite production in the bEnd.3 cells under high glucose condition. Taken together, we suggest that miR-Let7A may attenuate brain endothelial cell damage by controlling cell death signaling, loss of tight junction proteins, and proinflammatory response against high glucose stress. In the future, the manipulation of miR-Let7A may be a novel solution in controlling BBB disruption which leads to the central nervous system diseases.

  3. The blood-brain barrier fatty acid transport protein 1 (FATP1/SLC27A1) supplies docosahexaenoic acid to the brain, and insulin facilitates transport.

    Science.gov (United States)

    Ochiai, Yusuke; Uchida, Yasuo; Ohtsuki, Sumio; Tachikawa, Masanori; Aizawa, Sanshiro; Terasaki, Tetsuya

    2017-05-01

    We purposed to clarify the contribution of fatty acid transport protein 1 (FATP1/SLC 27A1) to the supply of docosahexaenoic acid (DHA) to the brain across the blood-brain barrier in this study. Transport experiments showed that the uptake rate of [ 14 C]-DHA in human FATP1-expressing HEK293 cells was significantly greater than that in empty vector-transfected (mock) HEK293 cells. The steady-state intracellular DHA concentration was nearly 2-fold smaller in FATP1-expressing than in mock cells, suggesting that FATP1 works as not only an influx, but also an efflux transporter for DHA. [ 14 C]-DHA uptake by a human cerebral microvascular endothelial cell line (hCMEC/D3) increased in a time-dependent manner, and was inhibited by unlabeled DHA and a known FATP1 substrate, oleic acid. Knock-down of FATP1 in hCMEC/D3 cells with specific siRNA showed that FATP1-mediated uptake accounts for 59.2-73.0% of total [ 14 C]-DHA uptake by the cells. Insulin treatment for 30 min induced translocation of FATP1 protein to the plasma membrane in hCMEC/D3 cells and enhanced [ 14 C]-DHA uptake. Immunohistochemical analysis of mouse brain sections showed that FATP1 protein is preferentially localized at the basal membrane of brain microvessel endothelial cells. We found that two neuroprotective substances, taurine and biotin, in addition to DHA, undergo FATP1-mediated efflux. Overall, our results suggest that FATP1 localized at the basal membrane of brain microvessels contributes to the transport of DHA, taurine and biotin into the brain, and insulin rapidly increases DHA supply to the brain by promoting translocation of FATP1 to the membrane. Read the Editorial Comment for this article on page 324. © 2016 International Society for Neurochemistry.

  4. Protective Effects of Scutellarin on Human Cardiac Microvascular Endothelial Cells against Hypoxia-Reoxygenation Injury and Its Possible Target-Related Proteins.

    Science.gov (United States)

    Shi, Meina; Liu, Yingting; Feng, Lixing; Cui, Yingbo; Chen, Yajuan; Wang, Peng; Wu, Wenjuan; Chen, Chen; Liu, Xuan; Yang, Weimin

    2015-01-01

    Scutellarin (SCU) is one of the main components of traditional Chinese medicine plant Erigeron breviscapus (Vant.) Hand.-Mazz. In this paper, we studied the protective effects of SCU on human cardiac microvascular endothelial cells (HCMECs) against hypoxia-reoxygenation (HR) injury and its possible target-related proteins. Results of MTT assay showed that pretreatment of SCU at doses of 1, 5, and 10 μM for 2 h could significantly inhibit the decrease in cell viability of HCMECs induced by HR injury. Subcellular fractions of cells treated with vehicle control, 1 μM SCU, HR injury, or 1 μM SCU + HR injury were separated by ultracentrifugation. The protein expression profiles of cytoplasm and membrane/nuclei fractions were checked using protein two-dimensional electrophoresis (2-DE). Proteins differentially expressed between control and SCU-treated group, control and HR group, or HR and SCU + HR group were identified using mass spectrometry (MS/MS). Possible interaction network of these target-related proteins was predicted using bioinformatic analysis. The influence of SCU on the expression levels of these proteins was confirmed using Western blotting assay. The results indicated that proteins such as p27BBP protein (EIF6), heat shock 60 kDa protein 1 (HSPD1), and chaperonin containing TCP1 subunit 6A isoform (CCT6A) might play important roles in the effects of SCU.

  5. Biosensor Technology Reveals the Disruption of the Endothelial Barrier Function and the Subsequent Death of Blood Brain Barrier Endothelial Cells to Sodium Azide and Its Gaseous Products.

    Science.gov (United States)

    Kho, Dan T; Johnson, Rebecca H; O'Carroll, Simon J; Angel, Catherine E; Graham, E Scott

    2017-09-21

    Herein we demonstrate the sensitive nature of human blood-brain barrier (BBB) endothelial cells to sodium azide and its gaseous product. Sodium azide is known to be acutely cytotoxic at low millimolar concentrations, hence its use as a biological preservative (e.g., in antibodies). Loss of barrier integrity was noticed in experiments using Electric Cell-substrate Impedance Sensing (ECIS) biosensor technology, to measure endothelial barrier integrity continuously in real-time. Initially the effect of sodium azide was observed as an artefact where it was present in antibodies being employed in neutralisation experiments. This was confirmed where antibody clones that were azide-free did not mediate loss of barrier function. A delayed loss of barrier function in neighbouring wells implied the influence of a liberated gaseous product. ECIS technology demonstrated that the BBB endothelial cells had a lower level of direct sensitivity to sodium azide of ~3 µM. Evidence of gaseous toxicity was consistently observed at 30 µM and above, with disrupted barrier function and cell death in neighbouring wells. We highlight the ability of this cellular biosensor technology to reveal both the direct and gaseous toxicity mediated by sodium azide. The sensitivity and temporal dimension of ECIS technology was instrumental in these observations. These findings have substantial implications for the wide use of sodium azide in biological reagents, raising issues of their application in live-cell assays and with regard to the protection of the user. This research also has wider relevance highlighting the sensitivity of brain endothelial cells to a known mitochondrial disruptor. It is logical to hypothesise that BBB endothelial dysfunction due to mitochondrial dys-regulation could have an important but underappreciated role in a range of neurological diseases.

  6. Three-dimensional culture conditions differentially affect astrocyte modulation of brain endothelial barrier function in response to transforming growth factor β1.

    Science.gov (United States)

    Hawkins, Brian T; Grego, Sonia; Sellgren, Katelyn L

    2015-05-22

    Blood-brain barrier (BBB) function is regulated by dynamic interactions among cell types within the neurovascular unit, including astrocytes and endothelial cells. Co-culture models of the BBB typically involve astrocytes seeded on two-dimensional (2D) surfaces, which recent studies indicate cause astrocytes to express a phenotype similar to that of reactive astrocytes in situ. We hypothesized that the culture conditions of astrocytes would differentially affect their ability to modulate BBB function in vitro. Brain endothelial cells were grown alone or in co-culture with astrocytes. Astrocytes were grown either as conventional (2D) monolayers, or in a collagen-based gel which allows them to grow in a three-dimensional (3D) construct. Astrocytes were viable in 3D conditions, and displayed a marked reduction in their expression of glial fibrillary acidic protein (GFAP), suggesting reduced activation. Stimulation of astrocytes with transforming growth factor (TGF)β1 decreased transendothelial electrical resistance (TEER) and reduced expression of claudin-5 in co-cultures, whereas treatment of endothelial cells in the absence of astrocytes was without effect. The effect of TGFβ1 on TEER was significantly more pronounced in endothelial cells cultured with 3D astrocytes compared to 2D astrocytes. These results demonstrate that astrocyte culture conditions differentially affect their ability to modulate brain endothelial barrier function, and suggest a direct relationship between reactive gliosis and BBB permeability. Moreover, these studies demonstrate the potential importance of physiologically relevant culture conditions to in vitro modeling of disease processes that affect the neurovascular unit. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Correlation of emmprin expression in vascular endothelial cells with blood-brain-barrier function: a study using magnetic resonance imaging enhanced by Gd-DTPA and immunohistochemistry in brain tumors.

    Science.gov (United States)

    Sameshima, Tetsuro; Nabeshima, Kazuki; Toole, Bryan P; Inoue, Teruhiko; Yokogami, Kiyotaka; Nakano, Shinichi; Ohi, Takekazu; Wakisaka, Shinichiro

    2003-06-01

    In a previous study, we demonstrated that the expression levels in tumor cells of emmprin (CD147) correlated with the grade of astrocytic tumors. Also, we found that emmprin was expressed in vascular endothelial cells of the non-neoplastic brain and hypothesized that emmprin expression could be associated with normal blood-brain-barrier (BBB) function of vascular endothelial cells. In this study, this possibility was examined in non-neoplastic brain, glioma and metastatic carcinoma tissues by comparing emmprin immunohistochemistry with gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) enhancement of magnetic resonance imaging (MRI), which is a clinical indicator of the BBB function. This study included 10 cases of non-neoplastic brain tissues, 7 of metastatic carcinoma, 7 of diffuse astrocytoma, 4 of anaplastic astrocytoma and 13 of glioblastoma multiforme. In all the cases, MRI with administration of Gd-DTPA was performed. The lesions were resected using the microdissection method with the help of ultrasonography and a neuronavigator. The tissues from Gd-DTPA-enhanced or non-enhanced areas were processed into frozen sections and subjected to immunohistochemistry with anti-emmprin antibody. The expression of emmprin in brain vascular endothelial cells inversely correlated with Gd-DTPA-enhancement of MRI: emmprin was positive in tissues not enhanced by Gd-DTPA and was negative in DTPA-enhanced tissues. Since BBB function presumably remains unimpaired in regions in which MR images are not Gd-DTPA-enhanced, emmprin expression appears to be associated with unimpaired BBB function. This is the first report to demonstrate a possible correlation between emmprin expression and BBB function in humans.

  8. Modelling the endothelial blood-CNS barriers: a method for the production of robust in vitro models of the rat blood-brain barrier and blood-spinal cord barrier.

    Science.gov (United States)

    Watson, P Marc D; Paterson, Judy C; Thom, George; Ginman, Ulrika; Lundquist, Stefan; Webster, Carl I

    2013-06-18

    Modelling the blood-CNS barriers of the brain and spinal cord in vitro continues to provide a considerable challenge for research studying the passage of large and small molecules in and out of the central nervous system, both within the context of basic biology and for pharmaceutical drug discovery. Although there has been considerable success over the previous two decades in establishing useful in vitro primary endothelial cell cultures from the blood-CNS barriers, no model fully mimics the high electrical resistance, low paracellular permeability and selective influx/efflux characteristics of the in vivo situation. Furthermore, such primary-derived cultures are typically labour-intensive and generate low yields of cells, limiting scope for experimental work. We thus aimed to establish protocols for the high yield isolation and culture of endothelial cells from both rat brain and spinal cord. Our aim was to optimise in vitro conditions for inducing phenotypic characteristics in these cells that were reminiscent of the in vivo situation, such that they developed into tight endothelial barriers suitable for performing investigative biology and permeability studies. Brain and spinal cord tissue was taken from the same rats and used to specifically isolate endothelial cells to reconstitute as in vitro blood-CNS barrier models. Isolated endothelial cells were cultured to expand the cellular yield and then passaged onto cell culture inserts for further investigation. Cell culture conditions were optimised using commercially available reagents and the resulting barrier-forming endothelial monolayers were characterised by functional permeability experiments and in vitro phenotyping by immunocytochemistry and western blotting. Using a combination of modified handling techniques and cell culture conditions, we have established and optimised a protocol for the in vitro culture of brain and, for the first time in rat, spinal cord endothelial cells. High yields of both CNS

  9. Sleep restriction impairs blood-brain barrier function.

    Science.gov (United States)

    He, Junyun; Hsuchou, Hung; He, Yi; Kastin, Abba J; Wang, Yuping; Pan, Weihong

    2014-10-29

    The blood-brain barrier (BBB) is a large regulatory and exchange interface between the brain and peripheral circulation. We propose that changes of the BBB contribute to many pathophysiological processes in the brain of subjects with chronic sleep restriction (CSR). To achieve CSR that mimics a common pattern of human sleep loss, we quantified a new procedure of sleep disruption in mice by a week of consecutive sleep recording. We then tested the hypothesis that CSR compromises microvascular function. CSR not only diminished endothelial and inducible nitric oxide synthase, endothelin1, and glucose transporter expression in cerebral microvessels of the BBB, but it also decreased 2-deoxy-glucose uptake by the brain. The expression of several tight junction proteins also was decreased, whereas the level of cyclooxygenase-2 increased. This coincided with an increase of paracellular permeability of the BBB to the small tracers sodium fluorescein and biotin. CSR for 6 d was sufficient to impair BBB structure and function, although the increase of paracellular permeability returned to baseline after 24 h of recovery sleep. This merits attention not only in neuroscience research but also in public health policy and clinical practice. Copyright © 2014 the authors 0270-6474/14/3414697-10$15.00/0.

  10. Decreased expression of serum and microvascular vascular endothelial growth factor receptor-2 in meningococcal sepsis*.

    NARCIS (Netherlands)

    Flier, M. van der; Baerveldt, E.M.; Miedema, A.; Hartwig, N.G.; Hazelzet, J.A.; Emonts, M.; Groot, R. de; Prens, E.P.; Vught, A.J. van; Jansen, N.J.

    2013-01-01

    OBJECTIVES: To determine the skin microvessel expression of vascular endothelial growth factor receptor 2 and serum-soluble vascular endothelial growth factor receptor 2 levels in children with meningococcal sepsis. DESIGN: Observational study. SETTING: Two tertiary academic children hospital PICUs.

  11. Ripk3 regulates cardiac microvascular reperfusion injury: The role of IP3R-dependent calcium overload, XO-mediated oxidative stress and F-action/filopodia-based cellular migration.

    Science.gov (United States)

    Zhou, Hao; Wang, Jin; Zhu, Pingjun; Hu, Shunying; Ren, Jun

    2018-05-01

    Ripk3-mediated cellular apoptosis is a major contributor to the pathogenesis of myocardial ischemia reperfusion (IR) injury. However, the mechanisms by which Ripk3 influences microvascular homeostasis and endothelial apoptosis are not completely understood. In this study, loss of Ripk3 inhibited endothelial apoptosis, alleviated luminal swelling, maintained microvasculature patency, reduced the expression of adhesion molecules and limited the myocardial inflammatory response. In vitro, Ripk3 deficiency protected endothelial cells from apoptosis and migratory arrest induced by HR injury. Mechanistically, Ripk3 had the ability to migrate onto the endoplasmic reticulum (ER), leading to ER damage, as evidenced by increased IP3R and XO expression. The higher IP3R content was associated with cellular calcium overload, and increased XO expression was involved in cellular oxidative injury. Furthermore, IP3R-mediated calcium overload and XO-dependent oxidative damage were able to initiate cellular apoptosis. More importantly, IP3R and XO also caused F-actin degradation into G-actin via post-transcriptional modification of cofilin, impairing the formation of the filopodia and limiting the migratory response of endothelial cells. Altogether, our data confirmed that Ripk3 was involved in microvascular IR injury via regulation of IP3R-mediated calcium overload, XO-dependent oxidative damage and filopodia-related cellular migration, ultimately leading to endothelial apoptosis and migratory inhibition. These findings provide a potential target for treating cardiac microcirculatory IR injury. Copyright © 2018 Elsevier Inc. All rights reserved.

  12. Constitutive production and thrombin-induced release of vascular endothelial growth factor by human megakaryocytes and platelets

    Science.gov (United States)

    Möhle, Robert; Green, David; Moore, Malcolm A. S.; Nachman, Ralph L.; Rafii, Shahin

    1997-01-01

    We have shown that coculture of bone marrow microvascular endothelial cells with hematopoietic progenitor cells results in proliferation and differentiation of megakaryocytes. In these long-term cultures, bone marrow microvascular endothelial cell monolayers maintain their cellular integrity in the absence of exogenous endothelial growth factors. Because this interaction may involve paracrine secretion of cytokines, we evaluated megakaryocytic cells for secretion of vascular endothelial growth factor (VEGF). Megakaryocytes (CD41a+) were generated by ex vivo expansion of hematopoietic progenitor cells with kit-ligand and thrombopoietin for 10 days and further purified with immunomagnetic microbeads. Using reverse transcription–PCR, we showed that megakaryocytic cell lines (Dami, HEL) and purified megakaryocytes expressed mRNA of the three VEGF isoforms (121, 165, and 189 amino acids). Large quantities of VEGF (>1 ng/106 cells/3 days) were detected in the supernatant of Dami cells, ex vivo-generated megakaryocytes, and CD41a+ cells isolated from bone marrow. The constitutive secretion of VEGF by CD41a+ cells was stimulated by growth factors of the megakaryocytic lineage (interleukin 3, thrombopoietin). Western blotting of heparin–Sepharose-enriched supernatant mainly detected the isoform VEGF165. In addition, immunohistochemistry showed intracytoplasmic VEGF in polyploid megakaryocytes. Thrombin stimulation of megakaryocytes and platelets resulted in rapid release of VEGF within 30 min. We conclude that human megakaryocytes produce and secrete VEGF in an inducible manner. Within the bone marrow microenvironment, VEGF secreted by megakaryocytes may contribute to the proliferation of endothelial cells. VEGF delivered to sites of vascular injury by activated platelets may initiate angiogenesis. PMID:9012841

  13. Endothelium-dependent vasodilatation, plasma markers of endothelial function, and adrenergic vasoconstrictor responses in type 1 diabetes under near-normoglycemic conditions

    NARCIS (Netherlands)

    Huvers, F C; De Leeuw, P W; Houben, A J; De Haan, C H; Hamulyak, K; Schouten, H; Wolffenbuttel, B H; Schaper, N C

    It is unknown whether and to what extent changes in various endothelial functions and adrenergic responsiveness are related to the development of microvascular complications in type 1 diabetes. Therefore, endothelium-dependent and endothelium-independent vasodilatation, endothelium-dependent

  14. Characterization of TEM1/endosialin in human and murine brain tumors

    International Nuclear Information System (INIS)

    Carson-Walter, Eleanor B; Walter, Kevin A; Winans, Bethany N; Whiteman, Melissa C; Liu, Yang; Jarvela, Sally; Haapasalo, Hannu; Tyler, Betty M; Huso, David L; Johnson, Mahlon D

    2009-01-01

    TEM1/endosialin is an emerging microvascular marker of tumor angiogenesis. We characterized the expression pattern of TEM1/endosialin in astrocytic and metastatic brain tumors and investigated its role as a therapeutic target in human endothelial cells and mouse xenograft models. In situ hybridization (ISH), immunohistochemistry (IH) and immunofluorescence (IF) were used to localize TEM1/endosialin expression in grade II-IV astrocytomas and metastatic brain tumors on tissue microarrays. Changes in TEM1/endosialin expression in response to pro-angiogenic conditions were assessed in human endothelial cells grown in vitro. Intracranial U87MG glioblastoma (GBM) xenografts were analyzed in nude TEM1/endosialin knockout (KO) and wildtype (WT) mice. TEM1/endosialin was upregulated in primary and metastatic human brain tumors, where it localized primarily to the tumor vasculature and a subset of tumor stromal cells. Analysis of 275 arrayed grade II-IV astrocytomas demonstrated TEM1/endosialin expression in 79% of tumors. Robust TEM1/endosialin expression occurred in 31% of glioblastomas (grade IV astroctyomas). TEM1/endosialin expression was inversely correlated with patient age. TEM1/endosialin showed limited co-localization with CD31, αSMA and fibronectin in clinical specimens. In vitro, TEM1/endosialin was upregulated in human endothelial cells cultured in matrigel. Vascular Tem1/endosialin was induced in intracranial U87MG GBM xenografts grown in mice. Tem1/endosialin KO vs WT mice demonstrated equivalent survival and tumor growth when implanted with intracranial GBM xenografts, although Tem1/endosialin KO tumors were significantly more vascular than the WT counterparts. TEM1/endosialin was induced in the vasculature of high-grade brain tumors where its expression was inversely correlated with patient age. Although lack of TEM1/endosialin did not suppress growth of intracranial GBM xenografts, it did increase tumor vascularity. The cellular localization of TEM1

  15. The TAM receptor Mertk protects against neuroinvasive viral infection by maintaining blood-brain barrier integrity.

    Science.gov (United States)

    Miner, Jonathan J; Daniels, Brian P; Shrestha, Bimmi; Proenca-Modena, Jose L; Lew, Erin D; Lazear, Helen M; Gorman, Matthew J; Lemke, Greg; Klein, Robyn S; Diamond, Michael S

    2015-12-01

    The TAM receptors Tyro3, Axl and Mertk are receptor tyrosine kinases that dampen host innate immune responses following engagement with their ligands Gas6 and Protein S, which recognize phosphatidylserine on apoptotic cells. In a form of apoptotic mimicry, many enveloped viruses display phosphatidylserine on the outer leaflet of their membranes, enabling TAM receptor activation and downregulation of antiviral responses. Accordingly, we hypothesized that a deficiency of TAM receptors would enhance antiviral responses and protect against viral infection. Unexpectedly, mice lacking Mertk and/or Axl, but not Tyro3, exhibited greater vulnerability to infection with neuroinvasive West Nile and La Crosse encephalitis viruses. This phenotype was associated with increased blood-brain barrier permeability, which enhanced virus entry into and infection of the brain. Activation of Mertk synergized with interferon-β to tighten cell junctions and prevent virus transit across brain microvascular endothelial cells. Because TAM receptors restrict pathogenesis of neuroinvasive viruses, these findings have implications for TAM antagonists that are currently in clinical development.

  16. Hypoxic stress up-regulates Kir2.1 expression and facilitates cell proliferation in brain capillary endothelial cells

    International Nuclear Information System (INIS)

    Yamamura, Hideto; Suzuki, Yoshiaki; Yamamura, Hisao; Asai, Kiyofumi; Imaizumi, Yuji

    2016-01-01

    The blood-brain barrier (BBB) is mainly composed of brain capillary endothelial cells (BCECs), astrocytes and pericytes. Brain ischemia causes hypoxic encephalopathy and damages BBB. However, it remains still unclear how hypoxia affects BCECs. In the present study, t-BBEC117 cells, an immortalized bovine brain endothelial cell line, were cultured under hypoxic conditions at 4–5% oxygen for 72 h. This hypoxic stress caused hyperpolarization of resting membrane potential. Patch-clamp recordings revealed a marked increase in Ba 2+ -sensitive inward rectifier K + current in t-BBEC117 cells after hypoxic culture. Western blot and real-time PCR analyses showed that Kir2.1 expression was significantly up-regulated at protein level but not at mRNA level after the hypoxic culture. Ca 2+ imaging study revealed that the hypoxic stress enhanced store-operated Ca 2+ (SOC) entry, which was significantly reduced in the presence of 100 μM Ba 2+ . On the other hand, the expression of SOC channels such as Orai1, Orai2, and transient receptor potential channels was not affected by hypoxic stress. MTT assay showed that the hypoxic stress significantly enhanced t-BBEC117 cell proliferation, which was inhibited by approximately 60% in the presence of 100 μM Ba 2+ . We first show here that moderate cellular stress by cultivation under hypoxic conditions hyperpolarizes membrane potential via the up-regulation of functional Kir2.1 expression and presumably enhances Ca 2+ entry, resulting in the facilitation of BCEC proliferation. These findings suggest potential roles of Kir2.1 expression in functional changes of BCECs in BBB following ischemia. -- Highlights: •Hypoxic culture of brain endothelial cells (BEC) caused membrane hyperpolarization. •This hyperpolarization was due to the increased expression of Kir2.1 channels. •Hypoxia enhanced store-operated Ca 2+ (SOC) entry via Kir2.1 up-regulation. •Expression levels of putative SOC channels were not affected by hypoxia.

  17. Fingolimod (FTY720-P Does Not Stabilize the Blood–Brain Barrier under Inflammatory Conditions in an in Vitro Model

    Directory of Open Access Journals (Sweden)

    Michael K. Schuhmann

    2015-12-01

    Full Text Available Breakdown of the blood-brain barrier (BBB is an early hallmark of multiple sclerosis (MS, a progressive inflammatory disease of the central nervous system. Cell adhesion in the BBB is modulated by sphingosine-1-phosphate (S1P, a signaling protein, via S1P receptors (S1P1. Fingolimod phosphate (FTY720-P a functional S1P1 antagonist has been shown to improve the relapse rate in relapsing-remitting MS by preventing the egress of lymphocytes from lymph nodes. However, its role in modulating BBB permeability—in particular, on the tight junction proteins occludin, claudin 5 and ZO-1—has not been well elucidated to date. In the present study, FTY720-P did not change the transendothelial electrical resistance in a rat brain microvascular endothelial cell (RBMEC culture exposed to inflammatory conditions and thus did not decrease endothelial barrier permeability. In contrast, occludin was reduced in RBMEC culture after adding FTY720-P. Additionally, FTY720-P did not alter the amount of endothelial matrix metalloproteinase (MMP-9 and MMP-2 in RBMEC cultures. Taken together, our observations support the assumption that S1P1 plays a dual role in vascular permeability, depending on its ligand. Thus, S1P1 provides a mechanistic basis for FTY720-P-associated disruption of endothelial barriers—such as the blood-retinal barrier—which might result in macular edema.

  18. Vascular endothelial growth factor-A determines detectability of experimental melanoma brain metastasis in GD-DTPA-enhanced MRI.

    NARCIS (Netherlands)

    Leenders, W.P.J.; Kusters, B.; Pikkemaat, J.A.; Wesseling, P.; Ruiter, D.J.; Heerschap, A.; Barentsz, J.O.; Waal, R.M.W. de

    2003-01-01

    We have previously shown that the dense vascular network in mouse brain allows for growth of human melanoma xenografts (Mel57) by co-option of preexisting vessels. Overexpression of recombinant vascular endothelial growth factor-A (VEGF-A) by such xenografts induced functional and morphologic

  19. Short-term hyperglycemia increases endothelial glycocalyx permeability and acutely decreases lineal density of capillaries with flowing red blood cells

    NARCIS (Netherlands)

    Zuurbier, Coert J.; Demirci, Cihan; Koeman, Anneke; Vink, Hans; Ince, Can

    2005-01-01

    Hyperglycemia is becoming recognized as an important risk factor for microvascular dysfunction. We hypothesized that short-term hyperglycemia, either on the scale of hours or weeks, alters the barrier function and the volume of the endothelial glycocalyx and decreases functional capillary density

  20. Radioprotection of mouse CNS endothelial cells in vivo

    International Nuclear Information System (INIS)

    Lyubimova, N.; Coultas, P.; Martin, R.

    1996-01-01

    Full text: Radioprotection using the minor groove binding DNA ligand Hoechst 33342 has been demonstrated in vitro, and more recently in vivo, in mouse lung. Intravenous administration was used for the lung studies, and both endothelial and alveolar epithelial cells-showed good up-take. Radiation damage to the endothelial cell population has also been postulated as important in late developing radionecrosis of spinal cord and brain. Endothelial cell density in brain can be readily determined by a fluorescent-histochemical technique. Treatment with a monoamine oxidase inhibitor and subsequent injection with L-DOPA results in an accumulation of dopamine (DA) in CNS endothelial cells. DA is converted to a fluorophore by exposure to paraformaldehyde, and cell numbers assayed by fluorescence microscopy. Earlier studies used this technique to monitor post-irradiation changes in endothelial cell density in rodent brain and showed the loss, within 24 hours, of a sensitive subpopulation comprising about 15% of the endothelial cells. Ten minutes after intravenous injection of Hoechst 33342 (80mg/kg) the ligand is confined by its limited penetration to the endothelial cells in mouse brain. When we irradiated at this time, there was protection against early endothelial cell loss. Ablation of the sensitive subpopulation in unprotected mice takes place over a dose range of 1 to 3 Gy γ-rays, but doses between 12 to 20 Gy are required in the presence of ligand. This protection equates to a very high dose modification factor of about 7 and possibly reflects a suppression of apoptosis in the sensitive endothelial subpopulation. The extent to which there is enhanced survival in the endothelial population as a whole and how the observed protection affects late CNS necrosis development has yet to be determined. However present results clearly show potential for the use of DNA-binding radioprotectors with limited penetration for investigations into the relative significance of

  1. Invasive assessment of coronary microvascular dysfunction in hypertrophic cardiomyopathy: the index of microvascular resistance

    International Nuclear Information System (INIS)

    Gutiérrez-Barrios, Alejandro; Camacho-Jurado, Francisco; Díaz-Retamino, Enrique; Gamaza-Chulián, Sergio; Agarrado-Luna, Antonio; Oneto-Otero, Jesús; Del Rio-Lechuga, Ana; Benezet-Mazuecos, Javier

    2015-01-01

    Summary: We present a review of microvascular dysfunction in hypertrophic cardiomyopathy (HCM) and an interesting case of a symptomatic familial HCM patient with inducible ischemia by single photon emission computed tomography. Coronary angiography revealed normal epicardial arteries. Pressure wire measurements of fractional flow reserve (FFR), coronary flow reserve (CFR) and index of microvascular resistance (IMR) demonstrated a significant microcirculatory dysfunction. This is the first such case that documents this abnormality invasively using the IMR. The measurement of IMR, a novel marker of microcirculatory dysfunction, provides novel insights into the pathophysiology of this condition. - Highlights: • Microvascular dysfunction is a common feature in hypertrophic cardiomyopathy (HCM) and represents a strong predictor of unfavorable outcome and cardiovascular mortality. • The index of microvascular resistance (IMR) is a new method for invasively assessing the state of the coronary microcirculation using a single pressure-temperature sensor-tipped coronary wire. • However assessment of IMR in HCM has not been previously reported. We report a case in which microvascular dysfunction is assessed by IMR. This index may be useful in future researches of HCM.

  2. Invasive assessment of coronary microvascular dysfunction in hypertrophic cardiomyopathy: the index of microvascular resistance

    Energy Technology Data Exchange (ETDEWEB)

    Gutiérrez-Barrios, Alejandro, E-mail: aleklos@hotmail.com [Cardiology Department, Jerez Hospital, Jerez (Spain); Camacho-Jurado, Francisco [Cardiology Department, Punta Europa Hospital, Algeciras (Spain); Díaz-Retamino, Enrique; Gamaza-Chulián, Sergio; Agarrado-Luna, Antonio; Oneto-Otero, Jesús; Del Rio-Lechuga, Ana; Benezet-Mazuecos, Javier [Cardiology Department, Jerez Hospital, Jerez (Spain)

    2015-10-15

    Summary: We present a review of microvascular dysfunction in hypertrophic cardiomyopathy (HCM) and an interesting case of a symptomatic familial HCM patient with inducible ischemia by single photon emission computed tomography. Coronary angiography revealed normal epicardial arteries. Pressure wire measurements of fractional flow reserve (FFR), coronary flow reserve (CFR) and index of microvascular resistance (IMR) demonstrated a significant microcirculatory dysfunction. This is the first such case that documents this abnormality invasively using the IMR. The measurement of IMR, a novel marker of microcirculatory dysfunction, provides novel insights into the pathophysiology of this condition. - Highlights: • Microvascular dysfunction is a common feature in hypertrophic cardiomyopathy (HCM) and represents a strong predictor of unfavorable outcome and cardiovascular mortality. • The index of microvascular resistance (IMR) is a new method for invasively assessing the state of the coronary microcirculation using a single pressure-temperature sensor-tipped coronary wire. • However assessment of IMR in HCM has not been previously reported. We report a case in which microvascular dysfunction is assessed by IMR. This index may be useful in future researches of HCM.

  3. Plasmalemmal Vesicle Associated Protein-1 (PV-1 is a marker of blood-brain barrier disruption in rodent models

    Directory of Open Access Journals (Sweden)

    Ali Zarina S

    2008-02-01

    Full Text Available Abstract Background Plasmalemmal vesicle associated protein-1 (PV-1 is selectively expressed in human brain microvascular endothelial cells derived from clinical specimens of primary and secondary malignant brain tumors, cerebral ischemia, and other central nervous system (CNS diseases associated with blood-brain barrier breakdown. In this study, we characterize the murine CNS expression pattern of PV-1 to determine whether localized PV-1 induction is conserved across species and disease state. Results We demonstrate that PV-1 is selectively upregulated in mouse blood vessels recruited by brain tumor xenografts at the RNA and protein levels, but is not detected in non-neoplastic brain. Additionally, PV-1 is induced in a mouse model of acute ischemia. Expression is confined to the cerebovasculature within the region of infarct and is temporally regulated. Conclusion Our results confirm that PV-1 is preferentially induced in the endothelium of mouse brain tumors and acute ischemic brain tissue and corresponds to blood-brain barrier disruption in a fashion analogous to human patients. Characterization of PV-1 expression in mouse brain is the first step towards development of rodent models for testing anti-edema and anti-angiogenesis therapeutic strategies based on this molecule.

  4. Transcranial diffuse optical monitoring of microvascular cerebral hemodynamics after thrombolysis in ischemic stroke

    Science.gov (United States)

    Zirak, Peyman; Delgado-Mederos, Raquel; Dinia, Lavinia; Carrera, David; Martí-Fàbregas, Joan; Durduran, Turgut

    2014-01-01

    The ultimate goal of therapeutic strategies for ischemic stroke is to reestablish the blood flow to the ischemic region of the brain. However, currently, the local cerebral hemodynamics (microvascular) is almost entirely inaccessible for stroke clinicians at the patient bed-side, and the recanalization of the major cerebral arteries (macrovascular) is the only available measure to evaluate the therapy, which does not always reflect the local conditions. Here we report the case of an ischemic stroke patient whose microvascular cerebral blood flow and oxygenation were monitored by a compact hybrid diffuse optical monitor during thrombolytic therapy. This monitor combined diffuse correlation spectroscopy and near-infrared spectroscopy. The reperfusion assessed by hybrid diffuse optics temporally correlated with the recanalization of the middle cerebral artery (assessed by transcranial-Doppler) and was in agreement with the patient outcome. This study suggests that upon further investigation, diffuse optics might have a potential for bed-side acute stroke monitoring and therapy guidance by providing hemodynamics information at the microvascular level.

  5. Cutaneous chronic graft-versus-host disease does not have the abnormal endothelial phenotype or vascular rarefaction characteristic of systemic sclerosis.

    Directory of Open Access Journals (Sweden)

    Jo Nadine Fleming

    2009-07-01

    Full Text Available The clinical and histologic appearance of fibrosis in cutaneous lesions in chronic graft-versus -host disease (c-GVHD resembles the appearance of fibrosis in scleroderma (SSc. Recent studies identified distinctive structural changes in the superficial dermal microvasculature and matrix of SSc skin. We compared the dermal microvasculature in human c-GVHD to SSc to determine if c-GVHD is a suitable model for SSc.We analyzed skin biopsies of normal controls (n = 24, patients with SSc (n = 30 and c-GVHD with dermal fibrosis (n = 133. Immunostaining was employed to identify vessels, vascular smooth muscle, dermal matrix, and cell proliferation. C-GVHD and SSc had similar dermal matrix composition and vascular smooth muscle pathology, including intimal hyperplasia. SSc, however, differed significantly from c-GVHD in three ways. First, there were significantly fewer (p = 0.00001 average vessels in SSc biopsies (9.8 when compared with c-GVHD (16.5. Second, in SSc, endothelial markers were decreased significantly (19/19 and 12/14 for VE cadherin and vWF (p = <0.0001 and <0.05, respectively. In contrast, 0/13 c-GVHD biopsies showed loss of staining with canonical endothelial markers. Third, c-GVHD contained areas of microvascular endothelial proliferation not present in the SSc biopsies.The sclerosis associated with c-GVHD appears to resemble wound healing. Focal capillary proliferation occurs in early c-GVHD. In contrast, loss of canonical endothelial markers and dermal capillaries is seen in SSc, but not in c-GVHD. The loss of VE cadherin in SSc, in particular, may be related to microvascular rarefaction because VE cadherin is necessary for angiogenesis. C-GVHD is a suitable model for studying dermal fibrosis but may not be applicable for studying the microvascular alterations characteristic of SSc.

  6. Regulation of cyclic AMP by extracellular ATP in cultured brain capillary endothelial cells

    Science.gov (United States)

    Anwar, Zubeya; Albert, Jennifer L; Gubby, Sharon E; Boyle, John P; Roberts, Jonathon A; Webb, Tania E; Boarder, Michael R

    1999-01-01

    In primary unpassaged rat brain capillary endothelial cell cultures (RBECs), using reverse-transcriptase PCR with primers specific for P2Y receptor subtypes, we detected mRNA for P2Y2, P2Y4 and P2Y6, but not P2Y1 receptors.None of the various nucleotides tested reduced forskolin elevated cyclic AMP levels in RBECs. ATP and ATPγS, as well as adenosine, enhanced cyclic AMP accumulation in the presence of forskolin.Comparison of the concentration response curves to ATPγS with those for ATP and adenosine, at different incubation times, indicated that the response to purine nucleotides was not wholly dependent on conversion to adenosine. Adenosine deaminase abolished the response to adenosine but only reduced the response to ATP by about 50%. These results suggest the participation of a receptor responsive to nucleotides.Isobutylmethylxanthine and 8-sulphophenyltheophylline prevented the cyclic AMP response, while neither 8-cyclopentyl-1,3-dipropylxanthine nor SCH58261 were effective antagonists. 2-chloradenosine gave a robust response, but neither 2-chloro-N6-cyclopentyladenosine nor CGS 21680 were agonists.These results show that adenosine and ATP can elevate the cyclic AMP levels of brain endothelial cells by acting on receptors which have a pharmacology apparently distinct from known P2Y and adenosine receptors. PMID:10510459

  7. 2,3,7,8-TCDD exposure, endothelial dysfunction and impaired microvascular reactivity

    Czech Academy of Sciences Publication Activity Database

    Pelclová, D.; Prázdný, M.; Škrha, J.; Fenclová, Z.; Kalousová, M.; Urban, P.; Navrátil, Tomáš; Šenholdová, Z.; Šmerhovský, Z.

    2007-01-01

    Roč. 26, - (2007), s. 705-713 ISSN 0960-3271 Institutional research plan: CEZ:AV0Z40400503 Keywords : 2,3,7,8-TCDD * endothelial dysfunction * oxidative stress * superoxide dismutase Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 1.335, year: 2007

  8. In-vivo assessment of microvascular functional dynamics by combination of cmOCT and wavelet transform

    Science.gov (United States)

    Smirni, Salvatore; MacDonald, Michael P.; Robertson, Catherine P.; McNamara, Paul M.; O'Gorman, Sean; Leahy, Martin J.; Khan, Faisel

    2018-02-01

    The cutaneous microcirculation represents an index of the health status of the cardiovascular system. Conventional methods to evaluate skin microvascular function are based on measuring blood flow by laser Doppler in combination with reactive tests such as post-occlusive reactive hyperaemia (PORH). Moreover, the spectral analysis of blood flow signals by continuous wavelet transform (CWT) reveals nonlinear oscillations reflecting the functionality of microvascular biological factors, e.g. endothelial cells (ECs). Correlation mapping optical coherence tomography (cmOCT) has been previously described as an efficient methodology for the morphological visualisation of cutaneous micro-vessels. Here, we show that cmOCT flow maps can also provide information on the functional components of the microcirculation. A spectral domain optical coherence tomography (SD-OCT) imaging system was used to acquire 90 sequential 3D OCT volumes from the forearm of a volunteer, while challenging the micro-vessels with a PORH test. The volumes were sampled in a temporal window of 25 minutes, and were processed by cmOCT to obtain flow maps at different tissue depths. The images clearly show changes of flow in response to the applied stimulus. Furthermore, a blood flow signal was reconstructed from cmOCT maps intensities to investigate the microvascular nonlinear dynamics by CWT. The analysis revealed oscillations changing in response to PORH, associated with the activity of ECs and the sympathetic innervation. The results demonstrate that cmOCT may be potentially used as diagnostic tool for the assessment of microvascular function, with the advantage of also providing spatial resolution and structural information compared to the traditional laser Doppler techniques.

  9. Protective Effects of Scutellarin on Human Cardiac Microvascular Endothelial Cells against Hypoxia-Reoxygenation Injury and Its Possible Target-Related Proteins

    Directory of Open Access Journals (Sweden)

    Meina Shi

    2015-01-01

    Full Text Available Scutellarin (SCU is one of the main components of traditional Chinese medicine plant Erigeron breviscapus (Vant. Hand.-Mazz. In this paper, we studied the protective effects of SCU on human cardiac microvascular endothelial cells (HCMECs against hypoxia-reoxygenation (HR injury and its possible target-related proteins. Results of MTT assay showed that pretreatment of SCU at doses of 1, 5, and 10 μM for 2 h could significantly inhibit the decrease in cell viability of HCMECs induced by HR injury. Subcellular fractions of cells treated with vehicle control, 1 μM SCU, HR injury, or 1 μM SCU + HR injury were separated by ultracentrifugation. The protein expression profiles of cytoplasm and membrane/nuclei fractions were checked using protein two-dimensional electrophoresis (2-DE. Proteins differentially expressed between control and SCU-treated group, control and HR group, or HR and SCU + HR group were identified using mass spectrometry (MS/MS. Possible interaction network of these target-related proteins was predicted using bioinformatic analysis. The influence of SCU on the expression levels of these proteins was confirmed using Western blotting assay. The results indicated that proteins such as p27BBP protein (EIF6, heat shock 60 kDa protein 1 (HSPD1, and chaperonin containing TCP1 subunit 6A isoform (CCT6A might play important roles in the effects of SCU.

  10. Peripheral Endothelial Function and Coronary Flow Velocity Reserve Are Not Associated in Women with Angina and No Obstructive Coronary Artery Disease

    DEFF Research Database (Denmark)

    Flintholm Raft, Kristoffer; Frestad, Daria; Michelsen, Marie Mide

    2017-01-01

    PURPOSE: We investigated whether impaired flow-mediated dilation (FMD) and plasma biomarkers reflecting endothelial dysfunction are associated with coronary microvascular dysfunction (CMD) in women with angina and no obstructive coronary artery disease (CAD). METHODS: Patients (n = 194) were rand...

  11. Transfection of primary brain capillary endothelial cells for protein synthesis and secretion of recombinant erythropoietin: a strategy to enable protein delivery to the brain.

    Science.gov (United States)

    Burkhart, Annette; Andresen, Thomas Lars; Aigner, Achim; Thomsen, Louiza Bohn; Moos, Torben

    2017-07-01

    Treatment of chronic disorders affecting the central nervous system (CNS) is complicated by the inability of drugs to cross the blood-brain barrier (BBB). Non-viral gene therapy applied to brain capillary endothelial cells (BCECs) denotes a novel approach to overcome the restraints in this passage, as turning BCECs into recombinant protein factories by transfection could result in protein secretion further into the brain. The present study aims to investigate the possibility of transfecting primary rat brain endothelial cells (RBECs) for recombinant protein synthesis and secretion of the neuroprotective protein erythropoietin (EPO). We previously showed that 4% of RBECs with BBB properties can be transfected without disrupting the BBB integrity in vitro, but it can be questioned whether this is sufficient to enable protein secretion at therapeutic levels. The present study examined various transfection vectors, with regard to increasing the transfection efficiency without disrupting the BBB integrity. Lipofectamine 3000™ was the most potent vector compared to polyethylenimine (PEI) and Turbofect. When co-cultured with astrocytes, the genetically modified RBECs secreted recombinant EPO into the cell culture medium both luminally and abluminally, and despite lower levels of EPO reaching the abluminal chamber, the amount of recombinant EPO was sufficient to evolve a biological effect on astrocytes cultured at the abluminal side in terms of upregulated gene expression of brain-derived neurotropic factor (BDNF). In conclusion, non-viral gene therapy to RBECs leads to protein secretion and signifies a method for therapeutic proteins to target cells inside the CNS otherwise omitted due to the BBB.

  12. High glucose induced oxidative stress and apoptosis in cardiac microvascular endothelial cells are regulated by FoxO3a.

    Directory of Open Access Journals (Sweden)

    Chaoming Peng

    Full Text Available Cardiac microvascular endothelial cells (CMECs dysfunction contributes to cardiovascular complications in diabetes, whereas, the underlying mechanism is not fully clarified. FoxO transcription factors are involved in apoptosis and reactive oxygen species (ROS production. Therefore, the present study was designed to elucidate the potential role of FoxO3a on the CMECs injury induced by high glucose.CMECs were isolated from hearts of adult rats and cultured in normal or high glucose medium for 6 h, 12 h and 24 h respectively. To down-regulate FoxO3a expression, CMECs were transfected with FoxO3a siRNA. ROS accumulation and apoptosis in CMECs were assessed by dihydroethidine (DHE staining and TUNEL assay respectively. Moreover, the expressions of Akt, FoxO3a, Bim and BclxL in CMECs were assessed by Western blotting assay.ROS accumulation in CMECs was significantly increased after high glucose incubation for 6 to 24 h. Meanwhile, high glucose also increased apoptosis in CMECs, correlated with decreased the phosphorylation expressions of Akt and FoxO3a. Moreover, high glucose incubation increased the expression of Bim, whereas increased anti-apoptotic protein BclxL. Furthermore, siRNA target FoxO3a silencing enhanced the ROS accumulation, whereas suppressed apoptosis in CMECs. FoxO3a silencing also abolished the disturbance of Bcl-2 proteins induced by high glucose in CMECs.Our data provide evidence that high glucose induced FoxO3a activation which suppressed ROS accumulation, and in parallel, resulted in apoptosis of CMECs.

  13. IL-17A potentiates TNFα-induced secretion from human endothelial cells and alters barrier functions controlling neutrophils rights of passage

    DEFF Research Database (Denmark)

    Bosteen, Markus H; Tritsaris, Katerina; Hansen, Anker J

    2014-01-01

    Interleukin-17A (IL-17A) is an important pro-inflammatory cytokine that regulates leukocyte mobilization and recruitment. To better understand how IL-17A controls leukocyte trafficking across capillaries in the peripheral blood circulation, we used primary human dermal microvascular endothelial...

  14. Endothelial juxtaposition of distinct adult stem cells activates angiogenesis signaling molecules in endothelial cells.

    Science.gov (United States)

    Mohammadi, Elham; Nassiri, Seyed Mahdi; Rahbarghazi, Reza; Siavashi, Vahid; Araghi, Atefeh

    2015-12-01

    Efficacy of therapeutic angiogenesis needs a comprehensive understanding of endothelial cell (EC) function and biological factors and cells that interplay with ECs. Stem cells are considered the key components of pro- and anti-angiogenic milieu in a wide variety of physiopathological states, and interactions of EC-stem cells have been the subject of controversy in recent years. In this study, the potential effects of three tissue-specific adult stem cells, namely rat marrow-derived mesenchymal stem cells (rBMSCs), rat adipose-derived stem cells (rADSCs) and rat muscle-derived satellite cells (rSCs), on the endothelial activation of key angiogenic signaling molecules, including VEGF, Ang-2, VEGFR-2, Tie-2, and Tie2-pho, were investigated. Human umbilical vein endothelial cells (HUVECs) and rat lung microvascular endothelial cells (RLMECs) were cocultured with the stem cells or incubated with the stem cell-derived conditioned media on Matrigel. Following HUVEC-stem cell coculture, CD31-positive ECs were flow sorted and subjected to western blotting to analyze potential changes in the expression of the pro-angiogenic signaling molecules. Elongation and co-alignment of the stem cells were seen along the EC tubes in the EC-stem cell cocultures on Matrigel, with cell-to-cell dye communication in the EC-rBMSC cocultures. Moreover, rBMSCs and rADSCs significantly improved endothelial tubulogenesis in both juxtacrine and paracrine manners. These two latter stem cells dynamically up-regulated VEGF, Ang-2, VREGR-2, and Tie-2 but down-regulated Tie2-pho and the Tie2-pho/Tie-2 ratio in HUVECs. Induction of pro-angiogenic signaling in ECs by marrow- and adipose-derived MSCs further indicates the significance of stem cell milieu in angiogenesis dynamics.

  15. TNAP and EHD1 are over-expressed in bovine brain capillary endothelial cells after the re-induction of blood-brain barrier properties.

    Directory of Open Access Journals (Sweden)

    Barbara Deracinois

    Full Text Available Although the physiological properties of the blood-brain barrier (BBB are relatively well known, the phenotype of the component brain capillary endothelial cells (BCECs has yet to be described in detail. Likewise, the molecular mechanisms that govern the establishment and maintenance of the BBB are largely unknown. Proteomics can be used to assess quantitative changes in protein levels and identify proteins involved in the molecular pathways responsible for cellular differentiation. Using the well-established in vitro BBB model developed in our laboratory, we performed a differential nano-LC MALDI-TOF/TOF-MS study of Triton X-100-soluble protein species from bovine BCECs displaying either limited BBB functions or BBB functions re-induced by glial cells. Due to the heterogeneity of the crude extract, we increased identification yields by applying a repeatable, reproducible fractionation process based on the proteins' relative hydrophobicity. We present proteomic and biochemical evidence to show that tissue non-specific alkaline phosphatase (TNAP and Eps15 homology domain-containing protein 1(EDH1 are over-expressed by bovine BCECs after the re-induction of BBB properties. We discuss the impact of these findings on current knowledge of endothelial and BBB permeability.

  16. Transcellular targeting of fiber- and hexon-modified adenovirus vectors across the brain microvascular endothelial cells in vitro.

    Directory of Open Access Journals (Sweden)

    Johanna P Laakkonen

    Full Text Available In central nervous system (CNS-directed gene therapy, efficient targeting of brain parenchyma through the vascular route is prevented by the endothelium and the epithelium of the blood-brain and the blood-cerebrospinal fluid barriers, respectively. In this study, we evaluated the feasibility of the combined genetic and chemical adenovirus capsid modification technology to enable transcellular delivery of targeted adenovirus (Ad vectors across the blood-brain barrier (BBB in vitro models. As a proof-of-principle ligand, maleimide-activated full-length human transferrin (hTf was covalently attached to cysteine-modified Ad serotype 5 vectors either to its fiber or hexon protein. In transcytosis experiments, hTf-coupled vectors were shown to be redirected across the BBB models, the transcytosis activity of the vectors being dependent on the location of the capsid modification and the in vitro model used. The transduction efficiency of hTf-targeted vectors decreased significantly in confluent, polarized cells, indicating that the intracellular route of the vectors differed between unpolarized and polarized cells. After transcellular delivery the majority of the hTf-modified vectors remained intact and partly capable of gene transfer. Altogether, our results demonstrate that i covalent attachment of a ligand to Ad capsid can mediate transcellular targeting across the cerebral endothelium in vitro, ii the attachment site of the ligand influences its transcytosis efficiency and iii combined genetic/chemical modification of Ad vector can be used as a versatile platform for the development of Ad vectors for transcellular targeting.

  17. Endothelial actions of atrial and B-type natriuretic peptides.

    Science.gov (United States)

    Kuhn, Michaela

    2012-05-01

    The cardiac hormone atrial natriuretic peptide (ANP) is critically involved in the maintenance of arterial blood pressure and intravascular volume homeostasis. Its cGMP-producing GC-A receptor is densely expressed in the microvascular endothelium of the lung and systemic circulation, but the functional relevance is controversial. Some studies reported that ANP stimulates endothelial cell permeability, whereas others described that the peptide attenuates endothelial barrier dysfunction provoked by inflammatory agents such as thrombin or histamine. Many studies in vitro addressed the effects of ANP on endothelial proliferation and migration. Again, both pro- and anti-angiogenic properties were described. To unravel the role of the endothelial actions of ANP in vivo, we inactivated the murine GC-A gene selectively in endothelial cells by homologous loxP/Cre-mediated recombination. Our studies in these mice indicate that ANP, via endothelial GC-A, increases endothelial albumin permeability in the microcirculation of the skin and skeletal muscle. This effect is critically involved in the endocrine hypovolaemic, hypotensive actions of the cardiac hormone. On the other hand the homologous GC-A-activating B-type NP (BNP), which is produced by cardiac myocytes and many other cell types in response to stressors such as hypoxia, possibly exerts more paracrine than endocrine actions. For instance, within the ischaemic skeletal muscle BNP released from activated satellite cells can improve the regeneration of neighbouring endothelia. This review will focus on recent advancements in our understanding of endothelial NP/GC-A signalling in the pulmonary versus systemic circulation. It will discuss possible mechanisms accounting for the discrepant observations made for the endothelial actions of this hormone-receptor system and distinguish between (patho)physiological and pharmacological actions. Lastly it will emphasize the potential therapeutical implications derived from the

  18. Habitual aerobic exercise does not protect against micro- or macrovascular endothelial dysfunction in healthy estrogen-deficient postmenopausal women.

    Science.gov (United States)

    Santos-Parker, Jessica R; Strahler, Talia R; Vorwald, Victoria M; Pierce, Gary L; Seals, Douglas R

    2017-01-01

    Aging causes micro- and macrovascular endothelial dysfunction, as assessed by endothelium-dependent dilation (EDD), which can be prevented and reversed by habitual aerobic exercise (AE) in men. However, in estrogen-deficient postmenopausal women, whole forearm microvascular EDD has not been studied, and a beneficial effect of AE on macrovascular EDD has not been consistently shown. We assessed forearm blood flow in response to brachial artery infusions of acetylcholine (FBF ACh ), a measure of whole forearm microvascular EDD, and brachial artery flow-mediated dilation (FMD), a measure of macrovascular EDD, in 12 premenopausal sedentary women (Pre-S; 24 ± 1 yr; V̇o 2max = 37.5 ± 1.6 ml·kg -1 ·min -1 ), 25 estrogen-deficient postmenopausal sedentary women (Post-S; 62 ± 1 yr; V̇o 2max = 24.7 ± 0.9 ml·kg -1 ·min -1 ), and 16 estrogen-deficient postmenopausal AE-trained women (Post-AE; 59 ± 1 yr; V̇o 2max = 40.4 ± 1.4 ml·kg -1 ·min -1 ). FBF ACh was lower in Post-S and Post-AE compared with Pre-S women (135 ± 9 and 116 ± 17 vs. 193 ± 21 AUC, respectively, both P stress. This is the first study to demonstrate that habitual aerobic exercise may not protect against age/menopause-related whole forearm microvascular endothelial dysfunction in healthy nonobese estrogen-deficient postmenopausal women, consistent with recent findings regarding macrovascular endothelial function. This is in contrast to what is observed in healthy middle-aged and older aerobic exercise-trained men. Copyright © 2017 the American Physiological Society.

  19. Cryptococcus neoformans-derived microvesicles enhance the pathogenesis of fungal brain infection.

    Directory of Open Access Journals (Sweden)

    Sheng-He Huang

    Full Text Available Cryptococcal meningoencephalitis is the most common fungal disease in the central nervous system. The mechanisms by which Cryptococcus neoformans invades the brain are largely unknown. In this study, we found that C. neoformans-derived microvesicles (CnMVs can enhance the traversal of the blood-brain barrier (BBB by C. neoformans invitro. The immunofluorescence imaging demonstrates that CnMVs can fuse with human brain microvascular endothelial cells (HBMECs, the constituents of the BBB. This activity is presumably due to the ability of the CnMVs to activate HBMEC membrane rafts and induce cell fusogenic activity. CnMVs also enhanced C. neoformans infection of the brain, found in both infected brains and cerebrospinal fluid. In infected mouse brains, CnMVs are distributed inside and around C. neoformans-induced cystic lesions. GFAP (glial fibrillary acidic protein-positive astrocytes were found surrounding the cystic lesions, overlapping with the 14-3-3-GFP (14-3-3-green fluorescence protein fusion signals. Substantial changes could be observed in areas that have a high density of CnMV staining. This is the first demonstration that C. neoformans-derived microvesicles can facilitate cryptococcal traversal across the BBB and accumulate at lesion sites of C. neoformans-infected brains. Results of this study suggested that CnMVs play an important role in the pathogenesis of cryptococcal meningoencephalitis.

  20. Nipah virus infection and glycoprotein targeting in endothelial cells

    Directory of Open Access Journals (Sweden)

    Maisner Andrea

    2010-11-01

    Full Text Available Abstract Background The highly pathogenic Nipah virus (NiV causes fatal respiratory and brain infections in animals and humans. The major hallmark of the infection is a systemic endothelial infection, predominantly in the CNS. Infection of brain endothelial cells allows the virus to overcome the blood-brain-barrier (BBB and to subsequently infect the brain parenchyma. However, the mechanisms of NiV replication in endothelial cells are poorly elucidated. We have shown recently that the bipolar or basolateral expression of the NiV surface glycoproteins F and G in polarized epithelial cell layers is involved in lateral virus spread via cell-to-cell fusion and that correct sorting depends on tyrosine-dependent targeting signals in the cytoplasmic tails of the glycoproteins. Since endothelial cells share many characteristics with epithelial cells in terms of polarization and protein sorting, we wanted to elucidate the role of the NiV glycoprotein targeting signals in endothelial cells. Results As observed in vivo, NiV infection of endothelial cells induced syncytia formation. The further finding that infection increased the transendothelial permeability supports the idea of spread of infection via cell-to-cell fusion and endothelial cell damage as a mechanism to overcome the BBB. We then revealed that both glycoproteins are expressed at lateral cell junctions (bipolar, not only in NiV-infected primary endothelial cells but also upon stable expression in immortalized endothelial cells. Interestingly, mutation of tyrosines 525 and 542/543 in the cytoplasmic tail of the F protein led to an apical redistribution of the protein in endothelial cells whereas tyrosine mutations in the G protein had no effect at all. This fully contrasts the previous results in epithelial cells where tyrosine 525 in the F, and tyrosines 28/29 in the G protein were required for correct targeting. Conclusion We conclude that the NiV glycoprotein distribution is responsible for

  1. Hypoxic stress up-regulates Kir2.1 expression and facilitates cell proliferation in brain capillary endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Yamamura, Hideto; Suzuki, Yoshiaki; Yamamura, Hisao [Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan); Asai, Kiyofumi [Department of Molecular Neurobiology, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Imaizumi, Yuji, E-mail: yimaizum@phar.nagoya-cu.ac.jp [Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan)

    2016-08-05

    The blood-brain barrier (BBB) is mainly composed of brain capillary endothelial cells (BCECs), astrocytes and pericytes. Brain ischemia causes hypoxic encephalopathy and damages BBB. However, it remains still unclear how hypoxia affects BCECs. In the present study, t-BBEC117 cells, an immortalized bovine brain endothelial cell line, were cultured under hypoxic conditions at 4–5% oxygen for 72 h. This hypoxic stress caused hyperpolarization of resting membrane potential. Patch-clamp recordings revealed a marked increase in Ba{sup 2+}-sensitive inward rectifier K{sup +} current in t-BBEC117 cells after hypoxic culture. Western blot and real-time PCR analyses showed that Kir2.1 expression was significantly up-regulated at protein level but not at mRNA level after the hypoxic culture. Ca{sup 2+} imaging study revealed that the hypoxic stress enhanced store-operated Ca{sup 2+} (SOC) entry, which was significantly reduced in the presence of 100 μM Ba{sup 2+}. On the other hand, the expression of SOC channels such as Orai1, Orai2, and transient receptor potential channels was not affected by hypoxic stress. MTT assay showed that the hypoxic stress significantly enhanced t-BBEC117 cell proliferation, which was inhibited by approximately 60% in the presence of 100 μM Ba{sup 2+}. We first show here that moderate cellular stress by cultivation under hypoxic conditions hyperpolarizes membrane potential via the up-regulation of functional Kir2.1 expression and presumably enhances Ca{sup 2+} entry, resulting in the facilitation of BCEC proliferation. These findings suggest potential roles of Kir2.1 expression in functional changes of BCECs in BBB following ischemia. -- Highlights: •Hypoxic culture of brain endothelial cells (BEC) caused membrane hyperpolarization. •This hyperpolarization was due to the increased expression of Kir2.1 channels. •Hypoxia enhanced store-operated Ca{sup 2+} (SOC) entry via Kir2.1 up-regulation. •Expression levels of putative SOC

  2. Macrophage Migration Inhibitory Factor-Induced Autophagy Contributes to Thrombin-Triggered Endothelial Hyperpermeability in Sepsis.

    Science.gov (United States)

    Chao, Chiao-Hsuan; Chen, Hong-Ru; Chuang, Yung-Chun; Yeh, Trai-Ming

    2018-07-01

    Vascular leakage contributes to the high morbidity and mortality associated with sepsis. Exposure of the endothelium to inflammatory mediators, such as thrombin and cytokines, during sepsis leads to hyperpermeability. We recently observed that autophagy, a cellular process for protein turnover, is involved in macrophage migration inhibitory factor (MIF)-induced endothelial hyperpermeability. Even though it is known that thrombin induces endothelial cells to secrete MIF and to increase vascular permeability, the possible role of autophagy in this process is unknown. In this study, we proposed and tested the hypothesis that MIF-induced autophagy plays an important role in thrombin-induced endothelial hyperpermeability. We evaluated the effects of thrombin on endothelial permeability, autophagy induction, and MIF secretion in vitro using the human microvascular endothelial cell line-1 and human umbilical vein endothelial cells. Several mechanisms/read outs of endothelial permeability and autophagy formation were examined. We observed that blocking autophagy attenuated thrombin-induced endothelial hyperpermeability. Furthermore, thrombin-induced MIF secretion was involved in this process because MIF inhibition reduced thrombin-induced autophagy and hyperpermeability. Finally, we showed that blocking MIF or autophagy effectively alleviated vascular leakage and mortality in endotoxemic mice. Thus, MIF-induced autophagy may represent a common mechanism causing vascular leakage in sepsis.

  3. Angiogenesis in the reparatory mucosa of the mandibular edentulous ridge is driven by endothelial tip cells.

    Science.gov (United States)

    Stănescu, Ruxandra; Didilescu, Andreea Cristiana; Jianu, Adelina Maria; Rusu, M C

    2012-01-01

    Sprouting angiogenesis is led by specialized cell--the endothelial tip cells (ETCs) which can be targeted by pro- or anti-angiogenic therapies. We aimed to perform a qualitative study in order to assess the guidance by tip cells of the endothelial sprouts in the repairing mucosa of the edentulous mandibular crest. Mucosa of the mandibular edentulous ridge was collected from six adult patients, prior to healing abutment placement (second surgery). Slides were prepared and immunostained with antibodies for CD34 and Ki67. The abundant vasculature of the lamina propria was observed on slides and the CD34 antibodies labeled endothelial tip cells in various stages of the endothelial sprouts. Ki67 identified positive endothelial cells, confirming the proliferative status of the microvascular bed. According to the results, the in situ sprouting angiogenesis is driven by tip cells in the oral mucosa of the edentulous ridge and these cells can be targeted by various therapies, as required by the local pathologic or therapeutic conditions.

  4. Differential effects of vascular endothelial growth factor A isoforms in a mouse brain metastasis model of human melanoma.

    NARCIS (Netherlands)

    Kusters, B.; Waal, R.M.W. de; Wesseling, P.; Verrijp, K.; Maass, C.N.; Heerschap, A.; Barentsz, J.O.; Sweep, C.G.J.; Ruiter, D.J.; Leenders, W.P.J.

    2003-01-01

    We reported previously that vascular endothelial growth factor isoform A (VEGF-A) expression by Mel57 human melanoma cells led to tumor progression in a murine brain metastasis model in an angiogenesis-independent fashion by dilation of co-opted, pre-existing vessels and concomitant enhanced blood

  5. Modeling of Cerebral Oxygen Transport Based on In vivo Microscopic Imaging of Microvascular Network Structure, Blood Flow, and Oxygenation.

    Science.gov (United States)

    Gagnon, Louis; Smith, Amy F; Boas, David A; Devor, Anna; Secomb, Timothy W; Sakadžić, Sava

    2016-01-01

    Oxygen is delivered to brain tissue by a dense network of microvessels, which actively control cerebral blood flow (CBF) through vasodilation and contraction in response to changing levels of neural activity. Understanding these network-level processes is immediately relevant for (1) interpretation of functional Magnetic Resonance Imaging (fMRI) signals, and (2) investigation of neurological diseases in which a deterioration of neurovascular and neuro-metabolic physiology contributes to motor and cognitive decline. Experimental data on the structure, flow and oxygen levels of microvascular networks are needed, together with theoretical methods to integrate this information and predict physiologically relevant properties that are not directly measurable. Recent progress in optical imaging technologies for high-resolution in vivo measurement of the cerebral microvascular architecture, blood flow, and oxygenation enables construction of detailed computational models of cerebral hemodynamics and oxygen transport based on realistic three-dimensional microvascular networks. In this article, we review state-of-the-art optical microscopy technologies for quantitative in vivo imaging of cerebral microvascular structure, blood flow and oxygenation, and theoretical methods that utilize such data to generate spatially resolved models for blood flow and oxygen transport. These "bottom-up" models are essential for the understanding of the processes governing brain oxygenation in normal and disease states and for eventual translation of the lessons learned from animal studies to humans.

  6. Reduced cortical microvascular oxygenation in multiple sclerosis: a blinded, case-controlled study using a novel quantitative near-infrared spectroscopy method

    Science.gov (United States)

    Yang, Runze; Dunn, Jeff F.

    2015-11-01

    Hypoxia (low oxygen) is associated with many brain disorders as well as inflammation, but the lack of widely available technology has limited our ability to study hypoxia in human brain. Multiple sclerosis (MS) is a poorly understood neurological disease with a significant inflammatory component which may cause hypoxia. We hypothesized that if hypoxia were to occur, there should be reduced microvascular hemoglobin saturation (StO2). In this study, we aimed to determine if reduced StO2 can be detected in MS using frequency domain near-infrared spectroscopy (fdNIRS). We measured fdNIRS data in cortex and assessed disability of 3 clinical isolated syndrome (CIS), 72 MS patients and 12 controls. Control StO2 was 63.5 ± 3% (mean ± SD). In MS patients, 42% of StO2 values were more than 2 × SD lower than the control mean. There was a significant relationship between StO2 and clinical disability. A reduced microvascular StO2 is supportive (although not conclusive) that there may be hypoxic regions in MS brain. This is the first study showing how quantitative NIRS can be used to detect reduced StO2 in patients with MS, opening the door to understanding how microvascular oxygenation impacts neurological conditions.

  7. Tailored delivery of analgesic ziconotide across a blood brain barrier model using viral nanocontainers

    Science.gov (United States)

    Anand, Prachi; O'Neil, Alison; Lin, Emily; Douglas, Trevor; Holford, Mandë

    2015-08-01

    The blood brain barrier (BBB) is often an insurmountable obstacle for a large number of candidate drugs, including peptides, antibiotics, and chemotherapeutic agents. Devising an adroit delivery method to cross the BBB is essential to unlocking widespread application of peptide therapeutics. Presented here is an engineered nanocontainer for delivering peptidic drugs across the BBB encapsulating the analgesic marine snail peptide ziconotide (Prialt®). We developed a bi-functional viral nanocontainer based on the Salmonella typhimurium bacteriophage P22 capsid, genetically incorporating ziconotide in the interior cavity, and chemically attaching cell penetrating HIV-Tat peptide on the exterior of the capsid. Virus like particles (VLPs) of P22 containing ziconotide were successfully transported in several BBB models of rat and human brain microvascular endothelial cells (BMVEC) using a recyclable noncytotoxic endocytic pathway. This work demonstrates proof in principle for developing a possible alternative to intrathecal injection of ziconotide using a tunable VLP drug delivery nanocontainer to cross the BBB.

  8. Endothelial perturbation: a link between non-dipping and retinopathy in type 2 diabetes?

    DEFF Research Database (Denmark)

    Knudsen, Søren Tang; Jeppesen, Peter; Frederiksen, Christian Alcaraz

    2007-01-01

    -six subjects, stratified to 4 gender-, age-, and duration-matched groups of 19 subjects each, were studied (group A: non-diabetic subjects; group B to D, type 2 diabetic subjects; group B: no retinopathy; group C: minimal background retinopathy; group D: diabetic maculopathy). All subjects underwent a 24-hour......Reduced diurnal blood pressure (BP) variation ("non-dipping") is associated with both micro- and macrovascular complications in patients with type 2 diabetes. The relation between endothelial perturbation and diurnal BP variation in diabetic subjects has not previously been studied. Seventy...... to endothelial cell activation as well as with retinopathy in subjects with type 2 diabetes. This finding suggests a possible mechanism linking non-dipping with microvascular complications in these subjects....

  9. Modeling the ischemic blood-brain barrier; the effects of oxygen-glucose deprivation (OGD) on endothelial cells in culture

    DEFF Research Database (Denmark)

    Tornabene, Erica; Helms, Hans Christian Cederberg; Berndt, Philipp

    Introduction - The blood-brain barrier (BBB) is a physical, transport and metabolic barrier which plays a key role in preventing uncontrolled exchanges between blood and brain, ensuring an optimal environment for neurons activity. This extent interface is created by the endothelial cells forming...... pathways across the barrier in ischemic and postischemic brain endothelium is important for developing new medical therapies capable to exploit the barrier changes occurring during/after ischemia to permeate in the brain and treat this devastating disease. Materials and Methods - Primary cultures...... the wall of brain capillaries. The restrictive nature of the BBB is due to the tight junctions (TJs), which seal the intercellular clefts, limiting the paracellular diffusion, efflux transporters, which extrude xenobiotics, and metabolizing enzymes, which may break down or convert molecules during...

  10. Effect of the glucagon-like peptide-1 analogue liraglutide on coronary microvascular function in patients with type 2 diabetes – a randomized, single-blinded, cross-over pilot study

    DEFF Research Database (Denmark)

    Faber, Rebekka; Zander, Mette; Pena, Adam

    2015-01-01

    dipyridamole induced stress. Peripheral microvascular endothelial function was assessed by Endo-PAT2000®. Interventions were compared by two-sample t-test after ensuring no carry over effect. RESULTS: A total of 24 patients were included. Twenty patients completed the study (15 male; mean age 57 ± 9; mean BMI...

  11. Measurement of the filtration coefficient (Kfc) in the lung of Gallus domesticus and the effects of increased microvascular permeability.

    Science.gov (United States)

    Weidner, W Jeffrey; Waddell, David S; Furlow, J David

    2006-08-01

    The filtration coefficient (Kfc) is a sensitive measure of microvascular hydraulic conductivity and has been reported for the alveolar lungs of many mammalian species, but not for the parabronchial avian lung. This study reports the Kfc in the isolated lungs of normal chickens and in the lungs of chickens given the edemogenic agents oleic acid (OA) or dimethyl amiloride (DMA). The control Kfc =0.04+/-0.01 ml min(-1) kPa(-1) g(-1). This parameter increased significantly following the administration of both OA (0.12+/-0.02 ml min(-1) kPa(-1) g(-1)) and DMA (0.07+/-0.01 ml min kPa(-1) g(-1)). As endothelial cadherins are thought to play a role in the dynamic response to acute lung injury, we utilized Western blot analysis to assess lung cadherin content and Northern blot analysis to assess pulmonary vascular endothelial (VE) cadherin expression following drug administration. Lung cadherin content decreases markedly following DMA, but not OA administration. VE cadherin expression increases as a result of DMA treatment, but is unchanged following OA. Our results suggest that the permeability characteristics of the avian lung are more closely consistent with those of the mammalian rather than the reptilian lung, and, that cadherins may play a significant role in the response to acute increases in avian pulmonary microvascular permeability.

  12. Vascular endothelial growth factors enhance the permeability of the mouse blood-brain barrier.

    Directory of Open Access Journals (Sweden)

    Shize Jiang

    Full Text Available The blood-brain barrier (BBB impedes entry of many drugs into the brain, limiting clinical efficacy. A safe and efficient method for reversibly increasing BBB permeability would greatly facilitate central nervous system (CNS drug delivery and expand the range of possible therapeutics to include water soluble compounds, proteins, nucleotides, and other large molecules. We examined the effect of vascular endothelial growth factor (VEGF on BBB permeability in Kunming (KM mice. Human VEGF165 was administered to treatment groups at two concentrations (1.6 or 3.0 µg/mouse, while controls received equal-volume saline. Changes in BBB permeability were measured by parenchymal accumulation of the contrast agent Gd-DTPA as assessed by 7 T magnetic resonance imaging (MRI. Mice were then injected with Evans blue, sacrificed 0.5 h later, and perfused transcardially. Brains were removed, fixed, and sectioned for histological study. Both VEGF groups exhibited a significantly greater signal intensity from the cerebral cortex and basal ganglia than controls (P<0.001. Evans blue fluorescence intensity was higher in the parenchyma and lower in the cerebrovasculature of VEGF-treated animals compared to controls. No significant brain edema was observed by diffusion weighted MRI (DWI or histological staining. Exogenous application of VEGF can increase the permeability of the BBB without causing brain edema. Pretreatment with VEGF may be a feasible method to facilitate drug delivery into the CNS.

  13. Targeted siRNA Delivery to Diseased Microvascular Endothelial Cells-Cellular and Molecular Concepts

    NARCIS (Netherlands)

    Kowalski, Piotr S.; Leus, Niek G. J.; Scherphof, Gerrit L.; Ruiters, Marcel H. J.; Kamps, Jan A. A. M.; Molema, Grietje

    Increased insight in the role of endothelial cells in the pathophysiology of cancer, inflammatory and cardiovascular diseases, has drawn great interest in pharmacological interventions aiming at the endothelium in diseased sites. Their location in the body makes them suitable targets for therapeutic

  14. HMGB1 Contributes to the Expression of P-Glycoprotein in Mouse Epileptic Brain through Toll-Like Receptor 4 and Receptor for Advanced Glycation End Products.

    Directory of Open Access Journals (Sweden)

    Yan Chen

    Full Text Available The objective of the present study was to investigate the role of high-mobility group box-1 (HMGB1 in the seizure-induced P-glycoprotein (P-gp overexpression and the underlying mechanism. Kainic acid (KA-induced mouse seizure model was used for in vivo experiments. Male C57BL/6 mice were divided into four groups: normal saline control (NS group, KA-induced epileptic seizure (EP group, and EP group pretreated with HMGB1 (EP+HMGB1 group or BoxA (HMGB1 antagonist, EP+BoxA group. Compared to the NS group, increased levels of HMGB1 and P-gp in the brain were observed in the EP group. Injection of HMGB1 before the induction of KA further increased the expression of P-gp while pre-treatment with BoxA abolished this up-regulation. Next, the regulatory role of HMGB1 and its potential involved signal pathways were investigated in mouse microvascular endothelial bEnd.3 cells in vitro. Cells were treated with HMGB1, HMGB1 plus lipopolysaccharide from Rhodobacter sphaeroides (LPS-RS [toll-like receptor 4 (TLR4 antagonist], HMGB1 plus FPS-ZM1 [receptor for advanced glycation end products (RAGE inhibitor], HMGB1 plus SN50 [nuclear factor-kappa B (NF-κB inhibitor], or vehicle. Treatment with HMGB1 increased the expression levels of P-gp, TLR4, RAGE and the activation of NF-κB in bEnd.3 cells. These effects were inhibited by the pre-treatment with either LPS-RS or FPS-ZM1, and were abolished by the pre-treatment of SN50 or a combination treatment of both LPS-RS and FPS-ZM1. Luciferase reporter assays showed that exogenous expression of NF-κB p65 increased the promoter activity of multidrug resistance 1a (P-gp-encoding gene in endothelial cells. These data indicate that HMGB1 contributes to the overexpression of P-gp in mouse epileptic brain tissues via activation of TLR4/RAGE receptors and the downstream transcription factor NF-κB in brain microvascular endothelial cells.

  15. Up-regulation of Kir2.1 by ER stress facilitates cell death of brain capillary endothelial cells

    International Nuclear Information System (INIS)

    Kito, Hiroaki; Yamazaki, Daiju; Ohya, Susumu; Yamamura, Hisao; Asai, Kiyofumi; Imaizumi, Yuji

    2011-01-01

    Highlights: → We found that application of endoplasmic reticulum (ER) stress with tunicamycin to brain capillary endothelial cells (BCECs) induced cell death. → The ER stress facilitated the expression of inward rectifier K + channel (K ir 2.1) and induced sustained membrane hyperpolarization. → The membrane hyperpolarization induced sustained Ca 2+ entry through voltage-independent nonspecific cation channels and consequently facilitated cell death. → The K ir 2.1 up-regulation by ER stress is, at least in part, responsible for cell death of BCECs under pathological conditions. -- Abstract: Brain capillary endothelial cells (BCECs) form blood brain barrier (BBB) to maintain brain homeostasis. Cell turnover of BCECs by the balance of cell proliferation and cell death is critical for maintaining the integrity of BBB. Here we found that stimuli with tunicamycin, endoplasmic reticulum (ER) stress inducer, up-regulated inward rectifier K + channel (K ir 2.1) and facilitated cell death in t-BBEC117, a cell line derived from bovine BCECs. The activation of K ir channels contributed to the establishment of deeply negative resting membrane potential in t-BBEC117. The deep resting membrane potential increased the resting intracellular Ca 2+ concentration due to Ca 2+ influx through non-selective cation channels and thereby partly but significantly regulated cell death in t-BBEC117. The present results suggest that the up-regulation of K ir 2.1 is, at least in part, responsible for cell death/cell turnover of BCECs induced by a variety of cellular stresses, particularly ER stress, under pathological conditions.

  16. Vascular Gene Expression in Nonneoplastic and Malignant Brain

    Science.gov (United States)

    Madden, Stephen L.; Cook, Brian P.; Nacht, Mariana; Weber, William D.; Callahan, Michelle R.; Jiang, Yide; Dufault, Michael R.; Zhang, Xiaoming; Zhang, Wen; Walter-Yohrling, Jennifer; Rouleau, Cecile; Akmaev, Viatcheslav R.; Wang, Clarence J.; Cao, Xiaohong; St. Martin, Thia B.; Roberts, Bruce L.; Teicher, Beverly A.; Klinger, Katherine W.; Stan, Radu-Virgil; Lucey, Brenden; Carson-Walter, Eleanor B.; Laterra, John; Walter, Kevin A.

    2004-01-01

    Malignant gliomas are uniformly lethal tumors whose morbidity is mediated in large part by the angiogenic response of the brain to the invading tumor. This profound angiogenic response leads to aggressive tumor invasion and destruction of surrounding brain tissue as well as blood-brain barrier breakdown and life-threatening cerebral edema. To investigate the molecular mechanisms governing the proliferation of abnormal microvasculature in malignant brain tumor patients, we have undertaken a cell-specific transcriptome analysis from surgically harvested nonneoplastic and tumor-associated endothelial cells. SAGE-derived endothelial cell gene expression patterns from glioma and nonneoplastic brain tissue reveal distinct gene expression patterns and consistent up-regulation of certain glioma endothelial marker genes across patient samples. We define the G-protein-coupled receptor RDC1 as a tumor endothelial marker whose expression is distinctly induced in tumor endothelial cells of both brain and peripheral vasculature. Further, we demonstrate that the glioma-induced gene, PV1, shows expression both restricted to endothelial cells and coincident with endothelial cell tube formation. As PV1 provides a framework for endothelial cell caveolar diaphragms, this protein may serve to enhance glioma-induced disruption of the blood-brain barrier and transendothelial exchange. Additional characterization of this extensive brain endothelial cell gene expression database will provide unique molecular insights into vascular gene expression. PMID:15277233

  17. MicroRNA-199a-5p Regulates the Proliferation of Pulmonary Microvascular Endothelial Cells in Hepatopulmonary Syndrome

    Directory of Open Access Journals (Sweden)

    Jing Zeng

    2015-10-01

    Full Text Available Background/Aims: Pulmonary microvascular endothelial cell (PMVEC proliferation and angiogenesis contribute to the development of hepatopulmonary syndrome (HPS. MicroRNA-199a-5p (miR-199a-5p has emerged as a potent regulator of angiogenesis, and its expression levels significantly decrease in the serum of patients with hepatopathy. However, it has not been reported about whether miR-199a-5p might control PMVEC proliferation. Here, we described the miR-199a-5p governing PMVEC proliferation in HPS. Methods: PMVECs were treated with rat serum from common bile duct ligation (CBDL or sham. MiR-199a-5p mimic or inhibitor was used to change the miR-199a-5p expression. Knockdown of caveolin-1 (Cav-1 was performed using siRNA. NSC-23766 was used to inhibit Rac1 activity. Gene and protein expressions were quantified by qRT-PCR and western blot. Cell proliferation was analyzed by 3H-TdR incorporation and CCK-8 assays. Stress fibers were detected by immunofluorescence. Results: CBDL rat serum induced the down-regulation of miR-199a-5p. Delivery of miR-199a-5p suppressed the CBDL rat serum-induced PMVEC proliferation whereas knockdown of miR-199a-5p promoted PMVEC proliferation. This was accompanied by a decrease and an increase in Cav-1 expression, respectively. Cav-1 siRNA abolished the enhancement of PMVEC proliferation induced by the miR-199a-5p inhibition. Although stress fibers were disrupted in Cav-1 deficient cells, NSC-23766 increased stress fibers and contributed to cell proliferation. Conclusions: CBDL rat serum induced down-regulation of miR-199a-5p in PMVECs, which led to an increase of Cav-1 gene expression. Increased Cav-1 expression, by inhibiting Rac1 activity, led to the formation of stress fibers, which contribute to PMVEC proliferation and thus the pathogenesis of HPS.

  18. Irradiation-induced up-regulation of HLA-E on macrovascular endothelial cells confers protection against killing by activated natural killer cells.

    Directory of Open Access Journals (Sweden)

    Isabelle Riederer

    Full Text Available BACKGROUND: Apart from the platelet/endothelial cell adhesion molecule 1 (PECAM-1, CD31, endoglin (CD105 and a positive factor VIII-related antigen staining, human primary and immortalized macro- and microvascular endothelial cells (ECs differ in their cell surface expression of activating and inhibitory ligands for natural killer (NK cells. Here we comparatively study the effects of irradiation on the phenotype of ECs and their interaction with resting and activated NK cells. METHODOLOGY/PRINCIPAL FINDINGS: Primary macrovascular human umbilical vein endothelial cells (HUVECs only express UL16 binding protein 2 (ULBP2 and the major histocompatibility complex (MHC class I chain-related protein MIC-A (MIC-A as activating signals for NK cells, whereas the corresponding immortalized EA.hy926 EC cell line additionally present ULBP3, membrane heat shock protein 70 (Hsp70, intercellular adhesion molecule ICAM-1 (CD54 and HLA-E. Apart from MIC-B, the immortalized human microvascular endothelial cell line HMEC, resembles the phenotype of EA.hy926. Surprisingly, primary HUVECs are more sensitive to Hsp70 peptide (TKD plus IL-2 (TKD/IL-2-activated NK cells than their immortalized EC counterpatrs. This finding is most likely due to the absence of the inhibitory ligand HLA-E, since the activating ligands are shared among the ECs. The co-culture of HUVECs with activated NK cells induces ICAM-1 (CD54 and HLA-E expression on the former which drops to the initial low levels (below 5% when NK cells are removed. Sublethal irradiation of HUVECs induces similar but less pronounced effects on HUVECs. Along with these findings, irradiation also induces HLA-E expression on macrovascular ECs and this correlates with an increased resistance to killing by activated NK cells. Irradiation had no effect on HLA-E expression on microvascular ECs and the sensitivity of these cells to NK cells remained unaffected. CONCLUSION/SIGNIFICANCE: These data emphasize that an irradiation

  19. Modulation of human dermal microvascular endothelial cell and human gingival fibroblast behavior by micropatterned silica coating surfaces for zirconia dental implant applications

    International Nuclear Information System (INIS)

    Laranjeira, Marta S; Carvalho, Ângela; Ferraz, Maria Pia; Monteiro, Fernando Jorge; Pelaez-Vargas, Alejandro; Hansford, Derek; Coimbra, Susana; Costa, Elísio; Santos-Silva, Alice; Fernandes, Maria Helena

    2014-01-01

    Dental ceramic implants have shown superior esthetic behavior and the absence of induced allergic disorders when compared to titanium implants. Zirconia may become a potential candidate to be used as an alternative to titanium dental implants if surface modifications are introduced. In this work, bioactive micropatterned silica coatings were produced on zirconia substrates, using a combined methodology of sol–gel processing and soft lithography. The aim of the work was to compare the in vitro behavior of human gingival fibroblasts (HGFs) and human dermal microvascular endothelial cells (HDMECs) on three types of silica-coated zirconia surfaces: flat and micropatterned (with pillars and with parallel grooves). Our results showed that cells had a higher metabolic activity (HGF, HDMEC) and increased gene expression levels of fibroblast-specific protein-1 (FSP-1) and collagen type I (COL I) on surfaces with pillars. Nevertheless, parallel grooved surfaces were able to guide cell growth. Even capillary tube-like networks of HDMEC were oriented according to the surface geometry. Zirconia and silica with different topographies have shown to be blood compatible and silica coating reduced bacteria adhesion. All together, the results indicated that microstructured bioactive coating seems to be an efficient strategy to improve soft tissue integration on zirconia implants, protecting implants from peri-implant inflammation and improving long-term implant stabilization. This new approach of micropatterned silica coating on zirconia substrates can generate promising novel dental implants, with surfaces that provide physical cues to guide cells and enhance their behavior. (paper)

  20. Photodynamic efficacy of liposome-delivered hypocrellin B in microvascular endothelial cells in vitro and chicken combs in vivo: a potential photosensitizer for port wine stain

    International Nuclear Information System (INIS)

    Chen, H X; Zou, X B; Yang, Z F; Zhu, J G; Gu, Y; Deng, H; Zhao, J Q

    2013-01-01

    Photodynamic therapy (PDT) has been proved a successful method for port wine stain (PWS), but the prolonged skin photosensitivity induced by the photosensitizers used currently seriously limits the clinical application of PDT. In this study, we investigate the feasibility of hypocrellin B (HB), a promising second-generation photosensitizer for the treatment of PWS. The photodynamic effect of liposome-delivered HB was evaluated in vitro with microvascular endothelial cells (MEC) and in vivo with chicken combs. The dark cytotoxicity and photocytotoxicity of liposomal HB in MEC were evaluated using the MTT assay. Gross and histological examinations were performed to investigate the selective occlusion of the superficial dermal microvasculature in the chicken comb. The result showed that photocytotoxicity of liposomal HB was dependent on both light dose and drug concentration. PDT with HB (0.5–1 mg kg −1 ) and a light dose of 120 J cm −2 showed selective destruction of the superficial dermal microvasculature of the chicken comb, leaving the overlying epidermis intact. This is the first study to investigate the potential efficacy of HB-PDT as a novel modality for the treatment of PWS. These findings suggest that liposomal HB is a safe and effective photosensitizer for PWS. (paper)

  1. Brain Endothelial Cells Control Fertility through Ovarian-Steroid–Dependent Release of Semaphorin 3A

    Science.gov (United States)

    Messina, Andrea; Casoni, Filippo; Vanacker, Charlotte; Langlet, Fanny; Hobo, Barbara; Cagnoni, Gabriella; Gallet, Sarah; Hanchate, Naresh Kumar; Mazur, Danièle; Taniguchi, Masahiko; Mazzone, Massimiliano; Verhaagen, Joost; Ciofi, Philippe; Bouret, Sébastien G.; Tamagnone, Luca; Prevot, Vincent

    2014-01-01

    Neuropilin-1 (Nrp1) guides the development of the nervous and vascular systems, but its role in the mature brain remains to be explored. Here we report that the expression of the 65 kDa isoform of Sema3A, the ligand of Nrp1, by adult vascular endothelial cells, is regulated during the ovarian cycle and promotes axonal sprouting in hypothalamic neurons secreting gonadotropin-releasing hormone (GnRH), the neuropeptide controlling reproduction. Both the inhibition of Sema3A/Nrp1 signaling and the conditional deletion of Nrp1 in GnRH neurons counteract Sema3A-induced axonal sprouting. Furthermore, the localized intracerebral infusion of Nrp1- or Sema3A-neutralizing antibodies in vivo disrupts the ovarian cycle. Finally, the selective neutralization of endothelial-cell Sema3A signaling in adult Sema3a loxP/loxP mice by the intravenous injection of the recombinant TAT-Cre protein alters the amplitude of the preovulatory luteinizing hormone surge, likely by perturbing GnRH release into the hypothalamo-hypophyseal portal system. Our results identify a previously unknown function for 65 kDa Sema3A-Nrp1 signaling in the induction of axonal growth, and raise the possibility that endothelial cells actively participate in synaptic plasticity in specific functional domains of the adult central nervous system, thus controlling key physiological functions such as reproduction. PMID:24618750

  2. Brain endothelial cells control fertility through ovarian-steroid-dependent release of semaphorin 3A.

    Science.gov (United States)

    Giacobini, Paolo; Parkash, Jyoti; Campagne, Céline; Messina, Andrea; Casoni, Filippo; Vanacker, Charlotte; Langlet, Fanny; Hobo, Barbara; Cagnoni, Gabriella; Gallet, Sarah; Hanchate, Naresh Kumar; Mazur, Danièle; Taniguchi, Masahiko; Mazzone, Massimiliano; Verhaagen, Joost; Ciofi, Philippe; Bouret, Sébastien G; Tamagnone, Luca; Prevot, Vincent

    2014-03-01

    Neuropilin-1 (Nrp1) guides the development of the nervous and vascular systems, but its role in the mature brain remains to be explored. Here we report that the expression of the 65 kDa isoform of Sema3A, the ligand of Nrp1, by adult vascular endothelial cells, is regulated during the ovarian cycle and promotes axonal sprouting in hypothalamic neurons secreting gonadotropin-releasing hormone (GnRH), the neuropeptide controlling reproduction. Both the inhibition of Sema3A/Nrp1 signaling and the conditional deletion of Nrp1 in GnRH neurons counteract Sema3A-induced axonal sprouting. Furthermore, the localized intracerebral infusion of Nrp1- or Sema3A-neutralizing antibodies in vivo disrupts the ovarian cycle. Finally, the selective neutralization of endothelial-cell Sema3A signaling in adult Sema3aloxP/loxP mice by the intravenous injection of the recombinant TAT-Cre protein alters the amplitude of the preovulatory luteinizing hormone surge, likely by perturbing GnRH release into the hypothalamo-hypophyseal portal system. Our results identify a previously unknown function for 65 kDa Sema3A-Nrp1 signaling in the induction of axonal growth, and raise the possibility that endothelial cells actively participate in synaptic plasticity in specific functional domains of the adult central nervous system, thus controlling key physiological functions such as reproduction.

  3. Brain endothelial cells control fertility through ovarian-steroid-dependent release of semaphorin 3A.

    Directory of Open Access Journals (Sweden)

    Paolo Giacobini

    2014-03-01

    Full Text Available Neuropilin-1 (Nrp1 guides the development of the nervous and vascular systems, but its role in the mature brain remains to be explored. Here we report that the expression of the 65 kDa isoform of Sema3A, the ligand of Nrp1, by adult vascular endothelial cells, is regulated during the ovarian cycle and promotes axonal sprouting in hypothalamic neurons secreting gonadotropin-releasing hormone (GnRH, the neuropeptide controlling reproduction. Both the inhibition of Sema3A/Nrp1 signaling and the conditional deletion of Nrp1 in GnRH neurons counteract Sema3A-induced axonal sprouting. Furthermore, the localized intracerebral infusion of Nrp1- or Sema3A-neutralizing antibodies in vivo disrupts the ovarian cycle. Finally, the selective neutralization of endothelial-cell Sema3A signaling in adult Sema3aloxP/loxP mice by the intravenous injection of the recombinant TAT-Cre protein alters the amplitude of the preovulatory luteinizing hormone surge, likely by perturbing GnRH release into the hypothalamo-hypophyseal portal system. Our results identify a previously unknown function for 65 kDa Sema3A-Nrp1 signaling in the induction of axonal growth, and raise the possibility that endothelial cells actively participate in synaptic plasticity in specific functional domains of the adult central nervous system, thus controlling key physiological functions such as reproduction.

  4. Magnetic alginate microfibers as scaffolding elements for the fabrication of microvascular-like structures.

    Science.gov (United States)

    Sun, Tao; Shi, Qing; Huang, Qiang; Wang, Huaping; Xiong, Xiaolu; Hu, Chengzhi; Fukuda, Toshio

    2018-01-15

    Traditional cell-encapsulating scaffolds may elicit adverse host responses and inhomogeneity in cellular distribution. Thus, fabrication techniques for cellular self-assembly with micro-scaffold incorporation have been used recently to generate toroidal cellular modules for the bottom-up construction of vascular-like structures. The micro-scaffolds show advantage in promoting tissue formation. However, owing to the lack of annular cell micro-scaffolds, it remains a challenge to engineer micro-scale toroidal cellular modules (micro-TCMs) to fabricate microvascular-like structures. Here, magnetic alginate microfibers (MAMs) are used as scaffolding elements, where a winding strategy enables them to be formed into micro-rings as annular cell micro-scaffolds. These micro-rings were investigated for NIH/3T3 fibroblast growth as a function of surface chemistry and MAM size. Afterwards, micro-TCMs were successfully fabricated with the formation of NIH/3T3 fibroblasts and extracellular matrix layers on the three-dimensional micro-ring surfaces. Simple non-contact magnetic assembly was used to stack the micro-TCMs along a micro-pillar, after which cell fusion rapidly connected the assembled micro-TCMs into a microvascular-like structure. Endothelial cells or drugs encapsulated in the MAMs could be included in the microvascular-like structures as in vitro cellular models for vascular tissue engineering, or as miniaturization platforms for pharmaceutical drug testing in the future. Magnetic alginate microfibers functioned as scaffolding elements for guiding cell growth in micro-scale toroidal cellular modules (micro-TCMs) and provided a magnetic functionality to the micro-TCMs for non-contact 3D assembly in external magnetic fields. By using the liquid/air interface, the non-contact spatial manipulation of the micro-TCMs in the liquid environment was performed with a cost-effective motorized electromagnetic needle. A new biofabrication paradigm of construct of microvascular

  5. [Blood-brain barrier part III: therapeutic approaches to cross the blood-brain barrier and target the brain].

    Science.gov (United States)

    Weiss, N; Miller, F; Cazaubon, S; Couraud, P-O

    2010-03-01

    Over the last few years, the blood-brain barrier has come to be considered as the main limitation for the treatment of neurological diseases caused by inflammatory, tumor or neurodegenerative disorders. In the blood-brain barrier, the close intercellular contact between cerebral endothelial cells due to tight junctions prevents the passive diffusion of hydrophilic components from the bloodstream into the brain. Several specific transport systems (via transporters expressed on cerebral endothelial cells) are implicated in the delivery of nutriments, ions and vitamins to the brain; other transporters expressed on cerebral endothelial cells extrude endogenous substances or xenobiotics, which have crossed the cerebral endothelium, out of the brain and into the bloodstream. Recently, several strategies have been proposed to target the brain, (i) by by-passing the blood-brain barrier by central drug administration, (ii) by increasing permeability of the blood-brain barrier, (iii) by modulating the expression and/or the activity of efflux transporters, (iv) by using the physiological receptor-dependent blood-brain barrier transport, and (v) by creating new viral or chemical vectors to cross the blood-brain barrier. This review focuses on the illustration of these different approaches. Copyright (c) 2009 Elsevier Masson SAS. All rights reserved.

  6. Ethanol suppression of peripheral blood mononuclear cell trafficking across brain endothelial cells in immunodeficiency virus infection

    Directory of Open Access Journals (Sweden)

    Lola C Hudson

    2010-01-01

    Full Text Available Lola C Hudson1, Brenda A Colby1, Rick B Meeker21Department of Molecular Biosciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA; 2Department of Neurology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USAAbstract: Earlier studies suggested that the combination of alcohol use and immunodeficiency virus infection resulted in more severe neurologic disease than either condition individually. These deleterious interactions could be due to increased immune cell and virus trafficking or may result from interactions between ethanol and human immunodeficiency virus (HIV-associated toxicity within the brain. To determine the extent to which increased trafficking played a role, we examined the effect of ethanol on the migration of different peripheral blood mononuclear cell (PBMCs subsets across a brain endothelial cell monolayer. We utilized combinations of feline brain endothelial cells with astrocytes, and/or microglia with either acute exposure to 0.08 g/dL ethanol, a combination of ethanol and feline immunodeficiency virus (FIV, or FIV alone. Adherence of PBMCs to endothelium was increased in all combinations of cells with the addition of ethanol. Despite increased PBMC adhesion with ethanol treatment, transmigration of B cells, monocytes, CD4 T cells and CD8 T cells was not increased and was actually decreased in the presence of astrocytes. Expression of three common adhesion molecules, intercellular adhesion molecule-1 (ICAM1, ICAM2, and vascular cell adhesion molecule, was unchanged or slightly decreased by ethanol. This indicated that although adherence is increased by ethanol it is not due to an increased expression of adhesion molecules. RANTES, MIP1α, MIP1β, and MCP-1 mRNA expression was also studied in brain endothelial cells, astrocytes and microglia by reverse transcriptase-polymerase chain reaction. Ethanol treatment of astrocytes resulted in modest changes of

  7. Cathepsin D non-proteolytically induces proliferation and migration in human omental microvascular endothelial cells via activation of the ERK1/2 and PI3K/AKT pathways.

    Science.gov (United States)

    Pranjol, Md Zahidul I; Gutowski, Nicholas J; Hannemann, Michael; Whatmore, Jacqueline L

    2018-01-01

    Epithelial ovarian cancer (EOC) frequently metastasises to the omentum, a process that requires pro-angiogenic activation of human omental microvascular endothelial cells (HOMECs) by tumour-secreted factors. We have previously shown that ovarian cancer cells secrete a range of factors that induce pro-angiogenic responses e.g. migration, in HOMECs including the lysosomal protease cathepsin D (CathD). However, the cellular mechanism by which CathD induces these cellular responses is not understood. The aim of this study was to further examine the pro-angiogenic effects of CathD in HOMECs i.e. proliferation and migration, to investigate whether these effects are dependent on CathD catalytic activity and to delineate the intracellular signalling kinases activated by CathD. We report, for the first time, that CathD significantly increases HOMEC proliferation and migration via a non-proteolytic mechanism resulting in activation of ERK1/2 and AKT. These data suggest that EOC cancer secreted CathD acts as an extracellular ligand and may play an important pro-angiogenic, and thus pro-metastatic, role by activating the omental microvasculature during EOC metastasis to the omentum. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Uptake and cytotoxicity of citrate-coated gold nanospheres: Comparative studies on human endothelial and epithelial cells

    Directory of Open Access Journals (Sweden)

    Freese Christian

    2012-07-01

    Full Text Available Abstract Background The use of gold nanoparticles (AuNPs for diagnostic applications and for drug and gene-delivery is currently under intensive investigation. For such applications, biocompatibility and the absence of cytotoxicity of AuNPs is essential. Although generally considered as highly biocompatible, previous in vitro studies have shown that cytotoxicity of AuNPs in certain human epithelial cells was observed. In particular, the degree of purification of AuNPs (presence of sodium citrate residues on the particles was shown to affect the proliferation and induce cytotoxicity in these cells. To expand these studies, we have examined if the effects are related to nanoparticle size (10, 11 nm, 25 nm, to the presence of sodium citrate on the particles' surface or they are due to a varying degree of internalization of the AuNPs. Since two cell types are present in the major barriers to the outside in the human body, we have also included endothelial cells from the vasculature and blood brain barrier. Results Transmission electron microscopy demonstrates that the internalized gold nanoparticles are located within vesicles. Increased cytotoxicity was observed after exposure to AuNPs and was found to be concentration-dependent. In addition, cell viability and the proliferation of both endothelial cells decreased after exposure to gold nanoparticles, especially at high concentrations. Moreover, in contrast to the size of the particles (10 nm, 11 nm, 25 nm, the presence of sodium citrate on the nanoparticle surface appeared to enhance these effects. The effects on microvascular endothelial cells from blood vessels were slightly enhanced compared to the effects on brain-derived endothelial cells. A quantification of AuNPs within cells by ICP-AES showed that epithelial cells internalized a higher quantity of AuNPs compared to endothelial cells and that the quantity of uptake is not correlated with the amount of sodium citrate on the

  9. UVB therapy decreases the adhesive interaction between peripheral blood mononuclear cells and dermal microvascular endothelium, and regulates the differential expression of CD54, VCAM-1, and E-selectin in psoriatic plaques

    International Nuclear Information System (INIS)

    Cai, J.-P.; Harris, K.; Chin, Y.H.

    1996-01-01

    A dermal lymphocytic infiltrate is a characteristic feature of psoriasis, and may be involved in the pathogenesis of the disease. We have previously shown that specialized dermal microvascular endothelial cells (DMEC) in psoriatic lesions promote the selective adherence of the CD4 CD45Ro helper T-cell subset. In this study, we examined the adhesive interaction between peripheral blood mononuclear cells and psoriatic DMEC in patients treated with ultraviolet B light (UVB), and correlated the results with the expression and function of endothelial adhesion molecules on DMEC. (author)

  10. [Evaluation of three-dimensional tumor microvascular architecture phenotype heterogeneity in non-small cell carcinoma and its significance].

    Science.gov (United States)

    Zhou, Hui; Liu, Jinkang; Chen, Shengxi; Xiong, Zeng; Zhou, Jianhua; Tong, Shiyu; Chen, Hao; Zhou, Moling

    2012-06-01

    To explore the degree, mechanism and clinical significance of three-dimensional tumor microvascular architecture phenotype heterogeneity (3D-TMAPH) in non-small cell carcinoma (NSCLC). Twenty-one samples of solitary pulmonary nodules were collected integrally. To establish two-dimensional tumor microvascular architecture phenotype (2D-TMAP) and three-dimensional tumor microvascular architecture phenotype (3D-TMAP), five layers of each nodule were selected and embedded in paraffin. Test indices included the expressions of vascular endothelial growth factor (VEGF), proliferating cell nuclear antigen (PCNA), EphB4, ephfinB2 and microvascular density marked by anti-CD34 (CD34-MVD). The degrees of 3D-TMAPH were evaluated by the coefficient of variation and extend of heterogeneity. Spearman rank correlation analysis was used to investigate the relationships between 2D-TMAP, 3D-TMAP and clinicopathological features. 3D-TMAPH showed that 2D-TMAP heterogeneity was expressed in the tissues of NSCLC. The heterogeneities in the malignant nodules were significantly higher than those in the active inflammatory nodules and tubercular nodules. In addition, different degrees of heterogeneity of CD34-MVD and PCNA were found in NSCLC tissues. The coefficients of variation of CD34- MVD and PCNA were positively related to the degree of differentiation (all P0.05). The level of heterogeneity of various expression indexes (ephrinB2, EphB4, VEGF) in NSCLC tissues were inconsistent, but there were no significant differences in heterogeneity in NSCLC tissues with different histological types (P>0.05). 3D-TMAPH exists widely in the microenvironment during the genesis and development of NSCLC and has a significant impact on its biological complexity.

  11. Regulation of Toll-like receptor 2 interaction with Ecgp96 controls Escherichia coli K1 invasion of brain endothelial cells

    OpenAIRE

    Krishnan, Subramanian; Chen, Shuang; Turcatel, Gianluca; Arditi, Moshe; Prasadarao, Nemani V.

    2012-01-01

    The interaction of outer membrane protein A (OmpA) with its receptor, Ecgp96 (a homologue of Hsp90β) is critical for the pathogenesis of E. coli K1 meningitis. Since Hsp90 chaperones Toll-like receptors (TLRs), we examined the role of TLRs in E. coli K1 infection. Herein, we show that newborn TLR2−/− mice are resistant to E. coli K1 meningitis, while TLR4−/− mice succumb to infection sooner. In vitro, OmpA+ E. coli infection selectively upregulates Ecgp96 and TLR2 in human brain microvascular...

  12. *NO and oxyradical metabolism in new cell lines of rat brain capillary endothelial cells forming the blood-brain barrier.

    Science.gov (United States)

    Blasig, I E; Giese, H; Schroeter, M L; Sporbert, A; Utepbergenov, D I; Buchwalow, I B; Neubert, K; Schönfelder, G; Freyer, D; Schimke, I; Siems, W E; Paul, M; Haseloff, R F; Blasig, R

    2001-09-01

    To investigate the relevance of *NO and oxyradicals in the blood-brain barrier (BBB), differentiated and well-proliferating brain capillary endothelial cells (BCEC) are required. Therefore, rat BCEC (rBCEC) were transfected with immortalizing genes. The resulting lines exhibited endothelial characteristics (factor VIII, angiotensin-converting enzyme, high prostacyclin/thromboxane release rates) and BBB markers (gamma-glutamyl transpeptidase, alkaline phosphatase). The control line rBCEC2 (mock transfected) revealed fibroblastoid morphology, less factor VIII, reduced gamma-glutamyl transpeptidase, weak radical defence, low prostanoid metabolism, and limited proliferation. Lines transfected with immortalizing genes (especially rBCEC4, polyoma virus large T antigen) conserved primary properties: epitheloid morphology, subcultivation with high proliferation rate under pure culture conditions, and powerful defence against reactive oxygen species (Mn-, Cu/Zn-superoxide dismutase, catalase, glutathione peroxidase, glutathione) effectively controlling radical metabolism. Only 100 microM H2O2 overcame this defence and stimulated the formation of eicosanoids similarly as in primary cells. Some BBB markers were expressed to a lower degree; however, cocultivation with astrocytes intensified these markers (e.g., alkaline phosphatase) and paraendothelial tightness, indicating induction of BBB properties. Inducible NO synthase was induced by a cytokine plus lipopolysaccharide mixture in all lines and primary cells, resulting in *NO release. Comparing the cell lines obtained, rBCEC4 are stable immortalized and reveal the best conservation of properties from primary cells, including enzymes producing or decomposing reactive species. These cells can be subcultivated in large amounts and, hence, they are suitable to study the role of radical metabolism in the BBB and in the cerebral microvasculature. Copyright 2001 Academic Press.

  13. Radiosensitization of human endothelial cells by IL-24

    International Nuclear Information System (INIS)

    Meyn, R.E.

    2003-01-01

    Radiation therapy remains an important cancer treatment modality but despite improvements in dose delivery many patients still fail at their primary tumor site. Therefore, new strategies designed to improve local control are needed. Protocols combining radiation with anti-angiogenic agents might be of particular advantage based on their documented low toxicity. In this regard, we have been conducting preclinical investigations of a novel cytokine, mda7/IL-24. Our collaborators have shown that mda7/IL-24 protein targets the endothelial cells of the tumor microvascular system and has potent anti-angiogenic properties in both in vitro and in vivo assays. Recently, we have demonstrated that recombinant mda7/IL-24 protein radiosensitizes human endothelial cells in vitro. Specifically, 10 ng/ml of recombinant human IL-24 protein for 12 hrs reduced the survival at 2 Gy for human umbilical vein endothelial cells (HUVECs) from 0.33 to 0.12. We are also working on understanding the molecular basis for this radiosensitizing effect. Preliminary data suggest a model whereby mda7/IL-24 engages a specific receptor on the surface of endothelial cells and initiates a signal transduction pathway that modulates the cell's propensity for radiation-induced apoptosis and capacity for repairing radiation-induced DNA double strand breaks. Mechanistic insight gained from these studies may have implications for the actions of other anti-angiogenic agents and may generally explain the regulation of radiosensitivity imparted by growth factors and cytokines

  14. Striated muscle microvascular response to silver implants: A comparative in vivo study with titanium and stainless steel.

    Science.gov (United States)

    Kraft, C N; Hansis, M; Arens, S; Menger, M D; Vollmar, B

    2000-02-01

    Local microvascular perfusion is the primary line of defense of tissue against microorganisms and plays a considerable role in reparative processes. The impairment of the microcirculation by a biomaterial may therefore have profound consequences. Silver is known to have excellent antimicrobial activity and, although regional and systemic toxic effects have been described, silver is regularly discussed as an implant material in bone surgery. Because little is known about the influence of silver implants on the adjacent host tissue microvasculature, we studied in vivo nutritive perfusion and leukocytic response, and compared these results with those of the conventionally used materials titanium and stainless steel. Using the hamster dorsal skinfold chamber preparation and intravital microscopy, the implantation of a commercially pure silver sample led to a distinct and persistent activation of leukocytes combined with a marked disruption of the microvascular endothelial integrity, massive leukocyte extravasation, and considerable venular dilation. Whereas animals with stainless-steel implants showed a moderate increase in these parameters with a tendency to recuperate, titanium implants caused only a transient increase of leukocyte-endothelial cell interaction within the first 120 min and no significant change in macromolecular leakage, leukocyte extravasation and venular diameter. After 3 days, five of six preparations with silver samples showed severe inflammation and massive edema. Thus, the use of silver as an implant material should be critically judged despite its bactericidal properties. The implant material titanium seems to be well tolerated by the local vascular system and currently represents the golden standard. Copyright 2000 John Wiley & Sons, Inc.

  15. CXCL10 can inhibit endothelial cell proliferation independently of CXCR3.

    Directory of Open Access Journals (Sweden)

    Gabriele S V Campanella

    2010-09-01

    Full Text Available CXCL10 (or Interferon-inducible protein of 10 kDa, IP-10 is an interferon-inducible chemokine with potent chemotactic activity on activated effector T cells and other leukocytes expressing its high affinity G protein-coupled receptor CXCR3. CXCL10 is also active on other cell types, including endothelial cells and fibroblasts. The mechanisms through which CXCL10 mediates its effects on non-leukocytes is not fully understood. In this study, we focus on the anti-proliferative effect of CXCL10 on endothelial cells, and demonstrate that CXCL10 can inhibit endothelial cell proliferation in vitro independently of CXCR3. Four main findings support this conclusion. First, primary mouse endothelial cells isolated from CXCR3-deficient mice were inhibited by CXCL10 as efficiently as wildtype endothelial cells. We also note that the proposed alternative splice form CXCR3-B, which is thought to mediate CXCL10's angiostatic activity, does not exist in mice based on published mouse CXCR3 genomic sequences as an in-frame stop codon would terminate the proposed CXCR3-B splice variant in mice. Second, we demonstrate that human umbilical vein endothelial cells and human lung microvascular endothelial cells that were inhibited by CXL10 did not express CXCR3 by FACS analysis. Third, two different neutralizing CXCR3 antibodies did not inhibit the anti-proliferative effect of CXCL10. Finally, fourth, utilizing a panel of CXCL10 mutants, we show that the ability to inhibit endothelial cell proliferation correlates with CXCL10's glycosaminoglycan binding affinity and not with its CXCR3 binding and signaling. Thus, using a very defined system, we show that CXCL10 can inhibit endothelial cell proliferation through a CXCR3-independent mechanism.

  16. Endothelial disruptive proinflammatory effects of nicotine and e-cigarette vapor exposures.

    Science.gov (United States)

    Schweitzer, Kelly S; Chen, Steven X; Law, Sarah; Van Demark, Mary; Poirier, Christophe; Justice, Matthew J; Hubbard, Walter C; Kim, Elena S; Lai, Xianyin; Wang, Mu; Kranz, William D; Carroll, Clinton J; Ray, Bruce D; Bittman, Robert; Goodpaster, John; Petrache, Irina

    2015-07-15

    The increased use of inhaled nicotine via e-cigarettes has unknown risks to lung health. Having previously shown that cigarette smoke (CS) extract disrupts the lung microvasculature barrier function by endothelial cell activation and cytoskeletal rearrangement, we investigated the contribution of nicotine in CS or e-cigarettes (e-Cig) to lung endothelial injury. Primary lung microvascular endothelial cells were exposed to nicotine, e-Cig solution, or condensed e-Cig vapor (1-20 mM nicotine) or to nicotine-free CS extract or e-Cig solutions. Compared with nicotine-containing extract, nicotine free-CS extract (10-20%) caused significantly less endothelial permeability as measured with electric cell-substrate impedance sensing. Nicotine exposures triggered dose-dependent loss of endothelial barrier in cultured cell monolayers and rapidly increased lung inflammation and oxidative stress in mice. The endothelial barrier disruptive effects were associated with increased intracellular ceramides, p38 MAPK activation, and myosin light chain (MLC) phosphorylation, and was critically mediated by Rho-activated kinase via inhibition of MLC-phosphatase unit MYPT1. Although nicotine at sufficient concentrations to cause endothelial barrier loss did not trigger cell necrosis, it markedly inhibited cell proliferation. Augmentation of sphingosine-1-phosphate (S1P) signaling via S1P1 improved both endothelial cell proliferation and barrier function during nicotine exposures. Nicotine-independent effects of e-Cig solutions were noted, which may be attributable to acrolein, detected along with propylene glycol, glycerol, and nicotine by NMR, mass spectrometry, and gas chromatography, in both e-Cig solutions and vapor. These results suggest that soluble components of e-Cig, including nicotine, cause dose-dependent loss of lung endothelial barrier function, which is associated with oxidative stress and brisk inflammation.

  17. Expansion of microvascular networks in vivo by phthalimide neovascular factor 1 (PNF1).

    Science.gov (United States)

    Wieghaus, Kristen A; Nickerson, Meghan M; Petrie Aronin, Caren E; Sefcik, Lauren S; Price, Richard J; Paige, Mikell A; Brown, Milton L; Botchwey, Edward A

    2008-12-01

    Phthalimide neovascular factor (PNF1, formerly SC-3-149) is a potent stimulator of proangiogenic signaling pathways in endothelial cells. In this study, we evaluated the in vivo effects of sustained PNF1 release to promote ingrowth and expansion of microvascular networks surrounding biomaterial implants. The dorsal skinfold window chamber was used to evaluate the structural remodeling response of the local microvasculature. PNF1 was released from poly(lactic-co-glycolic acid) (PLAGA) films, and a transport model was utilized to predict PNF1 penetration into the surrounding tissue. PNF1 significantly expanded microvascular networks within a 2mm radius from implants after 3 and 7 days by increasing microvessel length density and lumenal diameter of local arterioles and venules. Staining of histological sections with CD11b showed enhanced recruitment of circulating white blood cells, including monocytes, which are critical for the process of vessel enlargement through arteriogenesis. As PNF1 has been shown to modulate MT1-MMP, a facilitator of CCL2 dependent leukocyte transmigration, aspects of window chamber experiments were repeated in CCR2(-/-) (CCL2 receptor) mouse chimeras to more fully explore the critical nature of monocyte recruitment on the therapeutic benefits of PNF1 function in vivo.

  18. Acute cocoa flavanol supplementation improves muscle macro- and microvascular but not anabolic responses to amino acids in older men.

    Science.gov (United States)

    Phillips, Bethan E; Atherton, Philip J; Varadhan, Krishna; Limb, Marie C; Williams, John P; Smith, Kenneth

    2016-05-01

    The anabolic effects of nutrition on skeletal muscle may depend on adequate skeletal muscle perfusion, which is impaired in older people. Cocoa flavanols have been shown to improve flow-mediated dilation, an established measure of endothelial function. However, their effect on muscle microvascular blood flow is currently unknown. Therefore, the objective of this study was to explore links between the consumption of cocoa flavanols, muscle microvascular blood flow, and muscle protein synthesis (MPS) in response to nutrition in older men. To achieve this objective, leg blood flow (LBF), muscle microvascular blood volume (MBV), and MPS were measured under postabsorptive and postprandial (intravenous Glamin (Fresenius Kabi, Germany), dextrose to sustain glucose ∼7.5 mmol·L(-1)) conditions in 20 older men. Ten of these men were studied with no cocoa flavanol intervention and a further 10 were studied with the addition of 350 mg of cocoa flavanols at the same time that nutrition began. Leg (femoral artery) blood flow was measured by Doppler ultrasound, muscle MBV by contrast-enhanced ultrasound using Definity (Lantheus Medical Imaging, Mass., USA) perflutren contrast agent and MPS using [1, 2-(13)C2]leucine tracer techniques. Our results show that although older individuals do not show an increase in LBF or MBV in response to feeding, these absent responses are apparent when cocoa flavanols are given acutely with nutrition. However, this restoration in vascular responsiveness is not associated with improved MPS responses to nutrition. We conclude that acute cocoa flavanol supplementation improves muscle macro- and microvascular responses to nutrition, independently of modifying muscle protein anabolism.

  19. RhoA GTPase regulates radiation-induced alterations in endothelial cell adhesion and migration

    International Nuclear Information System (INIS)

    Rousseau, Matthieu; Gaugler, Marie-Hélène; Rodallec, Audrey; Bonnaud, Stéphanie; Paris, François; Corre, Isabelle

    2011-01-01

    Highlights: ► We explore the role of RhoA in endothelial cell response to ionizing radiation. ► RhoA is rapidly activated by single high-dose of radiation. ► Radiation leads to RhoA/ROCK-dependent actin cytoskeleton remodeling. ► Radiation-induced apoptosis does not require the RhoA/ROCK pathway. ► Radiation-induced alteration of endothelial adhesion and migration requires RhoA/ROCK. -- Abstract: Endothelial cells of the microvasculature are major target of ionizing radiation, responsible of the radiation-induced vascular early dysfunctions. Molecular signaling pathways involved in endothelial responses to ionizing radiation, despite being increasingly investigated, still need precise characterization. Small GTPase RhoA and its effector ROCK are crucial signaling molecules involved in many endothelial cellular functions. Recent studies identified implication of RhoA/ROCK in radiation-induced increase in endothelial permeability but other endothelial functions altered by radiation might also require RhoA proteins. Human microvascular endothelial cells HMEC-1, either treated with Y-27632 (inhibitor of ROCK) or invalidated for RhoA by RNA interference were exposed to 15 Gy. We showed a rapid radiation-induced activation of RhoA, leading to a deep reorganisation of actin cytoskeleton with rapid formation of stress fibers. Endothelial early apoptosis induced by ionizing radiation was not affected by Y-27632 pre-treatment or RhoA depletion. Endothelial adhesion to fibronectin and formation of focal adhesions increased in response to radiation in a RhoA/ROCK-dependent manner. Consistent with its pro-adhesive role, ionizing radiation also decreased endothelial cells migration and RhoA was required for this inhibition. These results highlight the role of RhoA GTPase in ionizing radiation-induced deregulation of essential endothelial functions linked to actin cytoskeleton.

  20. Wide-area mapping of resting state hemodynamic correlations at microvascular resolution with multi-contrast optical imaging (Conference Presentation)

    Science.gov (United States)

    Senarathna, Janaka; Hadjiabadi, Darian; Gil, Stacy; Thakor, Nitish V.; Pathak, Arvind P.

    2017-02-01

    Different brain regions exhibit complex information processing even at rest. Therefore, assessing temporal correlations between regions permits task-free visualization of their `resting state connectivity'. Although functional MRI (fMRI) is widely used for mapping resting state connectivity in the human brain, it is not well suited for `microvascular scale' imaging in rodents because of its limited spatial resolution. Moreover, co-registered cerebral blood flow (CBF) and total hemoglobin (HbT) data are often unavailable in conventional fMRI experiments. Therefore, we built a customized system that combines laser speckle contrast imaging (LSCI), intrinsic optical signal (IOS) imaging and fluorescence imaging (FI) to generate multi-contrast functional connectivity maps at a spatial resolution of 10 μm. This system comprised of three illumination sources: a 632 nm HeNe laser (for LSCI), a 570 nm ± 5 nm filtered white light source (for IOS), and a 473 nm blue laser (for FI), as well as a sensitive CCD camera operating at 10 frames per second for image acquisition. The acquired data enabled visualization of changes in resting state neurophysiology at microvascular spatial scales. Moreover, concurrent mapping of CBF and HbT-based temporal correlations enabled in vivo mapping of how resting brain regions were linked in terms of their hemodynamics. Additionally, we complemented this approach by exploiting the transit times of a fluorescent tracer (Dextran-FITC) to distinguish arterial from venous perfusion. Overall, we demonstrated the feasibility of wide area mapping of resting state connectivity at microvascular resolution and created a new toolbox for interrogating neurovascular function.

  1. Characterisation of an in vitro blood-brain barrier model based on primary porcine capillary endothelial cells in monoculture or co-culture with primary rat or porcine astrocytes and pericytes

    DEFF Research Database (Denmark)

    Thomsen, Louiza Bohn; Larsen, Annette Burkhart; Moos, Torben

    to in vivo such as efflux transporters, tight junction proteins, and high transendothelial electric resistance (TEER). Primary BCECs are isolated from a variety of mammals such as rats, mice, cattle and pigs. Often bovine and porcine BCECs are cultured in monoculture or in co-culture with rat astrocytes......In vitro blood-brain barrier (BBB) models based on primary brain capillary endothelial cells (BCECs) in monoculture or in co-culture with primary astrocytes and pericytes are often applied for studying physiology of the BBB. Primary BCECs retain many morphological and biochemical properties similar...... obtained from neonatal rats which have been shown to strengthen the barrier properties of the BCECs. In this study, brain endothelial cells (PBECs), astrocytes and pericytes are isolated from pig brains donated by the local abattoir. The brains are from 6 month old domestic pigs. The availability and high...

  2. Role of the Blood-Brain Barrier in the Formation of Brain Metastases

    Directory of Open Access Journals (Sweden)

    István A. Krizbai

    2013-01-01

    Full Text Available The majority of brain metastases originate from lung cancer, breast cancer and malignant melanoma. In order to reach the brain, parenchyma metastatic cells have to transmigrate through the endothelial cell layer of brain capillaries, which forms the morphological basis of the blood-brain barrier (BBB. The BBB has a dual role in brain metastasis formation: it forms a tight barrier protecting the central nervous system from entering cancer cells, but it is also actively involved in protecting metastatic cells during extravasation and proliferation in the brain. The mechanisms of interaction of cancer cells and cerebral endothelial cells are largely uncharacterized. Here, we provide a comprehensive review on our current knowledge about the role of junctional and adhesion molecules, soluble factors, proteolytic enzymes and signaling pathways mediating the attachment of tumor cells to brain endothelial cells and the transendothelial migration of metastatic cells. Since brain metastases represent a great therapeutic challenge, it is indispensable to understand the mechanisms of the interaction of tumor cells with the BBB in order to find targets of prevention of brain metastasis formation.

  3. In Vitro Modeling of Blood-Brain Barrier with Human iPSC-Derived Endothelial Cells, Pericytes, Neurons, and Astrocytes via Notch Signaling

    Directory of Open Access Journals (Sweden)

    Kohei Yamamizu

    2017-03-01

    Full Text Available The blood-brain barrier (BBB is composed of four cell populations, brain endothelial cells (BECs, pericytes, neurons, and astrocytes. Its role is to precisely regulate the microenvironment of the brain through selective substance crossing. Here we generated an in vitro model of the BBB by differentiating human induced pluripotent stem cells (hiPSCs into all four populations. When the four hiPSC-derived populations were co-cultured, endothelial cells (ECs were endowed with features consistent with BECs, including a high expression of nutrient transporters (CAT3, MFSD2A and efflux transporters (ABCA1, BCRP, PGP, MRP5, and strong barrier function based on tight junctions. Neuron-derived Dll1, which activates Notch signaling in ECs, was essential for the BEC specification. We performed in vitro BBB permeability tests and assessed ten clinical drugs by nanoLC-MS/MS, finding a good correlation with the BBB permeability reported in previous cases. This technology should be useful for research on human BBB physiology, pathology, and drug development.

  4. Heme oxygenase-1 protects endothelial cells from the toxicity of air pollutant chemicals

    International Nuclear Information System (INIS)

    Lawal, Akeem O.; Zhang, Min; Dittmar, Michael; Lulla, Aaron; Araujo, Jesus A.

    2015-01-01

    Diesel exhaust particles (DEPs) are a major component of diesel emissions, responsible for a large portion of their toxicity. In this study, we examined the toxic effects of DEPs on endothelial cells and the role of DEP-induced heme oxygenase-1 (HO-1) expression. Human microvascular endothelial cells (HMECs) were treated with an organic extract of DEPs from an automobile engine (A-DEP) or a forklift engine (F-DEP) for 1 and 4 h. ROS generation, cell viability, lactate dehydrogenase leakage, expression of HO-1, inflammatory genes, cell adhesion molecules and unfolded protein respone (UPR) gene were assessed. HO-1 expression and/or activity were inhibited by siRNA or tin protoporphyrin (Sn PPIX) and enhanced by an expression plasmid or cobalt protoporphyrin (CoPPIX). Exposure to 25 μg/ml of A-DEP and F-DEP significantly induced ROS production, cellular toxicity and greater levels of inflammatory and cellular adhesion molecules but to a different degree. Inhibition of HO-1 enzymatic activity with SnPPIX and silencing of the HO-1 gene by siRNA enhanced DEP-induced ROS production, further decreased cell viability and increased expression of inflammatory and cell adhesion molecules. On the other hand, overexpression of the HO-1 gene by a pcDNA 3.1D/V5-HO-1 plasmid significantly mitigated ROS production, increased cell survival and decreased the expression of inflammatory genes. HO-1 expression protected HMECs from DEP-induced prooxidative and proinflammatory effects. Modulation of HO-1 expression could potentially serve as a therapeutic target in an attempt to inhibit the cardiovascular effects of ambient PM. - Highlights: • We examined the role of HO-1 expression on diesel exhaust particle (DEP) in endothelial cells. • DEPs exert cytotoxic and inflammatory effects on human microvascular endothelial cells (HMECs). • DEPs induce HO-1 expression in HMECs. • HO-1 protects against the oxidative stress induced by DEps. • HO-1 attenuates the proinflammatory effects

  5. Heme oxygenase-1 protects endothelial cells from the toxicity of air pollutant chemicals

    Energy Technology Data Exchange (ETDEWEB)

    Lawal, Akeem O.; Zhang, Min; Dittmar, Michael [Division of Cardiology, David Geffen School of Medicine, University of California, Los Angeles, 10833 Le Conte Avenue, CHS 43-264, Los Angeles, CA 90095 (United States); Lulla, Aaron [Division of Cardiology, David Geffen School of Medicine, University of California, Los Angeles, 10833 Le Conte Avenue, CHS 43-264, Los Angeles, CA 90095 (United States); Molecular Toxicology Interdepartmental Program, University of California, Los Angeles (United States); Araujo, Jesus A., E-mail: JAraujo@mednet.ucla.edu [Division of Cardiology, David Geffen School of Medicine, University of California, Los Angeles, 10833 Le Conte Avenue, CHS 43-264, Los Angeles, CA 90095 (United States); Molecular Toxicology Interdepartmental Program, University of California, Los Angeles (United States); Molecular Biology Institute, University of California, Los Angeles (United States)

    2015-05-01

    Diesel exhaust particles (DEPs) are a major component of diesel emissions, responsible for a large portion of their toxicity. In this study, we examined the toxic effects of DEPs on endothelial cells and the role of DEP-induced heme oxygenase-1 (HO-1) expression. Human microvascular endothelial cells (HMECs) were treated with an organic extract of DEPs from an automobile engine (A-DEP) or a forklift engine (F-DEP) for 1 and 4 h. ROS generation, cell viability, lactate dehydrogenase leakage, expression of HO-1, inflammatory genes, cell adhesion molecules and unfolded protein respone (UPR) gene were assessed. HO-1 expression and/or activity were inhibited by siRNA or tin protoporphyrin (Sn PPIX) and enhanced by an expression plasmid or cobalt protoporphyrin (CoPPIX). Exposure to 25 μg/ml of A-DEP and F-DEP significantly induced ROS production, cellular toxicity and greater levels of inflammatory and cellular adhesion molecules but to a different degree. Inhibition of HO-1 enzymatic activity with SnPPIX and silencing of the HO-1 gene by siRNA enhanced DEP-induced ROS production, further decreased cell viability and increased expression of inflammatory and cell adhesion molecules. On the other hand, overexpression of the HO-1 gene by a pcDNA 3.1D/V5-HO-1 plasmid significantly mitigated ROS production, increased cell survival and decreased the expression of inflammatory genes. HO-1 expression protected HMECs from DEP-induced prooxidative and proinflammatory effects. Modulation of HO-1 expression could potentially serve as a therapeutic target in an attempt to inhibit the cardiovascular effects of ambient PM. - Highlights: • We examined the role of HO-1 expression on diesel exhaust particle (DEP) in endothelial cells. • DEPs exert cytotoxic and inflammatory effects on human microvascular endothelial cells (HMECs). • DEPs induce HO-1 expression in HMECs. • HO-1 protects against the oxidative stress induced by DEps. • HO-1 attenuates the proinflammatory effects

  6. Endothelial-monocyte activating polypeptide II alters fibronectin based endothelial cell adhesion and matrix assembly via alpha5 beta1 integrin

    International Nuclear Information System (INIS)

    Schwarz, Margaret A.; Zheng, Hiahua; Liu, Jie; Corbett, Siobhan; Schwarz, Roderich E.

    2005-01-01

    Mature Endothelial-Monocyte Activating Polypeptide (mEMAP) II functions as a potent antiangiogenic peptide. Although the anti-tumor effect of mEMAP II has been described, little is known regarding its mechanism of action. Observations that mEMAP II induced apoptosis only in a subset of migrating and proliferating endothelial cells (EC) suggests a targeted effect on cells engaged in angiogenic activities which are known to rely upon cell adhesion and migration. Indeed, we demonstrate that mEMAP II inhibited fibronectin (FN) dependent microvascular EC (MEC) adhesion and spreading and we show that this depends upon the alpha5 beta1 integrin. Immunofluorescence analysis demonstrated that mEMAP II-dependent blockade of FN-alpha5 beta1 interactions was associated with disassembly of both actin stress fiber networks and FN matrix. These findings suggest that mEMAP II blocks MEC adhesion and spreading on fibronectin, via a direct interaction with the integrin alpha5 beta1, thus implicating that alpha5 integrin may be a mediator of mEMAP II's antiangiogenic function

  7. The pleiotropic effects of simvastatin on retinal microvascular endothelium has important implications for ischaemic retinopathies.

    Directory of Open Access Journals (Sweden)

    Reinhold J Medina

    Full Text Available BACKGROUND: Current guidelines encourage the use of statins to reduce the risk of cardiovascular disease in diabetic patients; however the impact of these drugs on diabetic retinopathy is not well defined. Moreover, pleiotropic effects of statins on the highly specialised retinal microvascular endothelium remain largely unknown. The objective of this study was to investigate the effects of clinically relevant concentrations of simvastatin on retinal endothelium in vitro and in vivo. METHODS AND FINDINGS: Retinal microvascular endothelial cells (RMECs were treated with 0.01-10 microM simvastatin and a biphasic dose-related response was observed. Low concentrations enhanced microvascular repair with 0.1 microM simvastatin significantly increasing proliferation (p<0.05, and 0.01 microM simvastatin significantly promoting migration (p<0.05, sprouting (p<0.001, and tubulogenesis (p<0.001. High concentration of simvastatin (10 microM had the opposite effect, significantly inhibiting proliferation (p<0.01, migration (p<0.01, sprouting (p<0.001, and tubulogenesis (p<0.05. Furthermore, simvastatin concentrations higher than 1 microM induced cell death. The mouse model of oxygen-induced retinopathy was used to investigate the possible effects of simvastatin treatment on ischaemic retinopathy. Low dose simvastatin (0.2 mg/Kg promoted retinal microvascular repair in response to ischaemia by promoting intra-retinal re-vascularisation (p<0.01. By contrast, high dose simvastatin(20 mg/Kg significantly prevented re-vascularisation (p<0.01 and concomitantly increased pathological neovascularisation (p<0.01. We also demonstrated that the pro-vascular repair mechanism of simvastatin involves VEGF stimulation, Akt phosphorylation, and nitric oxide production; and the anti-vascular repair mechanism is driven by marked intracellular cholesterol depletion and related disorganisation of key intracellular structures. CONCLUSIONS: A beneficial effect of low

  8. Endothelial dysfunction and reduced heart rate variability in patients with metabolic syndrome

    Directory of Open Access Journals (Sweden)

    Elena Nikolaevna Smirnova

    2018-03-01

    Full Text Available According to experts of the World Health Organization (WHO, metabolic syndrome (MS can be considered as pandemy of the XXI century, because its prevalence among the population of developed countries is about 25-35%. In this study with the purpose of complex investigation of the autonomic nervous system and endothelial function we included 66 patients with MS between the ages of 25 and 61 (46.9±9.9 years. A comparison group of apparently healthy individuals (16 individuals, average age of 45.3±2.3 years; P>0.05 was studied. To evaluate the response of microvascular tone, we used the method of wavelet analysis of skin temperature oscillations during cooling of the limb. All patients underwent the study of heart rate variability. The levels of insulin, endothelin-1, and vascular endothelial growth factor were determined using enzyme immunoassay. Patients with MS had significant differences in all metabolic parameters. Our study showed that in the group of MS there is a decrease of the variability of heart rhythm compared with the healthy group. Conducting cold test revealed signs of endothelial dysfunction in the MS group, which was manifested by the decrease of the index of vasodilation in the endothelial and neurogenic frequency range. In the study group we determined the increase in biochemical markers of endothelial dysfunction, which correlated with parameters of vasodilation. Also, the presence of endothelial dysfunction significantly correlated with signs of reduction of the variability of the heart rhythm.

  9. What is the contribution of two genetic variants regulating VEGF levels to type 2 diabetes risk and to microvascular complications?

    DEFF Research Database (Denmark)

    Bonnefond, Amélie; Saulnier, Pierre-Jean; Stathopoulou, Maria G

    2013-01-01

    Vascular endothelial growth factor (VEGF) is a key chemokine involved in tissue growth and organ repair processes, particularly angiogenesis. Elevated circulating VEGF levels are believed to play a role in type 2 diabetes (T2D) microvascular complications, especially diabetic retinopathy. Recently...... for diabetic nephropathy (N(cases)¿=¿1,242-N(controls)¿=¿860) and the other for diabetic retinopathy (N(cases)¿=¿1,336-N(controls)¿=¿1,231). The effects of each SNP on quantitative traits were analyzed in a French general population-based cohort (N¿=¿4,760) and two French T2D studies (N¿=¿3,480). SNP...... on diabetic microvascular complications or the variation in related traits in T2D patients.In spite of their impact on the variance in circulating VEGF, we did not find any association between SNPs rs6921438 and rs10738760, and the risk of T2D, diabetic nephropathy or retinopathy. The link between VEGF and T2...

  10. Sodium-dependent vitamin C transporter 2 (SVCT2 expression and activity in brain capillary endothelial cells after transient ischemia in mice.

    Directory of Open Access Journals (Sweden)

    Burkhard Gess

    Full Text Available Expression and transport activity of Sodium-dependent Vitamin C Transporter 2 (SVCT2 was shown in various tissues and organs. Vitamin C was shown to be cerebroprotective in several animal models of stroke. Data on expression, localization and transport activity of SVCT2 after cerebral ischemia, however, has been scarce so far. Thus, we studied the expression of SVCT2 after middle cerebral artery occlusion (MCAO in mice by immunohistochemistry. We found an upregulation of SVCT2 after stroke. Co-stainings with Occludin, Von-Willebrand Factor and CD34 demonstrated localization of SVCT2 in brain capillary endothelial cells in the ischemic area after stroke. Time-course analyses of SVCT2 expression by immunohistochemistry and western blots showed upregulation in the subacute phase of 2-5 days. Radioactive uptake assays using (14C-labelled ascorbic acid showed a significant increase of ascorbic acid uptake into the brain after stroke. Taken together, these results provide evidence for the expression and transport activity of SVCT2 in brain capillary endothelial cells after transient ischemia in mice. These results may lead to the development of novel neuroprotective strategies in stroke therapy.

  11. Clinical significance of nailfold capillaroscopy in systemic lupus erythematosus: correlation with endothelial cell activation markers and disease activity.

    Science.gov (United States)

    Kuryliszyn-Moskal, A; Ciolkiewicz, M; Klimiuk, P A; Sierakowski, S

    2009-01-01

    To evaluate whether nailfold capillaroscopy (NC) changes are associated with the main serum endothelial cell activation markers and the disease activity of systemic lupus erythematosus (SLE). Serum levels of vascular endothelial growth factor (VEGF), endothelin-1 (ET-1), soluble E-selectin (sE-selectin), and soluble thrombomodulin (sTM) were determined by an enzyme-linked immunosorbent assay (ELISA) in 80 SLE patients and 33 healthy controls. Nailfold capillary abnormalities were seen in 74 out of 80 (92.5%) SLE patients. A normal capillaroscopic pattern or mild changes were found in 33 (41.25%) and moderate/severe abnormalities in 47 (58.75%) of all SLE patients. In SLE patients a capillaroscopic score >1 was more frequently associated with the presence of internal organ involvement (p 1 and controls. SLE patients with severe/moderate capillaroscopic abnormalities showed significantly higher VEGF serum levels than patients with mild changes (p < 0.001). Moreover, there was a significant positive correlation between the severity of capillaroscopic changes and the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) (p < 0.005) as well as between capillaroscopic score and VEGF serum levels (p < 0.001). Our findings confirm the usefulness of NC as a non-invasive technique for the evaluation of microvascular involvement in SLE patients. A relationship between changes in NC, endothelial cell activation markers and clinical features of SLE suggest an important role for microvascular abnormalities in clinical manifestation of the disease.

  12. Role of Transporters in Central Nervous System Drug Delivery and Blood-Brain Barrier Protection: Relevance to Treatment of Stroke

    Directory of Open Access Journals (Sweden)

    Hrvoje Brzica

    2017-03-01

    Full Text Available Ischemic stroke is a leading cause of morbidity and mortality in the United States. The only approved pharmacologic treatment for ischemic stroke is thrombolysis via recombinant tissue plasminogen activator (r-tPA. A short therapeutic window and serious adverse events (ie, hemorrhage, excitotoxicity greatly limit r-tPA therapy, which indicates an essential need to develop novel stroke treatment paradigms. Transporters expressed at the blood-brain barrier (BBB provide a significant opportunity to advance stroke therapy via central nervous system delivery of drugs that have neuroprotective properties. Examples of such transporters include organic anion–transporting polypeptides (Oatps and organic cation transporters (Octs. In addition, multidrug resistance proteins (Mrps are transporter targets in brain microvascular endothelial cells that can be exploited to preserve BBB integrity in the setting of stroke. Here, we review current knowledge on stroke pharmacotherapy and demonstrate how endogenous BBB transporters can be targeted for improvement of ischemic stroke treatment.

  13. In vitro models of the blood-brain barrier

    DEFF Research Database (Denmark)

    Helms, Hans Christian Cederberg; Abbott, N Joan; Burek, Malgorzata

    2016-01-01

    The endothelial cells lining the brain capillaries separate the blood from the brain parenchyma. The endothelial monolayer of the brain capillaries serves both as a crucial interface for exchange of nutrients, gases, and metabolites between blood and brain, and as a barrier for neurotoxic...... components of plasma and xenobiotics. This "blood-brain barrier" function is a major hindrance for drug uptake into the brain parenchyma. Cell culture models, based on either primary cells or immortalized brain endothelial cell lines, have been developed, in order to facilitate in vitro studies of drug...... transport to the brain and studies of endothelial cell biology and pathophysiology. In this review, we aim to give an overview of established in vitro blood-brain barrier models with a focus on their validation regarding a set of well-established blood-brain barrier characteristics. As an ideal cell culture...

  14. Microvascular Recruitment in Insulin Resistance

    DEFF Research Database (Denmark)

    Sjøberg, Kim Anker

    the resonating sound from the microbubbles in the systemic circulation were recorded for determination of microvascular recruitment in designated muscle segments. Results showed that microvascular recruitment increased with insulin stimulation by ~30% in rats and ~40% in humans (study I). Furthermore......, it was observed that muscle contractions increased muscle perfusion rapidly by 3-4 fold and by 1-2 fold compared to basal and insulin, respectively, in both rat and human skeletal muscle (study I). The real-time contrast-enhanced ultrasound method was applied to investigate the vaso-active effect of the incretin...... hormone glucagon-like-peptide-1 (GLP-1) in the microcirculation. Glucagon-like-peptide-1 analogs are drugs used for treatments of insulin resistance and type 2 diabetes but the vascular effects of GLP-1 in vivo are elusive. Here it was shown that GLP-1 rapidly increased the microvascular recruitment...

  15. Biocompatibility studies of endothelial cells on a novel calcium phosphate/SiO2-xerogel composite for bone tissue engineering

    International Nuclear Information System (INIS)

    Thimm, Benjamin W; Unger, Ronald E; Kirkpatrick, C James; Neumann, Hans-Georg

    2008-01-01

    The bone biomaterial BONITmatrix, a nanoporous, granular scaffold composed of hydroxylapatite, calcium phosphate and SiO 2 , linked by a dense collagen mesh, was tested for its biocompatibility using endothelial cells (EC) in the form of macrovascular HUVEC, microvascular HDMEC and the endothelial cell line ISOHAS-1. Cells were examined for their adherence and growth on the biomaterial and this was followed by confocal laser scanning microscopy after vital staining or immunocytochemical reactions, as well as by scanning electron microscopy. Macro- and microvascular ECs predominantly spread on BONITmatrix-collagen mesh-covered surfaces and fibres and maintained their typical morphology. As ECs in vivo must build up a functional vasculature, the seeded cells were further tested for proinflammatory expression markers and cytokine expression after lipopolysaccharide stimulation. Protein-coating studies revealed that BONITmatrix-collagen scaffolds needed human blood serum coating to successfully support the growth of ECs. All cells expressed endothelium-specific surface marker proteins such as PECAM-1, VE-cadherin and vWF. The in vitro data support recent in vivo studies and indicate that this calcium phosphate/SiO 2 -xerogel composite could be a useful scaffold material for tissue engineering

  16. Placenta growth factor and vascular endothelial growth factor B expression in the hypoxic lung

    LENUS (Irish Health Repository)

    Sands, Michelle

    2011-01-25

    Abstract Background Chronic alveolar hypoxia, due to residence at high altitude or chronic obstructive lung diseases, leads to pulmonary hypertension, which may be further complicated by right heart failure, increasing morbidity and mortality. In the non-diseased lung, angiogenesis occurs in chronic hypoxia and may act in a protective, adaptive manner. To date, little is known about the behaviour of individual vascular endothelial growth factor (VEGF) family ligands in hypoxia-induced pulmonary angiogenesis. The aim of this study was to examine the expression of placenta growth factor (PlGF) and VEGFB during the development of hypoxic pulmonary angiogenesis and their functional effects on the pulmonary endothelium. Methods Male Sprague Dawley rats were exposed to conditions of normoxia (21% O2) or hypoxia (10% O2) for 1-21 days. Stereological analysis of vascular structure, real-time PCR analysis of vascular endothelial growth factor A (VEGFA), VEGFB, placenta growth factor (PlGF), VEGF receptor 1 (VEGFR1) and VEGFR2, immunohistochemistry and western blots were completed. The effects of VEGF ligands on human pulmonary microvascular endothelial cells were determined using a wound-healing assay. Results Typical vascular remodelling and angiogenesis were observed in the hypoxic lung. PlGF and VEGFB mRNA expression were significantly increased in the hypoxic lung. Immunohistochemical analysis showed reduced expression of VEGFB protein in hypoxia although PlGF protein was unchanged. The expression of VEGFA mRNA and protein was unchanged. In vitro PlGF at high concentration mimicked the wound-healing actions of VEGFA on pulmonary microvascular endothelial monolayers. Low concentrations of PlGF potentiated the wound-healing actions of VEGFA while higher concentrations of PlGF were without this effect. VEGFB inhibited the wound-healing actions of VEGFA while VEGFB and PlGF together were mutually antagonistic. Conclusions VEGFB and PlGF can either inhibit or potentiate the

  17. Agrin in Alzheimer's Disease: Altered Solubility and Abnormal Distribution within Microvasculature and Brain Parenchyma

    Science.gov (United States)

    Donahue, John E.; Berzin, Tyler M.; Rafii, Michael S.; Glass, David J.; Yancopoulos, George D.; Fallon, Justin R.; Stopa, Edward G.

    1999-05-01

    Agrin is a heparan sulfate proteoglycan that is widely expressed in neurons and microvascular basal lamina in the rodent and avian central nervous system. Agrin induces the differentiation of nerve-muscle synapses, but its function in either normal or diseased brains is not known. Alzheimer's disease (AD) is characterized by loss of synapses, changes in microvascular architecture, and formation of neurofibrillary tangles and senile plaques. Here we have asked whether AD causes changes in the distribution and biochemical properties of agrin. Immunostaining of normal, aged human central nervous system revealed that agrin is expressed in neurons in multiple brain areas. Robust agrin immunoreactivity was observed uniformly in the microvascular basal lamina. In AD brains, agrin is highly concentrated in both diffuse and neuritic plaques as well as neurofibrillary tangles; neuronal expression of agrin also was observed. Furthermore, patients with AD had microvascular alterations characterized by thinning and fragmentation of the basal lamina. Detergent extraction and Western blotting showed that virtually all the agrin in normal brain is soluble in 1% SDS. In contrast, a large fraction of the agrin in AD brains is insoluble under these conditions, suggesting that it is tightly associated with β -amyloid. Together, these data indicate that the agrin abnormalities observed in AD are closely linked to β -amyloid deposition. These observations suggest that altered agrin expression in the microvasculature and the brain parenchyma contribute to the pathogenesis of AD.

  18. Transfection of primary brain capillary endothelial cells for protein synthesis and secretion of recombinant erythropoietin: a strategy to enable protein delivery to the brain

    DEFF Research Database (Denmark)

    Burkhart, Annette; Andresen, Thomas Lars; Aigner, Achim

    2017-01-01

    , as turning BCECs into recombinant protein factories by transfection could result in protein secretion further into the brain. The present study aims to investigate the possibility of transfecting primary rat brain endothelial cells (RBECs) for recombinant protein synthesis and secretion...... of the neuroprotective protein erythropoietin (EPO). We previously showed that 4% of RBECs with BBB properties can be transfected without disrupting the BBB integrity in vitro, but it can be questioned whether this is sufficient to enable protein secretion at therapeutic levels. The present study examined various......-derived neurotropic factor (BDNF). In conclusion, non-viral gene therapy to RBECs leads to protein secretion and signifies a method for therapeutic proteins to target cells inside the CNS otherwise omitted due to the BBB....

  19. Sex Differences Influencing Micro- and Macrovascular Endothelial Phenotype In Vitro.

    Science.gov (United States)

    Huxley, Virginia H; Kemp, Scott S; Schramm, Christine; Sieveking, Steve; Bingaman, Susan; Yu, Yang; Zaniletti, Isabella; Stockard, Kevin; Wang, Jianjie

    2018-06-09

    Endothelial dysfunction is an early hallmark of multiple disease states that also display sex differences with respect to age of onset, frequency, and severity. Results of in vivo studies of basal and stimulated microvascular barrier function revealed sex differences difficult to ascribe to specific cells or environmental factors. The present study evaluated endothelial cells (EC) isolated from macro- and/or microvessels of reproductively mature rats under the controlled conditions of low-passage culture to test the assumption that EC phenotype would be sex-independent. The primary finding was that EC, regardless of where they are derived, retain a sex-bias in low-passage culture, independent of varying levels of reproductive hormones. Implications of the work include the fallacy of expecting a universal set of mechanisms derived from study of EC from one sex and/or one vascular origin to apply uniformly to all EC under unstimulated conditions no less in the disease state. Vascular endothelial cells (EC) are heterogeneous with respect to phenotype reflecting at least organ of origin, location within the vascular network, and physical forces. Sex, as an independent influence on EC functions in health or etiology, susceptibility, and progression of dysfunction in numerous disease states, has been largely ignored. The current study focussed on EC isolated from aorta (macrovascular) and skeletal muscle vessels (microvascular) of age-matched male and female rats under identical conditions of short term (passage 4) culture. We tested the hypothesis that genomic sex would not influence endothelial growth, wound healing, morphology, lactate production, or messenger RNA and protein expression of key proteins (sex hormone receptors for androgen (AR) and oestrogen (ERα and ERβ); PECAM-1 and VE-CAD mediating barrier function; α v β 3 and N-Cadherin influencing matrix interactions; ICAM-1 and VCAM-1 mediating EC/white cell adhesion). The hypothesis was rejected as EC origin

  20. Methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxypyrovalerone (MDPV) induce differential cytotoxic effects in bovine brain microvessel endothelial cells.

    Science.gov (United States)

    Rosas-Hernandez, Hector; Cuevas, Elvis; Lantz, Susan M; Rice, Kenner C; Gannon, Brenda M; Fantegrossi, William E; Gonzalez, Carmen; Paule, Merle G; Ali, Syed F

    2016-08-26

    Designer drugs such as synthetic psychostimulants are indicative of a worldwide problem of drug abuse and addiction. In addition to methamphetamine (METH), these drugs include 3,4-methylenedioxy-methamphetamine (MDMA) and commercial preparations of synthetic cathinones including 3,4-methylenedioxypyrovalerone (MDPV), typically referred to as "bath salts." These psychostimulants exert neurotoxic effects by altering monoamine systems in the brain. Additionally, METH and MDMA adversely affect the integrity of the blood-brain barrier (BBB): there are no current reports on the effects of MDPV on the BBB. The aim of this study was to compare the effects of METH, MDMA and MDPV on bovine brain microvessel endothelial cells (bBMVECs), an accepted in vitro model of the BBB. Confluent bBMVEC monolayers were treated with METH, MDMA and MDPV (0.5mM-2.5mM) for 24h. METH and MDMA increased lactate dehydrogenase release only at the highest concentration (2.5mM), whereas MDPV induced cytotoxicity at all concentrations. MDMA and METH decreased cellular proliferation only at 2.5mM, with similar effects observed after MDPV exposures starting at 1mM. Only MDPV increased reactive oxygen species production at all concentrations tested whereas all 3 drugs increased nitric oxide production. Morphological analysis revealed different patterns of compound-induced cell damage. METH induced vacuole formation at 1mM and disruption of the monolayer at 2.5mM. MDMA induced disruption of the endothelial monolayer from 1mM without vacuolization. On the other hand, MDPV induced monolayer disruption at doses ≥0.5mM without vacuole formation; at 2.5mM, the few remaining cells lacked endothelial morphology. These data suggest that even though these synthetic psychostimulants alter monoaminergic systems, they each induce BBB toxicity by different mechanisms with MDPV being the most toxic. Published by Elsevier Ireland Ltd.

  1. The Endo-Lysosomal System of Brain Endothelial Cells Is Influenced by Astrocytes In Vitro.

    Science.gov (United States)

    Toth, Andrea E; Siupka, Piotr; P Augustine, Thomas J; Venø, Susanne T; Thomsen, Louiza B; Moos, Torben; Lohi, Hannes T; Madsen, Peder; Lykke-Hartmann, Karin; Nielsen, Morten S

    2018-03-20

    Receptor- and adsorptive-mediated transport through brain endothelial cells (BEC) of the blood-brain barrier (BBB) involves a complex array of subcellular vesicular structures, the endo-lysosomal system. It consists of several types of vesicles, such as early, recycling, and late endosomes, retromer-positive structures, and lysosomes. Since this system is important for receptor-mediated transcytosis of drugs across brain capillaries, our aim was to characterise the endo-lysosomal system in BEC with emphasis on their interactions with astrocytes. We used primary porcine BEC in monoculture and in co-culture with primary rat astrocytes. The presence of astrocytes changed the intraendothelial vesicular network and significantly impacted vesicular number, morphology, and distribution. Additionally, gene set enrichment analysis revealed that 60 genes associated with vesicular trafficking showed altered expression in co-cultured BEC. Cytosolic proteins involved in subcellular trafficking were investigated to mark transport routes, such as RAB25 for transcytosis. Strikingly, the adaptor protein called AP1-μ1B, important for basolateral sorting in epithelial cells, was not expressed in BEC. Altogether, our data pin-point unique features of BEC trafficking network, essentially mapping the endo-lysosomal system of in vitro BBB models. Consequently, our findings constitute a valuable basis for planning the optimal route across the BBB when advancing drug delivery to the brain.

  2. 26S Proteasome regulation of Ankrd1/CARP in adult rat ventricular myocytes and human microvascular endothelial cells

    International Nuclear Information System (INIS)

    Samaras, Susan E.; Chen, Billy; Koch, Stephen R.; Sawyer, Douglas B.; Lim, Chee Chew; Davidson, Jeffrey M.

    2012-01-01

    Highlights: ► The 26S proteasome regulates Ankrd1 levels in cardiomyocytes and endothelial cells. ► Ankrd1 protein degrades 60-fold faster in endothelial cells than cardiomyocytes. ► Differential degradation appears related to nuclear vs. sarcolemmal localization. ► Endothelial cell density shows uncoupling of Ankrd1 mRNA and protein levels. -- Abstract: Ankyrin repeat domain 1 protein (Ankrd1), also known as cardiac ankyrin repeat protein (CARP), increases dramatically after tissue injury, and its overexpression improves aspects of wound healing. Reports that Ankrd1/CARP protein stability may affect cardiovascular organization, together with our findings that the protein is crucial to stability of the cardiomyocyte sarcomere and increased in wound healing, led us to compare the contribution of Ankrd1/CARP stability to its abundance. We found that the 26S proteasome is the dominant regulator of Ankrd1/CARP degradation, and that Ankrd1/CARP half-life is significantly longer in cardiomyocytes (h) than endothelial cells (min). In addition, higher endothelial cell density decreased the abundance of the protein without affecting steady state mRNA levels. Taken together, our data and that of others indicate that Ankrd1/CARP is highly regulated at multiple levels of its expression. The striking difference in protein half-life between a muscle and a non-muscle cell type suggests that post-translational proteolysis is correlated with the predominantly structural versus regulatory role of the protein in the two cell types.

  3. Cyclooxygenase inhibition improves endothelial vasomotor dysfunction of visceral adipose arterioles in human obesity

    Science.gov (United States)

    Farb, Melissa G.; Tiwari, Stephanie; Karki, Shakun; Ngo, Doan TM; Carmine, Brian; Hess, Donald T.; Zuriaga, Maria A.; Walsh, Kenneth; Fetterman, Jessica L.; Hamburg, Naomi M.; Vita, Joseph A.; Apovian, Caroline M.; Gokce, Noyan

    2013-01-01

    Objective The purpose of this study was to determine whether cyclooxygenase inhibition improves vascular dysfunction of adipose microvessels from obese humans. Design and Methods In 20 obese subjects (age 37±12 yrs, BMI 47±8 kg/m2) we collected subcutaneous and visceral fat during bariatric surgery and characterized adipose depot-specific gene expression, endothelial cell phenotype, and microvascular function. Vasomotor function was assessed in response to endothelium-dependent agonists using videomicroscopy of small arterioles from fat. Results Arterioles from visceral fat exhibited impaired endothelium-dependent, acetylcholine-mediated vasodilation, compared to the subcutaneous depot (p<0.001). Expression of mRNA transcripts relevant to the cyclooxygenase pathway were upregulated in visceral compared to subcutaneous fat. Pharmacological inhibition of cyclooxygenase with indomethacin improved endothelium-dependent vasodilator function of arterioles from visceral fat by 2-fold (p=0.01), whereas indomethacin had no effect in the subcutaneous depot. Indomethacin increased activation via serine-1177 phosphorylation of endothelial nitric oxide synthase in response to acetylcholine in endothelial cells from visceral fat. Inhibition of endothelial nitric oxide synthase with Nω-nitro-L-arginine methyl ester abrogated the effects of cyclooxygenase-inhibition suggesting that vascular actions of indomethacin were related to increased nitric oxide bioavailability. Conclusions Our findings suggest that cyclooxygenase-mediated vasoconstrictor prostanoids partly contribute to endothelial dysfunction of visceral adipose arterioles in human obesity. PMID:23640904

  4. Solid lipid nanoparticles carrying chemotherapeutic drug across the blood-brain barrier through insulin receptor-mediated pathway.

    Science.gov (United States)

    Kuo, Yung-Chih; Shih-Huang, Chun-Yuan

    2013-09-01

    Carmustine (BCNU)-loaded solid lipid nanoparticles (SLNs) were grafted with 83-14 monoclonal antibody (MAb) (83-14 MAb/BCNU-SLNs) and applied to the brain-targeting delivery. Human brain-microvascular endothelial cells (HBMECs) incubated with 83-14 MAb/BCNU-SLNs were stained to demonstrate the interaction between the nanocarriers and expressed insulin receptors (IRs). The results revealed that the particle size of 83-14 MAb/BCNU-SLNs decreased with an increasing weight percentage of Dynasan 114 (DYN). Storage at 4 °C for 6 weeks slightly deformed the colloidal morphology. In addition, poloxamer 407 on 83-14 MAb/BCNU-SLNs induced cytotoxicity to RAW264.7 cells and inhibited phagocytosis by RAW264.7 cells. An increase in the weight percentage of DYN from 0% to 67% slightly reduced the viability of RAW264.7 cells and promoted phagocytosis. Moreover, the transport ability of 83-14 MAb/BCNU-SLNs across the blood-brain barrier (BBB) in vitro enhanced with an increasing weight percentage of Tween 80. 83-14 MAb on MAb/BCNU-SLNs stimulated endocytosis by HBMECs via IRs and enhanced the permeability of BCNU across the BBB. 83-14 MAb/BCNU-SLNs can be a promising antitumor drug delivery system for transporting BCNU to the brain.

  5. Pathogenic triad in bacterial meningitis: pathogen invasion, NF-κB activation and leukocyte transmigration that occur at the Blood-Brain Barrier

    Directory of Open Access Journals (Sweden)

    Sheng-He eHuang

    2016-02-01

    Full Text Available Bacterial meningitis remains the leading cause of disabilities worldwide. This life-threatening disease has a high mortality rate despite the availability of antibiotics and improved critical care. The interactions between bacterial surface components and host defense systems that initiate bacterial meningitis have been studied in molecular and cellular detail over the past several decades. Bacterial meningitis commonly exhibits triad hallmark features (THFs: pathogen penetration, nuclear factor-kappaB (NF-B activation in coordination with type 1 interferon (IFN signaling and leukocyte transmigration that occur at the blood-brain barrier (BBB, which consists mainly of brain microvascular endothelial cells (BMEC. This review outlines the progression of these early inter-correlated events contributing to the central nervous system (CNS inflammation and injury during the pathogenesis of bacterial meningitis. A better understanding of these issues is not only imperative to elucidating the pathogenic mechanism of bacterial meningitis, but may also provide the in-depth insight into the development of novel therapeutic interventions against this disease.

  6. Expression of PKA inhibitor (PKI) gene abolishes cAMP-mediated protection to endothelial barrier dysfunction.

    Science.gov (United States)

    Lum, H; Jaffe, H A; Schulz, I T; Masood, A; RayChaudhury, A; Green, R D

    1999-09-01

    We investigated the hypothesis that cAMP-dependent protein kinase (PKA) protects against endothelial barrier dysfunction in response to proinflammatory mediators. An E1-, E3-, replication-deficient adenovirus (Ad) vector was constructed containing the complete sequence of PKA inhibitor (PKI) gene (AdPKI). Infection of human microvascular endothelial cells (HMEC) with AdPKI resulted in overexpression of PKI. Treatment with 0.5 microM thrombin increased transendothelial albumin clearance rate (0.012 +/- 0.003 and 0.035 +/- 0.005 microl/min for control and thrombin, respectively); the increase was prevented with forskolin + 3-isobutyl-1-methylxanthine (F + I) treatment. Overexpression of PKI resulted in abrogation of the F + I-induced inhibition of the permeability increase. However, with HMEC infected with ultraviolet-inactivated AdPKI, the F + I-induced inhibition was present. Also, F + I treatment of HMEC transfected with reporter plasmid containing the cAMP response element-directed transcription of the luciferase gene resulted in an almost threefold increase in luciferase activity. Overexpression of PKI inhibited this induction of luciferase activity. The results show that Ad-mediated overexpression of PKI in endothelial cells abrogated the cAMP-mediated protection against increased endothelial permeability, providing direct evidence that cAMP-dependent protein kinase promotes endothelial barrier function.

  7. Glutamate Efflux at the Blood-Brain Barrier

    DEFF Research Database (Denmark)

    Cederberg-Helms, Hans Christian; Uhd-Nielsen, Carsten; Brodin, Birger

    2014-01-01

    is well known, however endothelial cells may also play an important role through mediating brain-to-blood L-glutamate efflux. Expression of excitatory amino acid transporters has been demonstrated in brain endothelial cells of bovine, human, murine, rat and porcine origin. These can account for high...... affinity concentrative uptake of L-glutamate from the brain interstitial fluid into the capillary endothelial cells. The mechanisms in between L-glutamate uptake in the endothelial cells and L-glutamate appearing in the blood are still unclear and may involve a luminal transporter for L......-glutamate, metabolism of L-glutamate and transport of metabolites or a combination of the two. However, both in vitro and in vivo studies have demonstrated blood-to-brain transport of L-glutamate, at least during pathological events. This review summarizes the current knowledge on the brain-to-blood L-glutamate efflux...

  8. Biocompatibility studies of endothelial cells on a novel calcium phosphate/SiO{sub 2}-xerogel composite for bone tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    Thimm, Benjamin W; Unger, Ronald E; Kirkpatrick, C James [Institute of Pathology, Johannes Gutenberg-University Mainz, Langenbeckstr.1, 55101 Mainz (Germany); Neumann, Hans-Georg [DOT GmbH, Charles-Darwin-Ring 1a, 18059 Rostock (Germany)], E-mail: runger@uni-mainz.de

    2008-03-01

    The bone biomaterial BONITmatrix, a nanoporous, granular scaffold composed of hydroxylapatite, calcium phosphate and SiO{sub 2}, linked by a dense collagen mesh, was tested for its biocompatibility using endothelial cells (EC) in the form of macrovascular HUVEC, microvascular HDMEC and the endothelial cell line ISOHAS-1. Cells were examined for their adherence and growth on the biomaterial and this was followed by confocal laser scanning microscopy after vital staining or immunocytochemical reactions, as well as by scanning electron microscopy. Macro- and microvascular ECs predominantly spread on BONITmatrix-collagen mesh-covered surfaces and fibres and maintained their typical morphology. As ECs in vivo must build up a functional vasculature, the seeded cells were further tested for proinflammatory expression markers and cytokine expression after lipopolysaccharide stimulation. Protein-coating studies revealed that BONITmatrix-collagen scaffolds needed human blood serum coating to successfully support the growth of ECs. All cells expressed endothelium-specific surface marker proteins such as PECAM-1, VE-cadherin and vWF. The in vitro data support recent in vivo studies and indicate that this calcium phosphate/SiO{sub 2}-xerogel composite could be a useful scaffold material for tissue engineering.

  9. An in vitro transport model for rapid screening and predicting the permeability of candidate compounds at blood-brain barrier.

    Science.gov (United States)

    Yang, Zhi-Hong; Sun, Xiao; Mei, Chao; Sun, Xiao-Bo; Liu, Xiao-Dong; Chang, Qi

    2011-12-01

    The aim of this study was to design and develop a simple in vitro blood-brain barrier (BBB) permeation model for elementarily and rapidly predicting the permeability of candidate compounds at BBB and further evaluating whether P-glycoprotein (P-gp) affects them across BBB. The model was mainly composed of cultured rat brain microvascular endothelial cells (rBMECs), glass contraption, and micropore membrane. First, we evaluated the model by morphological observation. Second, the restriction effects of paracellular transport were verified by measuring marker probes transport, and monitoring transendothelial electrical resistance (TEER) and leakage. Finally, protein expression and activity of P-gp were confirmed by carrying out Western blot analysis and polarized transport of rhodamine-123 (Rho123) in rBMECs. The rBMECs retained both endothelial cells and BBB features. The rBMECs model reproducibly attained approximately 130 Ω cm² on the steady-state TEER value, and displayed a barrier function to marker probes transport by decreasing the permeability. Protein band of 170 kDa manifested the existence of P-gp in the rBMECs, and the findings of cyclosporin A-sensitive decrease of Rho123 efflux confirmed the presence of P-gp activity. A simple, rapid, and convenient in vitro BBB permeation model was successfully established and applied to evaluate the BBB transport profiles of three natural flavonoids: quercetin, naringenin, and rutin.

  10. The adaptor CRADD/RAIDD controls activation of endothelial cells by proinflammatory stimuli.

    Science.gov (United States)

    Qiao, Huan; Liu, Yan; Veach, Ruth A; Wylezinski, Lukasz; Hawiger, Jacek

    2014-08-08

    A hallmark of inflammation, increased vascular permeability, is induced in endothelial cells by multiple agonists through stimulus-coupled assembly of the CARMA3 signalosome, which contains the adaptor protein BCL10. Previously, we reported that BCL10 in immune cells is targeted by the "death" adaptor CRADD/RAIDD (CRADD), which negatively regulates nuclear factor κB (NFκB)-dependent cytokine and chemokine expression in T cells (Lin, Q., Liu, Y., Moore, D. J., Elizer, S. K., Veach, R. A., Hawiger, J., and Ruley, H. E. (2012) J. Immunol. 188, 2493-2497). This novel anti-inflammatory CRADD-BCL10 axis prompted us to analyze CRADD expression and its potential anti-inflammatory action in non-immune cells. We focused our study on microvascular endothelial cells because they play a key role in inflammation. We found that CRADD-deficient murine endothelial cells display heightened BCL10-mediated expression of the pleotropic proinflammatory cytokine IL-6 and chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2) in response to LPS and thrombin. Moreover, these agonists also induce significantly increased permeability in cradd(-/-), as compared with cradd(+/+), primary murine endothelial cells. CRADD-deficient cells displayed more F-actin polymerization with concomitant disruption of adherens junctions. In turn, increasing intracellular CRADD by delivery of a novel recombinant cell-penetrating CRADD protein (CP-CRADD) restored endothelial barrier function and suppressed the induction of IL-6 and MCP-1 evoked by LPS and thrombin. Likewise, CP-CRADD enhanced barrier function in CRADD-sufficient endothelial cells. These results indicate that depletion of endogenous CRADD compromises endothelial barrier function in response to inflammatory signals. Thus, we define a novel function for CRADD in endothelial cells as an inducible suppressor of BCL10, a key mediator of responses to proinflammatory agonists. © 2014 by The American Society for Biochemistry and Molecular Biology

  11. Lectin-like oxidized low-density lipoprotein receptor (LOX-1) in sickle cell disease vasculopathy

    Science.gov (United States)

    Chen, Mingyi; Qiu, Hong; Lin, Xin; Nam, David; Ogbu-Nwobodo, Lucy; Archibald, Hannah; Joslin, Amelia; Wun, Ted; Sawamura, Tatsuya; Green, Ralph

    2017-01-01

    Lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1) is an endothelial receptor for oxidized LDL. Increased expression of LOX-1 has been demonstrated in atherosclerotic lesions and diabetic vasculopathy. In this study, we investigate the expression of LOX-1 receptor in sickle cell disease (SCD) vasculopathy. Expression of LOX-1 in brain vascular endothelium is markedly increased and LOX-1 gene expression is upregulated in cultured human brain microvascular endothelial cells by incubation with SCD erythrocytes. Also, the level of circulating soluble LOX-1 concentration is elevated in the plasma of SCD patients. Increased LOX-1 expression in endothelial cells is potentially involved in the pathogenesis of SCD vasculopathy. Soluble LOX-1 concentration in SCD may provide a novel biomarker for risk stratification of sickle cell vascular complications. PMID:27519944

  12. Rat Pial Microvascular Changes During Cerebral Blood Flow Decrease and Recovery: Effects of Cyanidin Administration

    Directory of Open Access Journals (Sweden)

    Teresa Mastantuono

    2018-05-01

    Full Text Available The reactive oxygen species (ROS are known to play a major role in many pathophysiological conditions, such as ischemia and reperfusion injury. The present study was aimed to evaluate the in vivo cyanidin (anthocyanin effects on damages induced by rat pial microvascular hypoperfusion-reperfusion injury by cerebral blood flow decrease (CBFD and subsequent cerebral blood flow recovery (CBFR. In particular, the main purpose was to detect changes in ROS production after cyanidin administration. Rat pial microvasculature was investigated using fluorescence microscopy through a cranial window (closed; Strahler's method was utilized to define the geometric features of pial vessels. ROS production was investigated in vivo by 2′-7′-dichlorofluorescein-diacetate assay and neuronal damage was measured on isolated brain sections by 2,3,5-triphenyltetrazolium chloride staining. After 30 min of CBFD, induced by bilateral common carotid artery occlusion, and 60 min of CBFR, rats showed decrease of arteriolar diameter and capillary perfusion; furthermore, increase in microvascular leakage and leukocyte adhesion was observed. Conversely, cyanidin administration induced dose-related arteriolar dilation, reduction in microvascular permeability as well as leukocyte adhesion when compared to animals subjected to restriction of cerebral blood flow; moreover, capillary perfusion was protected. ROS generation increase and marked neuronal damage were detected in animals subjected to CBFD and CBFR. On the other hand, cyanidin was able to reduce ROS generation and neuronal damage. In conclusion, cyanidin treatment showed dose-related protective effects on rat pial microcirculation during CBFD and subsequent CBFR, inducing arteriolar dilation by nitric oxide release and inhibiting ROS formation, consequently preserving the blood brain barrier integrity.

  13. Up-regulation of K{sub ir}2.1 by ER stress facilitates cell death of brain capillary endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Kito, Hiroaki [Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan); Yamazaki, Daiju [Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan); Department of Biological Chemistry, Kyoto University, Graduate School of Pharmaceutical Sciences, Kyoto (Japan); Department of Molecular Neurobiology, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Ohya, Susumu; Yamamura, Hisao [Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan); Asai, Kiyofumi [Department of Molecular Neurobiology, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Imaizumi, Yuji, E-mail: yimaizum@phar.nagoya-cu.ac.jp [Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan)

    2011-07-29

    Highlights: {yields} We found that application of endoplasmic reticulum (ER) stress with tunicamycin to brain capillary endothelial cells (BCECs) induced cell death. {yields} The ER stress facilitated the expression of inward rectifier K{sup +} channel (K{sub ir}2.1) and induced sustained membrane hyperpolarization. {yields} The membrane hyperpolarization induced sustained Ca{sup 2+} entry through voltage-independent nonspecific cation channels and consequently facilitated cell death. {yields} The K{sub ir}2.1 up-regulation by ER stress is, at least in part, responsible for cell death of BCECs under pathological conditions. -- Abstract: Brain capillary endothelial cells (BCECs) form blood brain barrier (BBB) to maintain brain homeostasis. Cell turnover of BCECs by the balance of cell proliferation and cell death is critical for maintaining the integrity of BBB. Here we found that stimuli with tunicamycin, endoplasmic reticulum (ER) stress inducer, up-regulated inward rectifier K{sup +} channel (K{sub ir}2.1) and facilitated cell death in t-BBEC117, a cell line derived from bovine BCECs. The activation of K{sub ir} channels contributed to the establishment of deeply negative resting membrane potential in t-BBEC117. The deep resting membrane potential increased the resting intracellular Ca{sup 2+} concentration due to Ca{sup 2+} influx through non-selective cation channels and thereby partly but significantly regulated cell death in t-BBEC117. The present results suggest that the up-regulation of K{sub ir}2.1 is, at least in part, responsible for cell death/cell turnover of BCECs induced by a variety of cellular stresses, particularly ER stress, under pathological conditions.

  14. Regulation of brain capillary endothelial cells by P2Y receptors coupled to Ca2+, phospholipase C and mitogen-activated protein kinase.

    Science.gov (United States)

    Albert, J L; Boyle, J P; Roberts, J A; Challiss, R A; Gubby, S E; Boarder, M R

    1997-11-01

    1. The blood-brain barrier is formed by capillary endothelial cells and is regulated by cell-surface receptors, such as the G protein-coupled P2Y receptors for nucleotides. Here we investigated some of the characteristics of control of brain endothelial cells by these receptors, characterizing the phospholipase C and Ca2+ response and investigating the possible involvement of mitogen-activated protein kinases (MAPK). 2. Using an unpassaged primary culture of rat brain capillary endothelial cells we showed that ATP, UTP and 2-methylthio ATP (2MeSATP) give similar and substantial increases in cytosolic Ca2+, with a rapid rise to peak followed by a slower decline towards basal or to a sustained plateau. Removal of extracellular Ca2+ had little effect on the peak Ca2+-response, but resulted in a more rapid decline to basal. There was no response to alpha,beta-MethylATP (alpha,beta MeATP) in these unpassaged cells, but a response to this P2X agonist was seen after a single passage. 3. ATP (log EC50 -5.1+/-0.2) also caused an increase in the total [3H]-inositol (poly)phosphates ([3H]-InsPx) in the presence of lithium with a rank order of agonist potency of ATP=UTP=UDP>ADP, with 2MeSATP and alpha,beta MeATP giving no detectable response. 4. Stimulating the cells with ATP or UTP gave a rapid rise in the level of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), with a peak at 10 s followed by a decline to a sustained plateau phase. 2MeSATP gave no detectable increase in the level of Ins(1,4,5)P3. 5. None of the nucleotides tested affected basal cyclic AMP, while ATP and ATPgammaS, but not 2MeSATP, stimulated cyclic AMP levels in the presence of 5 microM forskolin. 6. Both UTP and ATP stimulated tyrosine phosphorylation of p42 and p44 mitogen-activated protein kinase (MAPK), while 2MeSATP gave a smaller increase in this index of MAPK activation. By use of a peptide kinase assay, UTP gave a substantial increase in MAPK activity with a concentration-dependency consistent with

  15. Secoisolariciresinol diglucoside is a blood-brain barrier protective and anti-inflammatory agent: implications for neuroinflammation.

    Science.gov (United States)

    Rom, Slava; Zuluaga-Ramirez, Viviana; Reichenbach, Nancy L; Erickson, Michelle A; Winfield, Malika; Gajghate, Sachin; Christofidou-Solomidou, Melpo; Jordan-Sciutto, Kelly L; Persidsky, Yuri

    2018-01-27

    Secoisolariciresinol diglucoside (SDG), the main lignan in flaxseed, is known for its beneficial effects in inflammation, oxidative stress, heart disease, tumor progression, atherosclerosis, and diabetes. SDG might be an attractive natural compound that protects against neuroinflammation. Yet, there are no comprehensive studies to date investigating the effects of SDG on brain endothelium using relevant in vivo and in vitro models. We evaluated the effects of orally administered SDG on neuroinflammatory responses using in vivo imaging of the brain microvasculature during systemic inflammation and aseptic encephalitis. In parallel, the anti-inflammatory actions of SDG on brain endothelium and monocytes were evaluated in vitro blood-brain barrier (BBB) model. Multiple group comparisons were performed by one-way analysis of variance with Dunnet's post hoc tests. We found that SDG diminished leukocyte adhesion to and migration across the BBB in vivo in the setting of aseptic encephalitis (intracerebral TNFα injection) and prevented enhanced BBB permeability during systemic inflammatory response (LPS injection). In vitro SDG pretreatment of primary human brain microvascular endothelial cells (BMVEC) or human monocytes diminished adhesion and migration of monocytes across brain endothelial monolayers in conditions mimicking CNS inflammatory responses. Consistent with our in vivo observations, SDG decreased expression of the adhesion molecule, VCAM1, induced by TNFα, or IL-1β in BMVEC. SDG diminished expression of the active form of VLA-4 integrin (promoting leukocyte adhesion and migration) and prevented the cytoskeleton changes in primary human monocytes activated by relevant inflammatory stimuli. This study indicates that SDG directly inhibits BBB interactions with inflammatory cells and reduces the inflammatory state of leukocytes. Though more work is needed to determine the mechanism by which SDG mediates these effects, the ability of SDG to exert a multi

  16. Endothelium-Derived 5-Methoxytryptophan Protects Endothelial Barrier Function by Blocking p38 MAPK Activation.

    Directory of Open Access Journals (Sweden)

    Ling-Yun Chu

    Full Text Available The endothelial junction is tightly controlled to restrict the passage of blood cells and solutes. Disruption of endothelial barrier function by bacterial endotoxins, cytokines or growth factors results in inflammation and vascular damage leading to vascular diseases. We have identified 5-methoxytryptophan (5-MTP as an anti-inflammatory factor by metabolomic analysis of conditioned medium of human fibroblasts. Here we postulated that endothelial cells release 5-MTP to protect the barrier function. Conditioned medium of human umbilical vein endothelial cells (HUVECs prevented endothelial hyperpermeability and VE-cadherin downregulation induced by VEGF, LPS and cytokines. We analyzed the metabolomic profile of HUVEC conditioned medium and detected 5-MTP but not melatonin, serotonin or their catabolites, which was confirmed by enzyme-linked immunosorbent assay. Addition of synthetic pure 5-MTP preserved VE-cadherin and maintained barrier function despite challenge with pro-inflammatory mediators. Tryptophan hydroxylase-1, an enzyme required for 5-MTP biosynthesis, was downregulated in HUVECs by pro-inflammatory mediators and it was accompanied by reduction of 5-MTP. 5-MTP protected VE-cadherin and prevented endothelial hyperpermeability by blocking p38 MAPK activation. A chemical inhibitor of p38 MAPK, SB202190, exhibited a similar protective effect as 5-MTP. To determine whether 5-MTP prevents vascular hyperpermeability in vivo, we evaluated the effect of 5-MTP administration on LPS-induced murine microvascular permeability with Evans blue. 5-MTP significantly prevented Evans blue dye leakage. Our findings indicate that 5-MTP is a new class of endothelium-derived molecules which protects endothelial barrier function by blocking p38 MAPK.

  17. Uterine microvascular sensitivity to nanomaterial inhalation: An in vivo assessment

    Energy Technology Data Exchange (ETDEWEB)

    Stapleton, P.A.; McBride, C.R.; Yi, J.; Nurkiewicz, T.R., E-mail: tnurkiewicz@hsc.wvu.edu

    2015-11-01

    With the tremendous number and diverse applications of engineered nanomaterials incorporated in daily human activity, exposure can no longer be solely confined to occupational exposures of healthy male models. Cardiovascular and endothelial cell dysfunction have been established using in vitro and in situ preparations, but the translation to intact in vivo models is limited. Intravital microscopy has been used extensively to understand microvascular physiology while maintaining in vivo neurogenic, humoral, and myogenic control. However, a tissue specific model to assess the influences of nanomaterial exposure on female reproductive health has not been fully elucidated. Female Sprague Dawley (SD) rats were exposed to nano-TiO{sub 2} aerosols (171 ± 6 nm, 10.1 ± 0.39 mg/m{sup 3}, 5 h) 24-hours prior to experimentation, leading to a calculated deposition of 42.0 ± 1.65 μg. After verifying estrus status, vital signs were monitored and the right horn of the uterus was exteriorized, gently secured over an optical pedestal, and enclosed in a warmed tissue bath using intravital microscopy techniques. After equilibration, significantly higher leukocyte-endothelium interactions were recorded in the exposed group. Arteriolar responsiveness was assessed using ionophoretically applied agents: muscarinic agonist acetylcholine (0.025 M; ACh; 20, 40, 100, and 200 nA), and nitric oxide donor sodium nitroprusside (0.05 M; SNP; 20, 40, and 100 nA), or adrenergic agonist phenylephrine (0.05 M; PE; 20, 40, and 100 nA) using glass micropipettes. Passive diameter was established by tissue superfusion with 10{sup −4} M adenosine. Similar to male counterparts, female SD rats present systemic microvascular dysfunction; however the ramifications associated with female health and reproduction have yet to be elucidated. - Highlights: • Female reproductive health associated with nanomaterial exposure is understudied. • We examined uterine microvascular alterations 24-hours after nano

  18. Local heart irradiation of ApoE−/− mice induces microvascular and endocardial damage and accelerates coronary atherosclerosis

    International Nuclear Information System (INIS)

    Gabriels, Karen; Hoving, Saske; Seemann, Ingar; Visser, Nils L.; Gijbels, Marion J.; Pol, Jeffrey F.; Daemen, Mat J.; Stewart, Fiona A.; Heeneman, Sylvia

    2012-01-01

    Background and purpose: Radiotherapy of thoracic and chest-wall tumors increases the long-term risk of radiation-induced heart disease, like a myocardial infarct. Cancer patients commonly have additional risk factors for cardiovascular disease, such as hypercholesterolemia. The goal of this study is to define the interaction of irradiation with such cardiovascular risk factors in radiation-induced damage to the heart and coronary arteries. Material and methods: Hypercholesterolemic and atherosclerosis-prone ApoE −/− mice received local heart irradiation with a single dose of 0, 2, 8 or 16 Gy. Histopathological changes, microvascular damage and functional alterations were assessed after 20 and 40 weeks. Results: Inflammatory cells were significantly increased in the left ventricular myocardium at 20 and 40 weeks after 8 and 16 Gy. Microvascular density decreased at both follow-up time-points after 8 and 16 Gy. Remaining vessels had decreased alkaline phosphatase activity (2–16 Gy) and increased von Willebrand Factor expression (16 Gy), indicative of endothelial cell damage. The endocardium was extensively damaged after 16 Gy, with foam cell accumulations at 20 weeks, and fibrosis and protein leakage at 40 weeks. Despite an accelerated coronary atherosclerotic lesion development at 20 weeks after 16 Gy, gated SPECT and ultrasound measurements showed only minor changes in functional cardiac parameters at 20 weeks. Conclusions: The combination of hypercholesterolemia and local cardiac irradiation induced an inflammatory response, microvascular and endocardial damage, and accelerated the development of coronary atherosclerosis. Despite these pronounced effects, cardiac function of ApoE −/− mice was maintained.

  19. Optical clearing and fluorescence deep-tissue imaging for 3D quantitative analysis of the brain tumor microenvironment

    NARCIS (Netherlands)

    Lagerweij, Tonny; Dusoswa, Sophie A.; Negrean, Adrian; Hendrikx, Esther M.L.; de Vries, Helga E.; Kole, Jeroen; Garcia-Vallejo, Juan J.; Mansvelder, Huibert D; Vandertop, W. Peter; Noske, David P.; Tannous, Bakhos A.; Musters, René J P; van Kooyk, Yvette; Wesseling, Pieter; Zhao, Xi Wen; Wurdinger, Thomas

    2017-01-01

    Background: Three-dimensional visualization of the brain vasculature and its interactions with surrounding cells may shed light on diseases where aberrant microvascular organization is involved, including glioblastoma (GBM). Intravital confocal imaging allows 3D visualization of microvascular

  20. Hypoxia/reoxygenation increases the permeability of endothelial cell monolayers: Role of oxygen radicals

    International Nuclear Information System (INIS)

    Inauen, W.; Payne, D.K.; Kvietys, P.R.; Granger, D.N.

    1990-01-01

    We assessed the effect of hypoxia/reoxygenation on 14C-albumin flux across endothelial monolayers. Cultured bovine pulmonary artery endothelial cells were grown to confluence on nitrocellulose filters (pore size 12 microns). The endothelialized filters were mounted in Ussing-type chambers which were filled with cell culture medium (M 199). Equimolar amounts (33 nM) of 14C-labeled and unlabeled albumin were added to the hot and cold chambers, respectively. The monolayers were then exposed to successive periods (90 min) of normoxia (pO2 145 mmHg), hypoxia (pO2 20 mmHg), and reoxygenation (pO2 145 mmHg). A gas bubbling system was used to control media pO2 and to ensure adequate mixing. Four aliquots of culture media were taken during each period in order to calculate the 14C-albumin permeability across the endothelialized filter. In some experiments, either the xanthine oxidase inhibitor, oxypurinol (10 microM), or superoxide dismutase (600 U/mL), was added to the media immediately prior to the experiments. As compared to the normoxic control period, albumin permeability was 1.5 times higher during hypoxia (p less than 0.01) and 2.3 times higher during reoxygenation (p less than 0.01). The reoxygenation-induced increase in albumin permeability was prevented by either oxypurinol or superoxide dismutase. These data indicate that xanthine oxidase-derived oxygen radicals contribute to the hypoxia/reoxygenation-induced endothelial cell dysfunction. The altered endothelial barrier function induced by hypoxia/reoxygenation is consistent with the microvascular dysfunction observed following reperfusion of ischemic tissues

  1. Senescence from glioma stem cell differentiation promotes tumor growth

    International Nuclear Information System (INIS)

    Ouchi, Rie; Okabe, Sachiko; Migita, Toshiro; Nakano, Ichiro; Seimiya, Hiroyuki

    2016-01-01

    Glioblastoma (GBM) is a lethal brain tumor composed of heterogeneous cellular populations including glioma stem cells (GSCs) and differentiated non-stem glioma cells (NSGCs). While GSCs are involved in tumor initiation and propagation, NSGCs' role remains elusive. Here, we demonstrate that NSGCs undergo senescence and secrete pro-angiogenic proteins, boosting the GSC-derived tumor formation in vivo. We used a GSC model that maintains stemness in neurospheres, but loses the stemness and differentiates into NSGCs upon serum stimulation. These NSGCs downregulated telomerase, shortened telomeres, and eventually became senescent. The senescent NSGCs released pro-angiogenic proteins, including vascular endothelial growth factors and senescence-associated interleukins, such as IL-6 and IL-8. Conditioned medium from senescent NSGCs promoted proliferation of brain microvascular endothelial cells, and mixed implantation of GSCs and senescent NSGCs into mice enhanced the tumorigenic potential of GSCs. The senescent NSGCs seem to be clinically relevant, because both clinical samples and xenografts of GBM contained tumor cells that expressed the senescence markers. Our data suggest that senescent NSGCs promote malignant progression of GBM in part via paracrine effects of the secreted proteins. - Highlights: • Non-stem glioma cells (NSGCs) lose telomerase and eventually become senescent. • Senescent NSGCs secrete pro-angiogenic proteins, such as VEGFs, IL-6, and IL-8. • Senescent NSGCs enhance the growth of brain microvascular endothelial cells. • Senescent NSGCs enhance the tumorigenic potential of glioma stem cells in vivo.

  2. Senescence from glioma stem cell differentiation promotes tumor growth

    Energy Technology Data Exchange (ETDEWEB)

    Ouchi, Rie [Division of Molecular Biotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 3-8-31 Ariake, Koto-ku, Tokyo 135-8550 (Japan); Laboratory of Molecular Target Therapy of Cancer, Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, 3-8-31 Ariake, Koto-ku, Tokyo 135-8550 (Japan); Okabe, Sachiko; Migita, Toshiro [Division of Molecular Biotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 3-8-31 Ariake, Koto-ku, Tokyo 135-8550 (Japan); Nakano, Ichiro [Department of Neurosurgery, Comprehensive Cancer Center, University of Alabama at Birmingham, 1824 6th Avenue South, Birmingham, AL 35233 (United States); Seimiya, Hiroyuki, E-mail: hseimiya@jfcr.or.jp [Division of Molecular Biotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 3-8-31 Ariake, Koto-ku, Tokyo 135-8550 (Japan); Laboratory of Molecular Target Therapy of Cancer, Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, 3-8-31 Ariake, Koto-ku, Tokyo 135-8550 (Japan)

    2016-02-05

    Glioblastoma (GBM) is a lethal brain tumor composed of heterogeneous cellular populations including glioma stem cells (GSCs) and differentiated non-stem glioma cells (NSGCs). While GSCs are involved in tumor initiation and propagation, NSGCs' role remains elusive. Here, we demonstrate that NSGCs undergo senescence and secrete pro-angiogenic proteins, boosting the GSC-derived tumor formation in vivo. We used a GSC model that maintains stemness in neurospheres, but loses the stemness and differentiates into NSGCs upon serum stimulation. These NSGCs downregulated telomerase, shortened telomeres, and eventually became senescent. The senescent NSGCs released pro-angiogenic proteins, including vascular endothelial growth factors and senescence-associated interleukins, such as IL-6 and IL-8. Conditioned medium from senescent NSGCs promoted proliferation of brain microvascular endothelial cells, and mixed implantation of GSCs and senescent NSGCs into mice enhanced the tumorigenic potential of GSCs. The senescent NSGCs seem to be clinically relevant, because both clinical samples and xenografts of GBM contained tumor cells that expressed the senescence markers. Our data suggest that senescent NSGCs promote malignant progression of GBM in part via paracrine effects of the secreted proteins. - Highlights: • Non-stem glioma cells (NSGCs) lose telomerase and eventually become senescent. • Senescent NSGCs secrete pro-angiogenic proteins, such as VEGFs, IL-6, and IL-8. • Senescent NSGCs enhance the growth of brain microvascular endothelial cells. • Senescent NSGCs enhance the tumorigenic potential of glioma stem cells in vivo.

  3. Impaired microvascular reactivity and endothelial function in patients with Cushing's syndrome: Influence of arterial hypertension

    Czech Academy of Sciences Publication Activity Database

    Prázný, M.; Ježková, J.; Horová, E.; Lazárová, V.; Hána, V.; Kvasnička, J.; Pecen, Ladislav; Marek, J.; Škrha, J.; Kršek, M.

    2008-01-01

    Roč. 57, č. 1 (2008), s. 13-22 ISSN 0862-8408 Institutional research plan: CEZ:AV0Z10300504 Keywords : Cushing’s syndrome * vascular reactivity * endothelial function * oxidative stress * laser Doppler flowmetry Subject RIV: FB - Endocrinology, Diabetology, Metabolism, Nutrition Impact factor: 1.653, year: 2008

  4. Optical clearing and fluorescence deep-tissue imaging for 3D quantitative analysis of the brain tumor microenvironment

    NARCIS (Netherlands)

    Lagerweij, Tonny; Dusoswa, Sophie A.; Negrean, Adrian; Hendrikx, Esther M. L.; de Vries, Helga E.; Kole, Jeroen; Garcia-Vallejo, Juan J.; Mansvelder, Huibert D.; Vandertop, W. Peter; Noske, David P.; Tannous, Bakhos A.; Musters, René J. P.; van Kooyk, Yvette; Wesseling, Pieter; Zhao, Xi Wen; Wurdinger, Thomas

    2017-01-01

    Three-dimensional visualization of the brain vasculature and its interactions with surrounding cells may shed light on diseases where aberrant microvascular organization is involved, including glioblastoma (GBM). Intravital confocal imaging allows 3D visualization of microvascular structures and

  5. Experimental diode laser-assisted microvascular anastomosis.

    Science.gov (United States)

    Reali, U M; Gelli, R; Giannotti, V; Gori, F; Pratesi, R; Pini, R

    1993-05-01

    An experimental study to evaluate a diode-laser approach to microvascular end-to-end anastomoses is reported. Studies were carried out on the femoral arteries and veins of Wistar rats, and effective welding of vessel tissue was obtained at low laser power, by enhancing laser absorption with indocyanine green (Cardio-green) solution. The histologic and surgical effects of this laser technique were examined and compared with those of conventional microvascular sutured anastomoses.

  6. Scleroderma dermal microvascular endothelial cells exhibit defective response to pro-angiogenic chemokines

    Science.gov (United States)

    Rabquer, Bradley J.; Ohara, Ray A.; Stinson, William A.; Campbell, Phillip L.; Amin, M. Asif; Balogh, Beatrix; Zakhem, George; Renauer, Paul A.; Lozier, Ann; Arasu, Eshwar; Haines, G. Kenneth; Kahaleh, Bashar; Schiopu, Elena; Khanna, Dinesh; Koch, Alisa E.

    2016-01-01

    Objectives. Angiogenesis plays a critical role in SSc (scleroderma). The aim of this study was to examine the expression of growth-regulated protein-γ (Gro-γ/CXCL3), granulocyte chemotactic protein 2 (GCP-2/CXCL6) and their receptor CXCR2 in endothelial cells (ECs) isolated from SSc skin and determine whether these cells mount an angiogenic response towards pro-angiogenic chemokines. The downstream signalling pathways as well as the pro-angiogenic transcription factor inhibitor of DNA-binding protein 1 (Id-1) were also examined. Methods. Skin biopsies were obtained from patients with dcSSc. ECs were isolated via magnetic positive selection. Angiogenesis was measured by EC chemotaxis assay. Results. Gro-γ/CXCL3 and GCP-2/CXCL6 were minimally expressed in both skin types but elevated in SSc serum. Pro-angiogenic chemokine mRNA was greater in SSc ECs than in normal ECs. SSc ECs did not migrate to vascular endothelial growth factor (VEGF), Gro-γ/CXCL3, GCP-2/CXCL6 or CXCL16. The signalling pathways stimulated by these chemokines were also dysregulated. Id-1 mRNA in SSc ECs was lower compared with normal ECs, and overexpression of Id-1 in SSc ECs increased their ability to migrate towards VEGF and CXCL16. Conclusion. Our results show that SSc ECs are unable to respond to pro-angiogenic chemokines despite their increased expression in serum and ECs. This might be due to the differences in the signalling pathways activated by these chemokines in normal vs SSc ECs. In addition, the lower expression of Id-1 also decreases the angiogenic response. The inability of pro-angiogenic chemokines to promote EC migration provides an additional mechanism for the impaired angiogenesis that characterizes SSc. PMID:26705326

  7. DC8 and DC13 var genes associated with severe malaria bind avidly to diverse endothelial cells.

    Directory of Open Access Journals (Sweden)

    Marion Avril

    Full Text Available During blood stage infection, Plasmodium falciparum infected erythrocytes (IE bind to host blood vessels. This virulence determinant enables parasites to evade spleen-dependent killing mechanisms, but paradoxically in some cases may reduce parasite fitness by killing the host. Adhesion of infected erythrocytes is mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1, a family of polymorphic adhesion proteins encoded by var genes. Whereas cerebral binding and severe malaria are associated with parasites expressing DC8 and DC13 var genes, relatively little is known about the non-brain endothelial selection on severe malaria adhesive types. In this study, we selected P. falciparum-IEs on diverse endothelial cell types and demonstrate that DC8 and DC13 var genes were consistently among the major var transcripts selected on non-brain endothelial cells (lung, heart, bone marrow. To investigate the molecular basis for this avid endothelial binding activity, recombinant proteins were expressed from the predominant upregulated DC8 transcript, IT4var19. In-depth binding comparisons revealed that multiple extracellular domains from this protein bound brain and non-brain endothelial cells, and individual domains largely did not discriminate between different endothelial cell types. Additionally, we found that recombinant DC8 and DC13 CIDR1 domains exhibited a widespread endothelial binding activity and could compete for DC8-IE binding to brain endothelial cells, suggesting they may bind the same host receptor. Our findings provide new insights into the interaction of severe malaria adhesive types and host blood vessels and support the hypothesis that parasites causing severe malaria express PfEMP1 variants with a superior ability to adhere to diverse endothelial cell types, and may therefore endow these parasites with a growth and transmission advantage.

  8. Methylmercury Causes Blood-Brain Barrier Damage in Rats via Upregulation of Vascular Endothelial Growth Factor Expression.

    Directory of Open Access Journals (Sweden)

    Tetsuya Takahashi

    Full Text Available Clinical manifestations of methylmercury (MeHg intoxication include cerebellar ataxia, concentric constriction of visual fields, and sensory and auditory disturbances. The symptoms depend on the site of MeHg damage, such as the cerebellum and occipital lobes. However, the underlying mechanism of MeHg-induced tissue vulnerability remains to be elucidated. In the present study, we used a rat model of subacute MeHg intoxication to investigate possible MeHg-induced blood-brain barrier (BBB damage. The model was established by exposing the rats to 20-ppm MeHg for up to 4 weeks; the rats exhibited severe cerebellar pathological changes, although there were no significant differences in mercury content among the different brain regions. BBB damage in the cerebellum after MeHg exposure was confirmed based on extravasation of endogenous immunoglobulin G (IgG and decreased expression of rat endothelial cell antigen-1. Furthermore, expression of vascular endothelial growth factor (VEGF, a potent angiogenic growth factor, increased markedly in the cerebellum and mildly in the occipital lobe following MeHg exposure. VEGF expression was detected mainly in astrocytes of the BBB. Intravenous administration of anti-VEGF neutralizing antibody mildly reduced the rate of hind-limb crossing signs observed in MeHg-exposed rats. In conclusion, we demonstrated for the first time that MeHg induces BBB damage via upregulation of VEGF expression at the BBB in vivo. Further studies are required in order to determine whether treatment targeted at VEGF can ameliorate MeHg-induced toxicity.

  9. Endothelial-astrocytic interactions in acute liver failure.

    Science.gov (United States)

    Jayakumar, A R; Norenberg, M D

    2013-06-01

    Brain edema and the subsequent increase in intracranial pressure are major neurological complications of acute liver failure (ALF), and swelling of astrocytes (cytotoxic brain edema) is the most prominent neuropathological abnormality in ALF. Recent studies, however, have suggested the co-existence of cytotoxic and vasogenic mechanisms in the brain edema associated with ALF. This review 1) summarizes the nature of the brain edema in humans and experimental animals with ALF; 2) reviews in vitro studies supporting the presence of cytotoxic brain edema (cell swelling in cultured astrocytes); and 3) documents the role of brain endothelial cells in the development of astrocyte swelling/brain edema in ALF.

  10. Functional Response of Tumor Vasculature to PaCO2: Determination of Total and Microvascular Blood Volume by MRI

    Directory of Open Access Journals (Sweden)

    Scott D. Packard

    2003-07-01

    Full Text Available In order to identify differences in functional activity, we compared the reactivity of glioma vasculature and the native cerebral vasculature to both dilate and constrict in response to altered PaCO2. Gliomas were generated by unilateral implantation of U87MGdEGFR human glioma tumor cells into the striatum of adult female athymic rats. Relative changes in total and microvascular cerebral blood volume were determined by steady state contrast agent-enhanced magnetic resonance imaging for transitions from normocarbia to hypercarbia and hypocarbia. Although hypercarbia induced a significant increase in both total and microvascular blood volume in normal brain and glioma, reactivity of glioma vasculature was significantly blunted in comparison to normal striatum; glioma total CBV increased by 0.6±0.1%/mm Hg CO2 whereas normal striatum increased by 1.5±0.2%/mm Hg CO2, (P < .0001, group t-test. Reactivity of microvascular blood volume was also significantly blunted. In contrast, hypocarbia decreased both total and microvascular blood volumes more in glioma than in normal striatum. These results indicate that cerebral blood vessels derived by tumor-directed angiogenesis do retain reactivity to CO2. Furthermore, reduced reactivity of tumor vessels to a single physiological perturbation, such as hypercarbia, should not be construed as a generalized reduction of functional activity of the tumor vascular bed.

  11. Omeprazole induces heme oxygenase-1 in fetal human pulmonary microvascular endothelial cells via hydrogen peroxide-independent Nrf2 signaling pathway

    International Nuclear Information System (INIS)

    Patel, Ananddeep; Zhang, Shaojie; Shrestha, Amrit Kumar; Maturu, Paramahamsa; Moorthy, Bhagavatula; Shivanna, Binoy

    2016-01-01

    Omeprazole (OM) is an aryl hydrocarbon receptor (AhR) agonist and a proton pump inhibitor that is used to treat humans with gastric acid related disorders. Recently, we showed that OM induces NAD (P) H quinone oxidoreductase-1 (NQO1) via nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent mechanism. Heme oxygenase-1 (HO-1) is another cytoprotective and antioxidant enzyme that is regulated by Nrf2. Whether OM induces HO-1 in fetal human pulmonary microvascular endothelial cells (HPMEC) is unknown. Therefore, we tested the hypothesis that OM will induce HO-1 expression via Nrf2 in HPMEC. OM induced HO-1 mRNA and protein expression in a dose-dependent manner. siRNA-mediated knockdown of AhR failed to abrogate, whereas knockdown of Nrf2 abrogated HO-1 induction by OM. To identify the underlying molecular mechanisms, we determined the effects of OM on cellular hydrogen peroxide (H 2 O 2 ) levels since oxidative stress mediated by the latter is known to activate Nrf2. Interestingly, the concentration at which OM induced HO-1 also increased H 2 O 2 levels. Furthermore, H 2 O 2 independently augmented HO-1 expression. Although N-acetyl cysteine (NAC) significantly decreased H 2 O 2 levels in OM-treated cells, we observed that OM further increased HO-1 mRNA and protein expression in NAC-pretreated compared to vehicle-pretreated cells, suggesting that OM induces HO-1 via H 2 O 2 -independent mechanisms. In conclusion, we provide evidence that OM transcriptionally induces HO-1 via AhR - and H 2 O 2 - independent, but Nrf2 - dependent mechanisms. These results have important implications for human disorders where Nrf2 and HO-1 play a beneficial role. - Highlights: • Omeprazole induces HO-1 in human fetal lung cells. • AhR deficiency fails to abrogate omeprazole-mediated induction of HO-1. • Nrf2 knockdown abrogates omeprazole-mediated HO-1 induction in human lung cells. • Hydrogen peroxide depletion augments omeprazole-mediated induction of HO-1.

  12. Endothelial heterogeneity in the umbilico-placental unit: DNA methylation as an innuendo of epigenetic diversity

    Science.gov (United States)

    Casanello, Paola; Schneider, Daniela; Herrera, Emilio A.; Uauy, Ricardo; Krause, Bernardo J.

    2014-01-01

    The endothelium is a multifunctional heterogeneous tissue playing a key role in the physiology of every organ. To accomplish this role the endothelium presents a phenotypic diversity that is early prompted during vascular development, allowing it to cope with specific requirements in a time- and site-specific manner. During the last decade several reports show that endothelial diversity is also present in the umbilico-placental vasculature, with differences between macro- and microvascular vessels as well as arterial and venous endothelium. This diversity is evidenced in vitro as a higher angiogenic capacity in the microcirculation; or disparity in the levels of several molecules that control endothelial function (i.e., receptor for growth factors, vasoactive mediators, and adhesion molecules) which frequently are differentially expressed between arterial and venous endothelium. Emerging evidence suggests that endothelial diversity would be prominently driven by epigenetic mechanisms which also control the basal expression of endothelial-specific genes. This review outlines evidence for endothelial diversity since early stages of vascular development and how this heterogeneity is expressed in the umbilico-placental vasculature. Furthermore a brief picture of epigenetic mechanisms and their role on endothelial physiology emphasizing new data on umbilical and placental endothelial cells is presented. Unraveling the role of epigenetic mechanisms on long term endothelial physiology and its functional diversity would contribute to develop more accurate therapeutic interventions. Altogether these data show that micro- versus macro-vascular, or artery versus vein comparisons are an oversimplification of the complexity occurring in the endothelium at different levels, and the necessity for the future research to establish the precise source of cells which are under study. PMID:24723887

  13. Endothelial heterogeneity in the umbilico-placental unit: DNA methylation as an innuendo of epigenetic diversity

    Directory of Open Access Journals (Sweden)

    Paola eCasanello

    2014-03-01

    Full Text Available The endothelium is a multifunctional heterogeneous tissue playing a key role in the physiology of every organ. To accomplish this role the endothelium presents a phenotypic diversity that is early prompted during vascular development, allowing it to cope with specific requirements in a time- and site-specific manner. During the last decade several reports show that endothelial diversity is also present in the umbilico-placental vasculature, with differences between macro- and microvascular vessels as well as arterial and venous endothelium. This diversity is evidenced in vitro as a higher angiogenic capacity in the microcirculation; or disparity in the levels of several molecules that control endothelial function (i.e. receptor for growth factors, vasoactive mediators and adhesion molecules which frequently are differentially expressed between arterial and venous endothelium. Emerging evidence suggests that endothelial diversity would be prominently driven by epigenetic mechanisms which also control the basal expression of endothelial-specific genes. This review outlines evidence for endothelial diversity since early stages of vascular development and how this heterogeneity is expressed in the umbilico-placental vasculature. Furthermore a brief picture of epigenetic mechanisms and their role on endothelial physiology emphasising new data on umbilical and placental endothelial cells is presented. Unravelling the role of epigenetic mechanisms on long-term endothelial physiology and its functional diversity would contribute to develop more accurate therapeutic interventions. Altogether these data show that micro- versus macro-vascular, or artery versus vein comparisons are an oversimplification of the complexity occurring in the endothelium at different levels, and the necessity for the future research to establish the precise source of cells which are under study.

  14. Zingiber officinale attenuates retinal microvascular changes in diabetic rats via anti-inflammatory and antiangiogenic mechanisms

    Science.gov (United States)

    Dongare, Shirish; Mathur, Rajani; Saxena, Rohit; Mathur, Sandeep; Agarwal, Renu; Nag, Tapas C.; Srivastava, Sushma; Kumar, Pankaj

    2016-01-01

    Purpose Diabetic retinopathy is a common microvascular complication of long-standing diabetes. Several complex interconnecting biochemical pathways are activated in response to hyperglycemia. These pathways culminate into proinflammatory and angiogenic effects that bring about structural and functional damage to the retinal vasculature. Since Zingiber officinale (ginger) is known for its anti-inflammatory and antiangiogenic properties, we investigated the effects of its extract standardized to 5% 6-gingerol, the major active constituent of ginger, in attenuating retinal microvascular changes in rats with streptozotocin-induced diabetes. Methods Diabetic rats were treated orally with the vehicle or the ginger extract (75 mg/kg/day) over a period of 24 weeks along with regular monitoring of bodyweight and blood glucose and weekly fundus photography. At the end of the 24-week treatment, the retinas were isolated for histopathological examination under a light microscope, transmission electron microscopy, and determination of the retinal tumor necrosis factor-α (TNF-α), nuclear factor-kappa B (NF-κB), and vascular endothelial growth factor (VEGF) levels. Results Oral administration of the ginger extract resulted in significant reduction of hyperglycemia, the diameter of the retinal vessels, and vascular basement membrane thickness. Improvement in the architecture of the retinal vasculature was associated with significantly reduced expression of NF-κB and reduced activity of TNF-α and VEGF in the retinal tissue in the ginger extract–treated group compared to the vehicle-treated group. Conclusions The current study showed that ginger extract containing 5% of 6-gingerol attenuates the retinal microvascular changes in rats with streptozotocin-induced diabetes through anti-inflammatory and antiangiogenic actions. Although precise molecular targets remain to be determined, 6-gingerol seems to be a potential candidate for further investigation. PMID:27293376

  15. Mononuclear Phagocyte-Derived Microparticulate Caspase-1 Induces Pulmonary Vascular Endothelial Cell Injury.

    Directory of Open Access Journals (Sweden)

    Srabani Mitra

    Full Text Available Lung endothelial cell apoptosis and injury occurs throughout all stages of acute lung injury (ALI/ARDS and impacts disease progression. Lung endothelial injury has traditionally been focused on the role of neutrophil trafficking to lung vascular integrin receptors induced by proinflammatory cytokine expression. Although much is known about the pathogenesis of cell injury and death in ALI/ARDS, gaps remain in our knowledge; as a result of which there is currently no effective pharmacologic therapy. Enzymes known as caspases are essential for completion of the apoptotic program and secretion of pro-inflammatory cytokines. We hypothesized that caspase-1 may serve as a key regulator of human pulmonary microvascular endothelial cell (HPMVEC apoptosis in ALI/ARDS. Our recent experiments confirm that microparticles released from stimulated monocytic cells (THP1 induce lung endothelial cell apoptosis. Microparticles pretreated with the caspase-1 inhibitor, YVAD, or pan-caspase inhibitor, ZVAD, were unable to induce cell death of HPMVEC, suggesting the role of caspase-1 or its substrate in the induction of HPMVEC cell death. Neither un-induced microparticles (control nor direct treatment with LPS induced apoptosis of HPMVEC. Further experiments showed that caspase-1 uptake into HPMVEC and the induction of HPMVEC apoptosis was facilitated by caspase-1 interactions with microparticulate vesicles. Altering vesicle integrity completely abrogated apoptosis of HPMVEC suggesting an encapsulation requirement for target cell uptake of active caspase-1. Taken together, we confirm that microparticle centered caspase-1 can play a regulator role in endothelial cell injury.

  16. Exposure to traffic-generated air pollutants mediates alterations in brain microvascular integrity in wildtype mice on a high-fat diet.

    Science.gov (United States)

    Suwannasual, Usa; Lucero, JoAnn; McDonald, Jacob D; Lund, Amie K

    2018-01-01

    Air pollution-exposure is associated with detrimental outcomes in the central nervous system (CNS) such as cerebrovascular disorders, including stroke, and neurodegenerative diseases. While the mechanisms of these CNS-related outcomes involved have not been fully elucidated, exposure to traffic-generated air pollutants has been associated with altered blood brain barrier (BBB) integrity and permeability. The current study investigated whether inhalation exposure to mixed vehicle emissions (MVE) alters cerebral microvascular integrity in healthy 3 mo old C57BL/6 mice, as well as whether exposure-mediated effects were exacerbated by a high-fat (HF) vs. low-fat (LF) diet. Mice on each diet were randomly assigned to be exposed to either filtered air (FA) or MVE [100PM/m 3 vehicle emissions mixture: 30µg PM/m 3 gasoline engine + 70µg PM/m 3 diesel engine emissions; median size ~ 60nm; particle mass size distribution median of ~ 1µm (range: diet, results in altered BBB integrity and increased ox-LDL signaling in the cerebral vasculature in a wildtype animal model. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Functional expression of a proton-coupled organic cation (H+/OC antiporter in human brain capillary endothelial cell line hCMEC/D3, a human blood–brain barrier model

    Directory of Open Access Journals (Sweden)

    Shimomura Keita

    2013-01-01

    Full Text Available Abstract Background Knowledge of the molecular basis and transport function of the human blood–brain barrier (BBB is important for not only understanding human cerebral physiology, but also development of new central nervous system (CNS-acting drugs. However, few studies have been done using human brain capillary endothelial cells, because human brain materials are difficult to obtain. The purpose of this study is to clarify the functional expression of a proton-coupled organic cation (H+/OC antiporter in human brain capillary endothelial cell line hCMEC/D3, which has been recently developed as an in vitro human BBB model. Methods Diphenhydramine, [3H]pyrilamine and oxycodone were used as cationic drugs that proved to be H+/OC antiporter substrates. The in vitro uptake experiments by hCMEC/D3 cells were carried out under several conditions. Results Diphenhydramine and [3H]pyrilamine were both transported into hCMEC/D3 cells in a time- and concentration-dependent manner with Km values of 59 μM and 19 μM, respectively. Each inhibited uptake of the other in a competitive manner, suggesting that a common mechanism is involved in their transport. The diphenhydramine uptake was significantly inhibited by amantadine and quinidine, but not tetraethylammonium and 1-methyl-4-phenylpyridinium (substrates for well-known organic cation transporters. The uptake was inhibited by metabolic inhibitors, but was insensitive to extracellular sodium and membrane potential. Further, the uptake was increased by extracellular alkalization and intracellular acidification. These transport properties are completely consistent with those of previously characterized H+/OC antiporter in rat BBB. Conclusions The present results suggest that H+/OC antiporter is functionally expressed in hCMEC/D3 cells.

  18. Placenta growth factor and vascular endothelial growth factor B expression in the hypoxic lung

    Directory of Open Access Journals (Sweden)

    McLoughlin Paul

    2011-01-01

    Full Text Available Abstract Background Chronic alveolar hypoxia, due to residence at high altitude or chronic obstructive lung diseases, leads to pulmonary hypertension, which may be further complicated by right heart failure, increasing morbidity and mortality. In the non-diseased lung, angiogenesis occurs in chronic hypoxia and may act in a protective, adaptive manner. To date, little is known about the behaviour of individual vascular endothelial growth factor (VEGF family ligands in hypoxia-induced pulmonary angiogenesis. The aim of this study was to examine the expression of placenta growth factor (PlGF and VEGFB during the development of hypoxic pulmonary angiogenesis and their functional effects on the pulmonary endothelium. Methods Male Sprague Dawley rats were exposed to conditions of normoxia (21% O2 or hypoxia (10% O2 for 1-21 days. Stereological analysis of vascular structure, real-time PCR analysis of vascular endothelial growth factor A (VEGFA, VEGFB, placenta growth factor (PlGF, VEGF receptor 1 (VEGFR1 and VEGFR2, immunohistochemistry and western blots were completed. The effects of VEGF ligands on human pulmonary microvascular endothelial cells were determined using a wound-healing assay. Results Typical vascular remodelling and angiogenesis were observed in the hypoxic lung. PlGF and VEGFB mRNA expression were significantly increased in the hypoxic lung. Immunohistochemical analysis showed reduced expression of VEGFB protein in hypoxia although PlGF protein was unchanged. The expression of VEGFA mRNA and protein was unchanged. In vitro PlGF at high concentration mimicked the wound-healing actions of VEGFA on pulmonary microvascular endothelial monolayers. Low concentrations of PlGF potentiated the wound-healing actions of VEGFA while higher concentrations of PlGF were without this effect. VEGFB inhibited the wound-healing actions of VEGFA while VEGFB and PlGF together were mutually antagonistic. Conclusions VEGFB and PlGF can either inhibit or

  19. Injuries to the vascular endothelium: vascular wall and endothelial dysfunction.

    Science.gov (United States)

    Fisher, Mark

    2008-01-01

    Vascular endothelial injury has multiple elements, and this article focuses on ischemia-related processes that have particular relevance to ischemic stroke. Distinctions between necrotic and apoptotic cell death provide a basic science context in which to better understand the significance of classical core and penumbra concepts of acute stroke, with apoptotic processes particularly prominent in the penumbra. The mitochondria are understood to serve as a reservoir of proteins that mediate apoptosis. Oxidative stress pathways generating reactive oxygen species (ROS) are prominent in endothelial injury, both ischemic and nonischemic, with prominent roles of enzyme- and nonenzymemediated pathways; mitochondria once again have a critical role, particularly in the nonenzymatic pathways generating ROS. Inflammation also contributes to vascular endothelial injury, and endothelial cells have the capacity to rapidly increase expression of inflammatory mediators following ischemic challenge; this leads to enhanced leukocyte-endothelial interactions mediated by selectins and adhesion molecules. Preconditioning consists of a minor version of an injurious event, which in turn may protect vascular endothelium from injury following a more substantial event. Presence of the blood-brain barrier creates unique responses to endothelial injury, with permeability changes due to impairment of endothelial-matrix interactions compounding altered vasomotor tone and tissue perfusion mediated by nitric oxide. Pharmacological protection against vascular endothelial injury can be provided by several of the phosphodiesterases (cilostazol and dipyridamole), along with statins. Optimal clinical responses for protection of brain vascular endothelium may use preconditioning as a model, and will likely require combined protection against apoptosis, ROS, and inflammation.

  20. Targeted drug delivery to the brain using magnetic nanoparticles.

    Science.gov (United States)

    Thomsen, Louiza Bohn; Thomsen, Maj Schneider; Moos, Torben

    2015-01-01

    Brain capillary endothelial cells denote the blood-brain barrier (BBB), and conjugation of nanoparticles with antibodies that target molecules expressed by these endothelial cells may facilitate their uptake and transport into the brain. Magnetic nanoparticles can be encapsulated in liposomes and carry large molecules with therapeutic potential, for example, siRNA, cDNA and polypeptides. An additional approach to enhance the transport of magnetic nanoparticles across the BBB is the application of extracranially applied magnetic force. Stepwise targeting of magnetic nanoparticles to brain capillary endothelial cells followed by transport through the BBB using magnetic force may prove a novel mechanism for targeted therapy of macromolecules to the brain.

  1. Glyoxalase-1 overexpression reduces endothelial dysfunction and attenuates early renal impairment in a rat model of diabetes

    DEFF Research Database (Denmark)

    Brouwers, Olaf; Niessen, Petra M G; Miyata, Toshio

    2014-01-01

    AIMS/HYPOTHESIS: In diabetes, advanced glycation end-products (AGEs) and the AGE precursor methylglyoxal (MGO) are associated with endothelial dysfunction and the development of microvascular complications. In this study we used a rat model of diabetes, in which rats transgenically overexpressed...... the MGO-detoxifying enzyme glyoxalase-I (GLO-I), to determine the impact of intracellular glycation on vascular function and the development of early renal changes in diabetes. METHODS: Wild-type and Glo1-overexpressing rats were rendered diabetic for a period of 24 weeks by intravenous injection...... podocyte number and diabetes-induced elevation of urinary markers albumin, osteopontin, kidney-inflammation-molecule-1 and nephrin) were attenuated by Glo1 overexpression. In line with this, downregulation of Glo1 in cultured endothelial cells resulted in increased expression of inflammation...

  2. Correlates of time to microvascular complications among diabetes ...

    African Journals Online (AJOL)

    Socio-demographic and clinical factors have been known to affect the time to microvascular complications and survival probabilities of diabetes mellitus patients. The objective of this study was to identify risk factors and estimate average survival times for the time to the development of microvascular complications of ...

  3. Sodium valproate, a histone deacetylase inhibitor, modulates the vascular endothelial growth inhibitor-mediated cell death in human osteosarcoma and vascular endothelial cells.

    Science.gov (United States)

    Yamanegi, Koji; Kawabe, Mutsuki; Futani, Hiroyuki; Nishiura, Hiroshi; Yamada, Naoko; Kato-Kogoe, Nahoko; Kishimoto, Hiromitsu; Yoshiya, Shinichi; Nakasho, Keiji

    2015-05-01

    The level of vascular endothelial growth inhibitor (VEGI) has been reported to be negatively associated with neovascularization in malignant tumors. The soluble form of VEGI is a potent anti-angiogenic factor due to its effects in inhibiting endothelial cell proliferation. This inhibition is mediated by death receptor 3 (DR3), which contains a death domain in its cytoplasmic tail capable of inducing apoptosis that can be subsequently blocked by decoy receptor 3 (DcR3). We investigated the effects of sodium valproate (VPA) and trichostatin A (TSA), histone deacetylase inhibitors, on the expression of VEGI and its related receptors in human osteosarcoma (OS) cell lines and human microvascular endothelial (HMVE) cells. Consequently, treatment with VPA and TSA increased the VEGI and DR3 expression levels without inducing DcR3 production in the OS cell lines. In contrast, the effect on the HMVE cells was limited, with no evidence of growth inhibition or an increase in the DR3 and DcR3 expression. However, VPA-induced soluble VEGI in the OS cell culture medium markedly inhibited the vascular tube formation of HMVE cells, while VEGI overexpression resulted in enhanced OS cell death. Taken together, the HDAC inhibitor has anti-angiogenesis and antitumor activities that mediate soluble VEGI/DR3-induced apoptosis via both autocrine and paracrine pathways. This study indicates that the HDAC inhibitor may be exploited as a therapeutic strategy modulating the soluble VEGI/DR3 pathway in osteosarcoma patients.

  4. Studies of the endothelial origin of cells in systemic angioendotheliomatosis and other vascular lesions of the brain and meninges using ulex europaeus lectin stains.

    Science.gov (United States)

    Schelper, R L; Olson, S P; Carroll, T J; Hart, M N; Witters, E

    1986-01-01

    Ulex europaeus agglutinin I (UEA-I) is a plant lectin which binds specifically to alpha-L-fucose moieties on the surface glycoproteins of human endothelial cells. The binding is completely inhibited by preincubation of the lectin with fucose. UEA-I can be conjugated directly to fluorescein or peroxidase and can be used to stain endothelium of paraffin embedded tissues. UEA-I staining was evaluated on normal and infarcted brain, systemic angioendotheliomatosis, metastatic epidural angiosarcoma, hemangioendothelioma, hemangioblastoma, angioblastic meningioma of both the hemangioblastic and hemangiopericytic types, and vascular meningioma. The endothelium, but not neuropil of normal and infarcted brain was positive for UEA-I. The tumor cells of hemangioendothelioma and angiosarcoma also stained. However, no staining was seen in malignant intravascular cells of angioendotheliomatosis, the stromal cells of hemangioblastoma, or pericytes of angioblastic meningioma. It is concluded that the malignant cells in angioendotheliomatosis, the stromal cells of hemangioblastoma and the pericytes of angioblastic meningioma do not produce surface glycoproteins characteristic of endothelial cells.

  5. Netrin-1 Ameliorates Blood-Brain Barrier Impairment Secondary to Ischemic Stroke via the Activation of PI3K Pathway

    Directory of Open Access Journals (Sweden)

    Jian Yu

    2017-12-01

    Full Text Available Secondary impairment of blood-brain barrier (BBB occurs in the remote thalamus after ischemic stroke. Netrin-1, an axonal guidance molecule, presents bifunctional effects on blood vessels through receptor-dependent pathways. This study investigates whether netrin-1 protects BBB against secondary injury. Netrin-1 (600 ng/d for 7 days was intracerebroventricularly infused 24 h after middle cerebral artery occlusion (MCAO in hypertensive rats. Neurological function was assessed 8 and 14 days after MCAO, and the permeability of BBB in the ipsilateral thalamus was detected. The viability of brain microvascular endothelial cells was determined after being disposed with netrin-1 (50 ng/mL before oxygen-glucose deprivation (OGD. The role of netrin-1 was further explored by examining its receptors and their function. We found that netrin-1 infusion improved neurological function, attenuated secondary impairment of BBB by up-regulating the levels of tight junction proteins and diminishing extravasation of albumin, with autophagy activation 14 days after MCAO. Netrin-1 also enhanced cell survival and autophagy activity in OGD-treated cells, inhibited by UNC5H2 siRNA transfection. Furthermore, the beneficial effects of netrin-1 were suppressed by PI3K inhibitors 3-Methyladenine and LY294002. Our results showed that netrin-1 ameliorated BBB impairment secondary to ischemic stroke by promoting tight junction function and endothelial survival. PI3K-mediated autophagy activation depending on UNC5H2 receptor could be an underlying mechanism.

  6. Graft microvascular disease in solid organ transplantation.

    Science.gov (United States)

    Jiang, Xinguo; Sung, Yon K; Tian, Wen; Qian, Jin; Semenza, Gregg L; Nicolls, Mark R

    2014-08-01

    Alloimmune inflammation damages the microvasculature of solid organ transplants during acute rejection. Although immunosuppressive drugs diminish the inflammatory response, they do not directly promote vascular repair. Repetitive microvascular injury with insufficient regeneration results in prolonged tissue hypoxia and fibrotic remodeling. While clinical studies show that a loss of the microvascular circulation precedes and may act as an initiating factor for the development of chronic rejection, preclinical studies demonstrate that improved microvascular perfusion during acute rejection delays and attenuates tissue fibrosis. Therefore, preservation of a functional microvasculature may represent an effective therapeutic strategy for preventing chronic rejection. Here, we review recent advances in our understanding of the role of the microvasculature in the long-term survival of transplanted solid organs. We also highlight microvessel-centered therapeutic strategies for prolonging the survival of solid organ transplants.

  7. Heme-Mediated Induction of CXCL10 and Depletion of CD34+ Progenitor Cells Is Toll-Like Receptor 4 Dependent.

    Directory of Open Access Journals (Sweden)

    Carmen M Dickinson-Copeland

    Full Text Available Plasmodium falciparum infection can cause microvascular dysfunction, cerebral encephalopathy and death if untreated. We have previously shown that high concentrations of free heme, and C-X-C motif chemokine 10 (CXCL10 in sera of malaria patients induce apoptosis in microvascular endothelial and neuronal cells contributing to vascular dysfunction, blood-brain barrier (BBB damage and mortality. Endothelial progenitor cells (EPC are microvascular endothelial cell precursors partly responsible for repair and regeneration of damaged BBB endothelium. Studies have shown that EPC's are depleted in severe malaria patients, but the mechanisms mediating this phenomenon are unknown. Toll-like receptors recognize a wide variety of pathogen-associated molecular patterns generated by pathogens such as bacteria and parasites. We tested the hypothesis that EPC depletion during malaria pathogenesis is a function of heme-induced apoptosis mediated by CXCL10 induction and toll-like receptor (TLR activation. Heme and CXCL10 concentrations in plasma obtained from malaria patients were elevated compared with non-malaria subjects. EPC numbers were significantly decreased in malaria patients (P < 0.02 and TLR4 expression was significantly elevated in vivo. These findings were confirmed in EPC precursors in vitro; where it was determined that heme-induced apoptosis and CXCL10 expression was TLR4-mediated. We conclude that increased serum heme mediates depletion of EPC during malaria pathogenesis.

  8. Increased expression of vascular endothelial growth factor attenuates contusion necrosis without influencing contusion edema after traumatic brain injury in rats.

    Science.gov (United States)

    Tado, Masahiro; Mori, Tatsuro; Fukushima, Masamichi; Oshima, Hideki; Maeda, Takeshi; Yoshino, Atsuo; Aizawa, Shin; Katayama, Yoichi

    2014-04-01

    To clarify the role of vascular endothelial growth factor (VEGF) in the formation of contusion edema and necrosis after traumatic brain injury, we examined the time course of changes in the VEGF expression (enzyme-linked immunosorbent assay), cerebrovascular permeability (extravasation of Evans blue), and water content (dry-wet weight method) of the contused brain tissue in a cortical impact injury model using rats. In addition, we tested the effects of administration of bevacizumab (VEGF monoclonal antibody) on changes in the cerebrovascular permeability and water content of the contused brain tissue, as well as the neurological deficits (rota rod test) and volume of contusion necrosis. Increased VEGF expression was maximal at 72 h after injury (pnecrosis at 21 days (pnecrosis. This is probably because of an increased angiogenesis and improved microcirculation in the areas surrounding the core of contusion.

  9. Microvascular response of striated muscle to metal debris. A comparative in vivo study with titanium and stainless steel.

    Science.gov (United States)

    Kraft, C N; Diedrich, O; Burian, B; Schmitt, O; Wimmer, M A

    2003-01-01

    Wear products of metal implants are known to induce biological events which may have profound consequences for the microcirculation of skeletal muscle. Using the skinfold chamber model and intravital microscopy we assessed microcirculatory parameters in skeletal muscle after confrontation with titanium and stainless-steel wear debris, comparing the results with those of bulk materials. Implantation of stainless-steel bulk and debris led to a distinct activation of leukocytes combined with a disruption of the microvascular endothelial integrity and massive leukocyte extravasation. While animals with bulk stainless steel showed a tendency to recuperation, stainless-steel wear debris induced such severe inflammation and massive oedema that the microcirculation broke down within 24 hours after implantation. Titanium bulk caused only a transient increase in leukocyte-endothelial cell interaction within the first 120 minutes and no significant change in macromolecular leakage, leukocyte extravasation or venular diameter. Titanium wear debris produced a markedly lower inflammatory reaction than stainless-steel bulk, indicating that a general benefit of bulk versus debris could not be claimed. Depending on its constituents, wear debris is capable of eliciting acute inflammation which may result in endothelial damage and subsequent failure of microperfusion. Our results indicate that not only the bulk properties of orthopaedic implants but also the microcirculatory implications of inevitable wear debris play a pivotal role in determining the biocompatibility of an implant.

  10. Intravenous Lipid Infusion Induces Endoplasmic Reticulum Stress in Endothelial Cells and Blood Mononuclear Cells of Healthy Adults.

    Science.gov (United States)

    Tampakakis, Emmanouil; Tabit, Corey E; Holbrook, Monika; Linder, Erika A; Berk, Brittany D; Frame, Alissa A; Bretón-Romero, Rosa; Fetterman, Jessica L; Gokce, Noyan; Vita, Joseph A; Hamburg, Naomi M

    2016-01-11

    Endoplasmic reticulum (ER) stress and the subsequent unfolded protein response may initially be protective, but when prolonged, have been implicated in atherogenesis in diabetic conditions. Triglycerides and free fatty acids (FFAs) are elevated in patients with diabetes and may contribute to ER stress. We sought to evaluate the effect of acute FFA elevation on ER stress in endothelial and circulating white cells. Twenty-one healthy subjects were treated with intralipid (20%; 45 mL/h) plus heparin (12 U/kg/h) infusion for 5 hours. Along with increased triglyceride and FFA levels, intralipid/heparin infusion reduced the calf reactive hyperemic response without a change in conduit artery flow-mediated dilation consistent with microvascular dysfunction. To investigate the short-term effects of elevated triglycerides and FFA, we measured markers of ER stress in peripheral blood mononuclear cells (PBMCs) and vascular endothelial cells (VECs). In VECs, activating transcription factor 6 (ATF6) and phospho-inositol requiring kinase 1 (pIRE1) proteins were elevated after infusion (both P<0.05). In PBMCs, ATF6 and spliced X-box-binding protein 1 (XBP-1) gene expression increased by 2.0- and 2.5-fold, respectively (both P<0.05), whereas CHOP and GADD34 decreased by ≈67% and 74%, respectively (both P<0.01). ATF6 and pIRE1 protein levels also increased (both P<0.05), and confocal microscopy revealed the nuclear localization of ATF6 after infusion, suggesting activation. Along with microvascular dysfunction, intralipid infusion induced an early protective ER stress response evidenced by activation of ATF6 and IRE1 in both leukocytes and endothelial cells. Our results suggest a potential link between metabolic disturbances and ER stress that may be relevant to vascular disease. © 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

  11. Significance of endothelial dysfunction in the pathogenesis of early and delayed radiation enteropathy

    Institute of Scientific and Technical Information of China (English)

    Junru Wang; Marjan Boerma; Qiang Fu; Martin Hauer-Jensen

    2007-01-01

    This review summarizes the current state of knowledge regarding the role of endothelial dysfunction in the pathogenesis of early and delayed intestinal radiation toxicity and discusses various endothelial-oriented interventions aimed at reducing the risk of radiation enteropathy. Studies published in the biomedical literature during the past four decades and cited in PubMed, as well as clinical and laboratory data from our own research program are reviewed. The risk of injury to normal tissues limits the cancer cure rates that can be achieved with radiation therapy. During treatment of abdominal and pelvic tumors, the intestine is frequently a major dose-limiting factor. Microvascular injury is a prominent feature of both early (inflammatory), as well as delayed (fibroproliferative) radiation injuries in the intestine and in many other normal tissues. Evidence from our and other laboratories suggests that endothelial dysfunction, notably a deficiency of endothelial thrombomodulin, plays a key role in the pathogenesis of these radiation responses. Deficient levels of thrombomodulin cause loss of vascular thromboresistance, excessive activation of cellular thrombin receptors by thrombin, and insufficient activation of protein C, a plasma protein with anticoagulant, anti-inflammatory, and cytoprotective properties. These changes are presumed to be critically involved in many aspects of early intestinal radiation toxicity and may sustain the fibroproliferative processes that lead to delayed intestinal dysfunction, fibrosis, and clinical complications. In conclusion, injury of vascular endothelium is important in the pathogenesis of the intestinal radiation response. Endothelial-oriented interventions are appealing strategies to prevent or treat normal tissue toxicity associated with radiation treatment of cancer.

  12. Ornithine decarboxylase and extracellular polyamines regulate microvascular sprouting and actin cytoskeleton dynamics in endothelial cells

    International Nuclear Information System (INIS)

    Kucharzewska, Paulina; Welch, Johanna E.; Svensson, Katrin J.; Belting, Mattias

    2010-01-01

    The polyamines are essential for cancer cell proliferation during tumorigenesis. Targeted inhibition of ornithine decarboxylase (ODC), i.e. a key enzyme of polyamine biosynthesis, by α-difluoromethylornithine (DFMO) has shown anti-neoplastic activity in various experimental models. This activity has mainly been attributed to the anti-proliferative effect of DFMO in cancer cells. Here, we provide evidence that unperturbed ODC activity is a requirement for proper microvessel sprouting ex vivo as well as the migration of primary human endothelial cells. DFMO-mediated ODC inhibition was reversed by extracellular polyamine supplementation, showing that anti-angiogenic effects of DFMO were specifically related to polyamine levels. ODC inhibition was associated with an abnormal morphology of the actin cytoskeleton during cell spreading and migration. Moreover, our data suggest that de-regulated actin cytoskeleton dynamics in DFMO treated endothelial cells may be related to constitutive activation of the small GTPase CDC42, i.e. a well-known regulator of cell motility and actin cytoskeleton remodeling. These insights into the potential role of polyamines in angiogenesis should stimulate further studies testing the combined anti-tumor effect of polyamine inhibition and established anti-angiogenic therapies in vivo.

  13. Targeting Phosphatidylserine for Radioimmunotherapy of Breast Cancer Brain Metastasis

    Science.gov (United States)

    2015-12-01

    response. e. Correlate imaging findings with histological studies of vascular damage, tumor cell and endothelial cell apoptosis or necrosis and vascular ...phosphatidylserine (PS) is exposed exclusively on tumor vascular endothelium of brain metastases in mouse models. A novel PS-targeting antibody, PGN635... vascular endothelial cells in multi-focal brain metastases throughout the whole mouse brain. Vascular endothelium in normal brain tissues is negative

  14. Apolipoprotein B level and diabetic microvascular complications ( is there a correlation?

    Directory of Open Access Journals (Sweden)

    Mary N. Rizk

    2013-01-01

    Conclusion Apo B levels are strongly correlated to diabetic microvascular complications. The higher the degree of nephropathy, the higher the Apo B level. The presence of more than one microvascular complication correlates positively with high levels of Apo B. This suggests the possible use of Apo B as a sensitive biomarker of the presence of early diabetic microvascular complications.

  15. The inner CSF-brain barrier

    DEFF Research Database (Denmark)

    Whish, Sophie; Dziegielewska, Katarzyna M; Møllgård, Kjeld

    2015-01-01

    outlining the inner CSF-brain interface from E16; most of these markers were not present in the adult ependyma. Claudin-5 was present in the apical-most part of radial glial cells and in endothelial cells in embryos, but only in endothelial cells including plexus endothelial cells in adults. Claudin-11...

  16. Involvement of insulin-degrading enzyme in insulin- and atrial natriuretic peptide-sensitive internalization of amyloid-β peptide in mouse brain capillary endothelial cells.

    Science.gov (United States)

    Ito, Shingo; Ohtsuki, Sumio; Murata, Sho; Katsukura, Yuki; Suzuki, Hiroya; Funaki, Miho; Tachikawa, Masanori; Terasaki, Tetsuya

    2014-01-01

    Cerebral clearance of amyloid-β peptide (Aβ), which is implicated in Alzheimer's disease, involves elimination across the blood-brain barrier (BBB), and we previously showed that an insulin-sensitive process is involved in the case of Aβ1-40. The purpose of this study was to clarify the molecular mechanism of the insulin-sensitive Aβ1-40 elimination across mouse BBB. An in vivo cerebral microinjection study demonstrated that [125I]hAβ1-40 elimination from mouse brain was inhibited by human natriuretic peptide (hANP), and [125I]hANP elimination was inhibited by hAβ1-40, suggesting that hAβ1-40 and hANP share a common elimination process. Internalization of [125I]hAβ1-40 into cultured mouse brain capillary endothelial cells (TM-BBB4) was significantly inhibited by either insulin, hANP, other natriuretic peptides or insulin-degrading enzyme (IDE) inhibitors, but was not inhibited by phosphoramidon or thiorphan. Although we have reported the involvement of natriuretic peptide receptor C (Npr-C) in hANP internalization, cells stably expressing Npr-C internalized [125I]hANP but not [125I]hAβ1-40, suggesting that there is no direct interaction between Npr-C and hAβ1-40. IDE was detected in plasma membrane of TM-BBB4 cells, and internalization of [125I]hAβ1-40 by TM-BBB4 cells was reduced by IDE-targeted siRNAs. We conclude that elimination of hAβ1-40 from mouse brain across the BBB involves an insulin- and ANP-sensitive process, mediated by IDE expressed in brain capillary endothelial cells.

  17. Evaluation of microvascular endothelial function in patients with infective endocarditis using laser speckle contrast imaging and skin video-capillaroscopy: research proposal of a case control prospective study.

    Science.gov (United States)

    Barcelos, Amanda; Lamas, Cristiane; Tibiriça, Eduardo

    2017-07-28

    Infective endocarditis is a severe condition with high in-hospital and 5-year mortality. There is increasing incidence of infective endocarditis, which may be related to healthcare and changes in prophylaxis recommendations regarding oral procedures. Few studies have evaluated the microcirculation in patients with infective endocarditis, and so far, none have utilized laser-based technology or evaluated functional capillary density. The aim of the study is to evaluate the changes in the systemic microvascular bed of patients with both acute and subacute endocarditis. This is a cohort study that will include adult patients with confirmed active infective endocarditis according to the modified Duke criteria who were admitted to our center for treatment. A control group of sex- and age-matched healthy volunteers will be included. Functional capillary density, which is defined as the number of spontaneously perfused capillaries per square millimeter of skin, will be assessed by video-microscopy with an epi-illuminated fiber optic microscope. Capillary recruitment will be evaluated using post-occlusive reactive hyperemia. Microvascular flow will be evaluated in the forearm using a laser speckle contrast imaging system for the noninvasive and continuous measurement of cutaneous microvascular perfusion changes. Laser speckle contrast imaging will be used in combination with skin iontophoresis of acetylcholine, an endothelium-dependent vasodilator, or sodium nitroprusside (endothelium independent) to test microvascular reactivity. The present study will contribute to the investigation of microcirculatory changes in infective endocarditis and possibly lead to an earlier diagnosis of the condition and/or determination of its severity and complications. Trial registration ClinicalTrials.gov ID: NCT02940340.

  18. Efficient nanoparticle mediated sustained RNA interference in human primary endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Mukerjee, Anindita; Shankardas, Jwalitha; Ranjan, Amalendu P; Vishwanatha, Jamboor K, E-mail: Jamboor.vishwanatha@unthsc.edu [Department of Molecular Biology and Immunology and Institute for Cancer Research, Graduate School of Biomedical Sciences, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States)

    2011-11-04

    Endothelium forms an important target for drug and/or gene therapy since endothelial cells play critical roles in angiogenesis and vascular functions and are associated with various pathophysiological conditions. RNA mediated gene silencing presents a new therapeutic approach to overcome many such diseases, but the major challenge of such an approach is to ensure minimal toxicity and effective transfection efficiency of short hairpin RNA (shRNA) to primary endothelial cells. In the present study, we formulated shAnnexin A2 loaded poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles which produced intracellular small interfering RNA (siRNA) against Annexin A2 and brought about the downregulation of Annexin A2. The per cent encapsulation of the plasmid within the nanoparticle was found to be 57.65%. We compared our nanoparticle based transfections with Lipofectamine mediated transfection, and our studies show that nanoparticle based transfection efficiency is very high ({approx}97%) and is more sustained compared to conventional Lipofectamine mediated transfections in primary retinal microvascular endothelial cells and human cancer cell lines. Our findings also show that the shAnnexin A2 loaded PLGA nanoparticles had minimal toxicity with almost 95% of cells being viable 24 h post-transfection while Lipofectamine based transfections resulted in only 30% viable cells. Therefore, PLGA nanoparticle based transfection may be used for efficient siRNA transfection to human primary endothelial and cancer cells. This may serve as a potential adjuvant treatment option for diseases such as diabetic retinopathy, retinopathy of prematurity and age related macular degeneration besides various cancers.

  19. Efficient nanoparticle mediated sustained RNA interference in human primary endothelial cells

    Science.gov (United States)

    Mukerjee, Anindita; Shankardas, Jwalitha; Ranjan, Amalendu P.; Vishwanatha, Jamboor K.

    2011-11-01

    Endothelium forms an important target for drug and/or gene therapy since endothelial cells play critical roles in angiogenesis and vascular functions and are associated with various pathophysiological conditions. RNA mediated gene silencing presents a new therapeutic approach to overcome many such diseases, but the major challenge of such an approach is to ensure minimal toxicity and effective transfection efficiency of short hairpin RNA (shRNA) to primary endothelial cells. In the present study, we formulated shAnnexin A2 loaded poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles which produced intracellular small interfering RNA (siRNA) against Annexin A2 and brought about the downregulation of Annexin A2. The per cent encapsulation of the plasmid within the nanoparticle was found to be 57.65%. We compared our nanoparticle based transfections with Lipofectamine mediated transfection, and our studies show that nanoparticle based transfection efficiency is very high (~97%) and is more sustained compared to conventional Lipofectamine mediated transfections in primary retinal microvascular endothelial cells and human cancer cell lines. Our findings also show that the shAnnexin A2 loaded PLGA nanoparticles had minimal toxicity with almost 95% of cells being viable 24 h post-transfection while Lipofectamine based transfections resulted in only 30% viable cells. Therefore, PLGA nanoparticle based transfection may be used for efficient siRNA transfection to human primary endothelial and cancer cells. This may serve as a potential adjuvant treatment option for diseases such as diabetic retinopathy, retinopathy of prematurity and age related macular degeneration besides various cancers.

  20. Efficient nanoparticle mediated sustained RNA interference in human primary endothelial cells

    International Nuclear Information System (INIS)

    Mukerjee, Anindita; Shankardas, Jwalitha; Ranjan, Amalendu P; Vishwanatha, Jamboor K

    2011-01-01

    Endothelium forms an important target for drug and/or gene therapy since endothelial cells play critical roles in angiogenesis and vascular functions and are associated with various pathophysiological conditions. RNA mediated gene silencing presents a new therapeutic approach to overcome many such diseases, but the major challenge of such an approach is to ensure minimal toxicity and effective transfection efficiency of short hairpin RNA (shRNA) to primary endothelial cells. In the present study, we formulated shAnnexin A2 loaded poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles which produced intracellular small interfering RNA (siRNA) against Annexin A2 and brought about the downregulation of Annexin A2. The per cent encapsulation of the plasmid within the nanoparticle was found to be 57.65%. We compared our nanoparticle based transfections with Lipofectamine mediated transfection, and our studies show that nanoparticle based transfection efficiency is very high (∼97%) and is more sustained compared to conventional Lipofectamine mediated transfections in primary retinal microvascular endothelial cells and human cancer cell lines. Our findings also show that the shAnnexin A2 loaded PLGA nanoparticles had minimal toxicity with almost 95% of cells being viable 24 h post-transfection while Lipofectamine based transfections resulted in only 30% viable cells. Therefore, PLGA nanoparticle based transfection may be used for efficient siRNA transfection to human primary endothelial and cancer cells. This may serve as a potential adjuvant treatment option for diseases such as diabetic retinopathy, retinopathy of prematurity and age related macular degeneration besides various cancers.

  1. (-)-Epicatechin administration and exercising skeletal muscle vascular control and microvascular oxygenation in healthy rats.

    Science.gov (United States)

    Copp, Steven W; Inagaki, Tadakatsu; White, Michael J; Hirai, Daniel M; Ferguson, Scott K; Holdsworth, Clark T; Sims, Gabrielle E; Poole, David C; Musch, Timothy I

    2013-01-15

    Consumption of the dietary flavanol (-)-epicatechin (EPI) is associated with enhanced endothelial function and augmented skeletal muscle capillarity and mitochondrial volume density. The potential for EPI to improve peripheral vascular function and muscle oxygenation during exercise is unknown. We tested the hypothesis that EPI administration in healthy rats would improve treadmill exercise performance secondary to elevated skeletal muscle blood flow and vascular conductance [VC, blood flow/mean arterial pressure (MAP)] and improved skeletal muscle microvascular oxygenation. Rats received water (control, n = 12) or 4 mg/kg EPI (n = 12) via oral gavage daily for 24 days. Exercise endurance capacity and peak O(2) uptake (Vo(2) peak) were measured via treadmill runs to exhaustion. MAP (arterial catheter) and blood flow (radiolabeled microspheres) were measured and VC was calculated during submaximal treadmill exercise (25 m/min, 5% grade). Spinotrapezius muscle microvascular O(2) pressure (Po(2mv)) was measured (phosphorescence quenching) during electrically induced twitch (1 Hz) contractions. In conscious rats, EPI administration resulted in lower (↓~5%) resting (P = 0.03) and exercising (P = 0.04) MAP. There were no differences in exercise endurance capacity, Vo(2) peak, total exercising hindlimb blood flow (control, 154 ± 13; and EPI, 159 ± 8 ml·min(-1)·100 g(-1), P = 0.68), or VC (control, 1.13 ± 0.10; and EPI, 1.24 ± 0.08 ml·min(-1)·100 g(-1)·mmHg(-1), P = 0.21) between groups. Following anesthesia, EPI resulted in lower MAP (↓~16%) but did not impact resting Po(2mv) or any kinetics parameters (P > 0.05 for all) during muscle contractions compared with control. EPI administration (4 mg·kg(-1)·day(-1)) improved modestly cardiovascular function (i.e., ↓MAP) with no impact on exercise performance, total exercising skeletal muscle blood flow and VC, or contracting muscle microvascular oxygenation in healthy rats.

  2. Pharmacological enhancement of leg and muscle microvascular blood flow does not augment anabolic responses in skeletal muscle of young men under fed conditions.

    Science.gov (United States)

    Phillips, Bethan E; Atherton, Philip J; Varadhan, Krishna; Wilkinson, Daniel J; Limb, Marie; Selby, Anna L; Rennie, Michael J; Smith, Kenneth; Williams, John P

    2014-01-15

    Skeletal muscle anabolism associated with postprandial plasma aminoacidemia and insulinemia is contingent upon amino acids (AA) and insulin crossing the microcirculation-myocyte interface. In this study, we hypothesized that increasing muscle microvascular blood volume (flow) would enhance fed-state anabolic responses in muscle protein turnover. We studied 10 young men (23.2 ± 2.1 yr) under postabsorptive and fed [iv Glamin (∼10 g AA), glucose ∼7.5 mmol/l] conditions. Methacholine was infused into the femoral artery of one leg to determine, via bilateral comparison, the effects of feeding alone vs. feeding plus pharmacological vasodilation. We measured leg blood flow (LBF; femoral artery) by Doppler ultrasound, muscle microvascular blood volume (MBV) by contrast-enhanced ultrasound (CEUS), muscle protein synthesis (MPS) and breakdown (MPB; a-v balance modeling), and net protein balance (NPB) using [1,2-(13)C2]leucine and [(2)H5]phenylalanine tracers via gas chromatography-mass spectrometry (GC-MS). Indexes of anabolic signaling/endothelial activation (e.g., Akt/mTORC1/NOS) were assessed using immunoblotting techniques. Under fed conditions, LBF (+12 ± 5%, P anabolism.

  3. Unraveling the role of hypoxia-inducible factor (HIF)-1α and HIF-2α in the adaption process of human microvascular endothelial cells (HMEC-1) to hypoxia: Redundant HIF-dependent regulation of macrophage migration inhibitory factor.

    Science.gov (United States)

    Hahne, Martin; Schumann, Peggy; Mursell, Mathias; Strehl, Cindy; Hoff, Paula; Buttgereit, Frank; Gaber, Timo

    2018-03-01

    Hypoxia driven angiogenesis is a prominent feature of tissue regeneration, inflammation and tumor growth and is regulated by hypoxia-inducible factor (HIF)-1 and -2. The distinct functions of HIFs in the hypoxia-induced angiogenesis and metabolic switch of endothelial cells are still unknown and therefore aim of this study. We investigated the role of HIF-1 and -2 in the adaptation of immortalized human microvascular endothelial cells (HMEC-1) to hypoxic conditions (1% O 2 ) in terms of angiogenesis, cytokine secretion, gene expression and ATP/ADP-ratio using shRNA-mediated reduction of the oxygen sensitive α-subunits of either HIF-1 or HIF-2 or the combination of both. Reduction of HIF-1α diminished cellular energy, hypoxia-induced glycolytic gene expression, and angiogenesis not altering pro-angiogenic factors. Reduction of HIF-2α diminished hypoxia-induced pro-angiogenic factors, enhanced anti-angiogenic factors and attenuated angiogenesis not altering glycolytic gene expression. Reduction of both HIFs reduced cell survival, gene expression of glycolytic enzymes and pro-angiogenic factors as compared to the corresponding control. Finally, we identified the macrophage migration inhibitory factor (MIF) to be redundantly regulated by HIF-1 and HIF-2 and to be essential in the process of hypoxia-driven angiogenesis. Our results demonstrate a major impact of HIF-1 and HIF-2 on hypoxia-induced angiogenesis indicating distinct but also overlapping functions of HIF-1 and HIF-2. These findings open new possibilities for therapeutic approaches by specifically targeting the HIF-1 and HIF-2 or their target MIF. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Experimental inflammation following dural application of complete Freund's adjuvant or inflammatory soup does not alter brain and trigeminal microvascular passage.

    Science.gov (United States)

    Lundblad, Cornelia; Haanes, Kristian A; Grände, Gustaf; Edvinsson, Lars

    2015-01-01

    Migraine is a paroxysmal, disabling primary headache that affects 16 % of the adult population. In spite of decades of intense research, the origin and the pathophysiology mechanisms involved are still not fully known. Although triptans and gepants provide effective relief from acute migraine for many patients, their site of action remains unidentified. It has been suggested that during migraine attacks the leakiness of the blood-brain barrier (BBB) is altered, increasing the passage of anti-migraine drugs. This study aimed to investigate the effect of experimental inflammation, following dural application of complete Freund's adjuvant (CFA) or inflammatory soup (IS) on brain and trigeminal microvascular passage. In order to address this issue, we induced local inflammation in male Sprague-Dawley-rats dura mater by the addition of CFA or IS directly on the dural surface. Following 2, 24 or 48 h of inflammation we calculated permeability-surface area product (PS) for [(51)Cr]-EDTA in the trigeminal ganglion (TG), spinal trigeminal nucleus, cortex, periaqueductal grey and cerebellum. We observed that [(51)Cr]-EDTA did not pass into the central nervous system (CNS) in a major way. However, [(51)Cr]-EDTA readily passed the TG by >30 times compared to the CNS. Application of CFA or IS did not show altered transfer constants. With these experiments we show that dural IS/CFA triggered TG inflammation, did not increase the BBB passage, and that the TG is readily exposed to circulating molecules. The TG could provide a site of anti-migraine drug interaction with effect on the trigeminal system.

  5. An in vitro and in vivo study of peptide-functionalized nanoparticles for brain targeting : The importance of selective blood–brain barrier uptake

    NARCIS (Netherlands)

    Bode, Gerard H.; Coué, G.M.J.P.C.; Freese, Christian; Pickl, Karin E.; Sanchez-Purrà, Maria; Albaiges, Berta; Borrós, Salvador; van Winden, Ewoud C.; Tziveleka, Leto Aikaterini; Sideratou, Zili; Engbersen, Johan F.J.; Singh, Smriti; Albrecht, Krystyna; Groll, Jürgen; Möller, Martin; Pötgens, Andy J.G.; Schmitz, Christoph; Fröhlich, Eleonore; Grandfils, Christian; Sinner, Frank M.; Kirkpatrick, C. James; Steinbusch, Harry W.M.; Frank, Hans Georg; Unger, Ronald E.; Martinez-Martinez, Pilar

    2017-01-01

    Targeted delivery of drugs across endothelial barriers remains a formidable challenge, especially in the case of the brain, where the blood–brain barrier severely limits entry of drugs into the central nervous system. Nanoparticle-mediated transport of peptide/protein-based drugs across endothelial

  6. Impact of nanostructured surfaces on endothelial β-catenin signaling

    International Nuclear Information System (INIS)

    Schernthaner, M.

    2013-01-01

    In this thesis, we set out to investigate the influence of differently structured polyethylene terephtalate (PET) surfaces on human microvascular endothelial cells (HMEC). First, we tested so called ripples with a periodicity of 300 nm and second, wall structures with a periodicity of 1.5 µm. both generated by laser irradiation of PET substrates. We investigated cell adhesion and orientation by electron microscopy and determined cell proliferation levels. Endothelial proliferation is controlled by the junctional protein β-catenin, which translocates to the nucleus to activate target gene expression when released from is normal localization in cell-cell contacts. We observed that β-catenin translocates to the nucleus of endothelial cells grown on both types of structured substrates. However, the expression levels of the target gene cyclin D1 were enhanced in cells grown on ripples only. The use of different tyrosine kinase inhibitors revealed Src and Abl kinases as upstream signaling elements of β-catenin-mediated cell proliferation on ripples, while Src was found not involved in cell proliferation on walls. Since Src activation is strongly linked to Ca 2+ signaling, we tested for differences in store-operated Ca 2+ -entry (SOCE) and mechanically induced Ca 2+ -entry. Cells grown on ripples displayed significantly enhanced SOCE compared to cells grown on walls. Moreover, SOCE and shear-induced Ca 2+ -entry showed clear gadolinium (Gd 3+ ) sensitivity in cells grown on ripples only. Taken together, our results demonstrated that both structures induce endothelial β-catenin signaling by enhanced tyrosine phosphorylation leading to increased cell proliferation. The signaling process on ripples further involves Src activation and SOCE signaling. The proliferation enhancing features of the nanostructured PET substrates investigated in this thesis might be used for tissue engineering strategies.(author) [de

  7. Targeted adenovirus mediated inhibition of NF-κB-dependent inflammatory gene expression in endothelial cells in vitro and in vivo.

    Science.gov (United States)

    Kułdo, J M; Ásgeirsdóttir, S A; Zwiers, P J; Bellu, A R; Rots, M G; Schalk, J A C; Ogawara, K I; Trautwein, C; Banas, B; Haisma, H J; Molema, G; Kamps, J A A M

    2013-02-28

    In chronic inflammatory diseases the endothelium expresses mediators responsible for harmful leukocyte infiltration. We investigated whether targeted delivery of a therapeutic transgene that inhibits nuclear factor κB signal transduction could silence the proinflammatory activation status of endothelial cells. For this, an adenovirus encoding dominant-negative IκB (dnIκB) as a therapeutic transgene was employed. Selectivity for the endothelial cells was achieved by introduction of antibodies specific for inflammatory endothelial adhesion molecules E-selectin or VCAM-1 chemically linked to the virus via polyethylene glycol. In vitro, the retargeted adenoviruses selectively infected cytokine-activated endothelial cells to express functional transgene. The comparison of transductional capacity of both retargeted viruses revealed that E-selectin based transgene delivery exerted superior pharmacological effects. Targeted delivery mediated dnIκB transgene expression in endothelial cells inhibited the induced expression of several inflammatory genes, including adhesion molecules, cytokines, and chemokines. In vivo, in mice suffering from glomerulonephritis, E-selectin-retargeted adenovirus selectively homed in the kidney to microvascular glomerular endothelium. Subsequent downregulation of endothelial adhesion molecule expression 2 days after induction of inflammation demonstrated the pharmacological potential of this gene therapy approach. The data justify further studies towards therapeutic virus design and optimization of treatment schedules to investigate their capacity to interfere with inflammatory disease progression. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. A large-scale electrophoresis- and chromatography-based determination of gene expression profiles in bovine brain capillary endothelial cells after the re-induction of blood-brain barrier properties

    Directory of Open Access Journals (Sweden)

    Duban-Deweer Sophie

    2010-11-01

    Full Text Available Abstract Background Brain capillary endothelial cells (BCECs form the physiological basis of the blood-brain barrier (BBB. The barrier function is (at least in part due to well-known proteins such as transporters, tight junctions and metabolic barrier proteins (e.g. monoamine oxidase, gamma glutamyltranspeptidase and P-glycoprotein. Our previous 2-dimensional gel proteome analysis had identified a large number of proteins and revealed the major role of dynamic cytoskeletal remodelling in the differentiation of bovine BCECs. The aim of the present study was to elaborate a reference proteome of Triton X-100-soluble species from bovine BCECs cultured in the well-established in vitro BBB model developed in our laboratory. Results A total of 215 protein spots (corresponding to 130 distinct proteins were identified by 2-dimensional gel electrophoresis, whereas over 350 proteins were identified by a shotgun approach. We classified around 430 distinct proteins expressed by bovine BCECs. Our large-scale gene expression analysis enabled the correction of mistakes referenced into protein databases (e.g. bovine vinculin and constitutes valuable evidence for predictions based on genome annotation. Conclusions Elaboration of a reference proteome constitutes the first step in creating a gene expression database dedicated to capillary endothelial cells displaying BBB characteristics. It improves of our knowledge of the BBB and the key proteins in cell structures, cytoskeleton organization, metabolism, detoxification and drug resistance. Moreover, our results emphasize the need for both appropriate experimental design and correct interpretation of proteome datasets.

  9. Iron uptake and transport at the blood-brain barrier

    DEFF Research Database (Denmark)

    Larsen, Annette Burkhart; Thomsen, Louiza Bohn; Moos, Torben

    The mechanism by which iron is transported across the blood-brain barrier (BBB) remains controversial, and in this study we aimed to further clarify mechanisms by which iron is transported into the brain. We analyzed and compared the mRNA and protein expression of a variety of proteins involved...... in the transport of iron (transferrin receptor, divalent metal transporter I (DMT1), steap 2, steap 3, ceruloplasmin, hephaestin and ferroportin) in both primary rat brain capillary endothelial cells (BCEC) and immortalized rat brain capillary endothelial cell line (RBE4) grown in co-culture with defined polarity....... The mRNA expression of the iron-related molecules was also investigated in isolated brain capillaries from iron deficiency, iron reversible and normal rats. We also performed iron transport studies to analyze the routes by which iron is transported through the brain capillary endothelial cells: i) We...

  10. Targeted transfection increases siRNA uptake and gene silencing of primary endothelial cells in vitro--a quantitative study.

    Science.gov (United States)

    Asgeirsdóttir, Sigridur A; Talman, Eduard G; de Graaf, Inge A; Kamps, Jan A A M; Satchell, Simon C; Mathieson, Peter W; Ruiters, Marcel H J; Molema, Grietje

    2010-01-25

    Applications of small-interfering RNA (siRNA) call for specific and efficient delivery of siRNA into particular cell types. We developed a novel, non-viral targeting system to deliver siRNA specifically into inflammation-activated endothelial cells. This was achieved by conjugating the cationic amphiphilic lipid SAINT to antibodies recognizing the inflammatory cell adhesion molecule E-selectin. These anti-E-selectin-SAINT lipoplexes (SAINTarg) maintained antigen recognition capacity of the parental antibody in vitro, and ex vivo in human kidney tissue slices subjected to inflammatory conditions. Regular SAINT mediated transfection resulted in efficient gene silencing in human microvascular endothelial cells (HMEC-1) and conditionally immortalized glomerular endothelial cells (ciGEnC). However, primary human umbilical vein endothelial cells (HUVEC) transfected poorly, a phenomenon that we could quantitatively correlate with a cell-type specific capacity to facilitate siRNA uptake. Importantly, SAINTarg increased siRNA uptake and transfection specificity for activated endothelial cells. Transfection with SAINTarg delivered significantly more siRNA into activated HUVEC, compared to transfection with non-targeted SAINT. The enhanced uptake of siRNA was corroborated by improved silencing of both gene- and protein expression of VE-cadherin in activated HUVEC, indicating that SAINTarg delivered functionally active siRNA into endothelial cells. The obtained results demonstrate a successful design of a small nucleotide carrier system with improved and specific siRNA delivery into otherwise difficult-to-transfect primary endothelial cells, which in addition reduced considerably the amount of siRNA needed for gene silencing. Copyright 2009 Elsevier B.V. All rights reserved.

  11. Erythropoietin promotes network formation of transplanted adipose tissue-derived microvascular fragments

    Directory of Open Access Journals (Sweden)

    P Karschnia

    2018-05-01

    Full Text Available The seeding of tissue constructs with adipose tissue-derived microvascular fragments (ad-MVF is an emerging pre-vascularisation strategy. Ad-MVF rapidly reassemble into new microvascular networks after in vivo implantation. Herein it was analysed whether this process was improved by erythropoietin (EPO. Ad-MVF were isolated from green fluorescent protein (GFP+ as well as wild-type C57BL/6 mice and cultivated for 24 h in medium supplemented with EPO (20 IU/mL or vehicle. Freshly isolated, non-cultivated ad-MVF served as controls. Protein expression, cell viability and proliferation of ad-MVF were assessed by proteome profiler array and fluorescence microscopy. GFP+ ad-MVF were seeded on collagen-glycosaminoglycan matrices, which were implanted into dorsal skinfold chambers of C57BL/6 mice, to analyse their vascularisation over 14 d by intravital fluorescence microscopy, histology and immunohistochemistry. Cultivation up-regulated the expression of pro- and anti-angiogenic factors within both vehicle- and EPO-treated ad-MVF when compared with non-cultivated controls. Moreover, EPO treatment suppressed cultivation-associated apoptosis and significantly increased the number of proliferating endothelial cells in ad-MVF when compared with vehicle-treated and non-cultivated ad-MVF. Accordingly, implanted matrices seeded with EPO-treated ad-MVF exhibited an improved vascularisation, as indicated by a significantly higher functional microvessel density. The matrices of the three groups contained a comparably large fraction of GFP+ microvessels originating from the ad-MVF, whereas the tissue surrounding the matrices seeded with EPO-treated ad-MVF exhibited a significantly increased microvessel density when compared with the other two groups. These findings indicated that EPO represents a promising cytokine to further boost the excellent vascularisation properties of ad-MVF in tissue-engineering applications.

  12. Omeprazole induces heme oxygenase-1 in fetal human pulmonary microvascular endothelial cells via hydrogen peroxide-independent Nrf2 signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Patel, Ananddeep; Zhang, Shaojie; Shrestha, Amrit Kumar; Maturu, Paramahamsa; Moorthy, Bhagavatula; Shivanna, Binoy, E-mail: shivanna@bcm.edu

    2016-11-15

    Omeprazole (OM) is an aryl hydrocarbon receptor (AhR) agonist and a proton pump inhibitor that is used to treat humans with gastric acid related disorders. Recently, we showed that OM induces NAD (P) H quinone oxidoreductase-1 (NQO1) via nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent mechanism. Heme oxygenase-1 (HO-1) is another cytoprotective and antioxidant enzyme that is regulated by Nrf2. Whether OM induces HO-1 in fetal human pulmonary microvascular endothelial cells (HPMEC) is unknown. Therefore, we tested the hypothesis that OM will induce HO-1 expression via Nrf2 in HPMEC. OM induced HO-1 mRNA and protein expression in a dose-dependent manner. siRNA-mediated knockdown of AhR failed to abrogate, whereas knockdown of Nrf2 abrogated HO-1 induction by OM. To identify the underlying molecular mechanisms, we determined the effects of OM on cellular hydrogen peroxide (H{sub 2}O{sub 2}) levels since oxidative stress mediated by the latter is known to activate Nrf2. Interestingly, the concentration at which OM induced HO-1 also increased H{sub 2}O{sub 2} levels. Furthermore, H{sub 2}O{sub 2} independently augmented HO-1 expression. Although N-acetyl cysteine (NAC) significantly decreased H{sub 2}O{sub 2} levels in OM-treated cells, we observed that OM further increased HO-1 mRNA and protein expression in NAC-pretreated compared to vehicle-pretreated cells, suggesting that OM induces HO-1 via H{sub 2}O{sub 2}-independent mechanisms. In conclusion, we provide evidence that OM transcriptionally induces HO-1 via AhR - and H{sub 2}O{sub 2} - independent, but Nrf2 - dependent mechanisms. These results have important implications for human disorders where Nrf2 and HO-1 play a beneficial role. - Highlights: • Omeprazole induces HO-1 in human fetal lung cells. • AhR deficiency fails to abrogate omeprazole-mediated induction of HO-1. • Nrf2 knockdown abrogates omeprazole-mediated HO-1 induction in human lung cells. • Hydrogen peroxide depletion augments

  13. Arsenic toxicity induced endothelial dysfunction and dementia: Pharmacological interdiction by histone deacetylase and inducible nitric oxide synthase inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Sharma, Bhupesh, E-mail: drbhupeshresearch@gmail.com; Sharma, P.M.

    2013-11-15

    Arsenic toxicity has been reported to damage all the major organs including the brain and vasculature. Dementia including Alzheimer's disease (AD) and vascular dementia (VaD) are posing greater risk to the world population as it is now increasing at a faster rate. We have investigated the role of sodium butyrate, a selective histone deacetylase (HDAC) inhibitor and aminoguanidine, a selective inducible nitric oxide synthase (iNOS) inhibitor in pharmacological interdiction of arsenic toxicity induced vascular endothelial dysfunction and dementia in rats. Arsenic toxicity was done by administering arsenic drinking water to rats. Morris water-maze (MWM) test was used for assessment of learning and memory. Endothelial function was assessed using student physiograph. Oxidative stress (aortic superoxide anion, serum and brain thiobarbituric acid reactive species, brain glutathione) and nitric oxide levels (serum nitrite/nitrate) were also measured. Arsenic treated rats have shown impairment of endothelial function, learning and memory, reduction in serum nitrite/nitrate and brain GSH levels along with increase in serum and brain TBARS. Sodium butyrate as well as aminoguanidine significantly convalesce arsenic induced impairment of learning, memory, endothelial function, and alterations in various biochemical parameters. It may be concluded that arsenic induces endothelial dysfunction and dementia, whereas, sodium butyrate, a HDAC inhibitor as well as aminoguanidine, a selective iNOS inhibitor may be considered as potential agents for the management of arsenic induced endothelial dysfunction and dementia. - Highlights: • As has induced endothelial dysfunction (Edf) and vascular dementia (VaD). • As has increased oxidative stress, AChE activity and decreased serum NO. • Inhibitors of HDAC and iNOS have attenuated As induced Edf and VaD. • Both the inhibitors have attenuated As induced biochemical changes. • Inhibitor of HDAC and iNOS has shown good potential

  14. Arsenic toxicity induced endothelial dysfunction and dementia: Pharmacological interdiction by histone deacetylase and inducible nitric oxide synthase inhibitors

    International Nuclear Information System (INIS)

    Sharma, Bhupesh; Sharma, P.M.

    2013-01-01

    Arsenic toxicity has been reported to damage all the major organs including the brain and vasculature. Dementia including Alzheimer's disease (AD) and vascular dementia (VaD) are posing greater risk to the world population as it is now increasing at a faster rate. We have investigated the role of sodium butyrate, a selective histone deacetylase (HDAC) inhibitor and aminoguanidine, a selective inducible nitric oxide synthase (iNOS) inhibitor in pharmacological interdiction of arsenic toxicity induced vascular endothelial dysfunction and dementia in rats. Arsenic toxicity was done by administering arsenic drinking water to rats. Morris water-maze (MWM) test was used for assessment of learning and memory. Endothelial function was assessed using student physiograph. Oxidative stress (aortic superoxide anion, serum and brain thiobarbituric acid reactive species, brain glutathione) and nitric oxide levels (serum nitrite/nitrate) were also measured. Arsenic treated rats have shown impairment of endothelial function, learning and memory, reduction in serum nitrite/nitrate and brain GSH levels along with increase in serum and brain TBARS. Sodium butyrate as well as aminoguanidine significantly convalesce arsenic induced impairment of learning, memory, endothelial function, and alterations in various biochemical parameters. It may be concluded that arsenic induces endothelial dysfunction and dementia, whereas, sodium butyrate, a HDAC inhibitor as well as aminoguanidine, a selective iNOS inhibitor may be considered as potential agents for the management of arsenic induced endothelial dysfunction and dementia. - Highlights: • As has induced endothelial dysfunction (Edf) and vascular dementia (VaD). • As has increased oxidative stress, AChE activity and decreased serum NO. • Inhibitors of HDAC and iNOS have attenuated As induced Edf and VaD. • Both the inhibitors have attenuated As induced biochemical changes. • Inhibitor of HDAC and iNOS has shown good potential in

  15. Myeloperoxidase-derived oxidants induce blood-brain barrier dysfunction in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Andreas Üllen

    Full Text Available Peripheral leukocytes can exacerbate brain damage by release of cytotoxic mediators that disrupt blood-brain barrier (BBB function. One of the oxidants released by activated leukocytes is hypochlorous acid (HOCl formed via the myeloperoxidase (MPO-H2O2-Cl(- system. In the present study we examined the role of leukocyte activation, leukocyte-derived MPO and MPO-generated oxidants on BBB function in vitro and in vivo. In a mouse model of lipopolysaccharide (LPS-induced systemic inflammation, neutrophils that had become adherent released MPO into the cerebrovasculature. In vivo, LPS-induced BBB dysfunction was significantly lower in MPO-deficient mice as compared to wild-type littermates. Both, fMLP-activated leukocytes and the MPO-H2O2-Cl(- system inflicted barrier dysfunction of primary brain microvascular endothelial cells (BMVEC that was partially rescued with the MPO inhibitor 4-aminobenzoic acid hydrazide. BMVEC treatment with the MPO-H2O2-Cl(- system or activated neutrophils resulted in the formation of plasmalogen-derived chlorinated fatty aldehydes. 2-chlorohexadecanal (2-ClHDA severely compromised BMVEC barrier function and induced morphological alterations in tight and adherens junctions. In situ perfusion of rat brain with 2-ClHDA increased BBB permeability in vivo. 2-ClHDA potently activated the MAPK cascade at physiological concentrations. An ERK1/2 and JNK antagonist (PD098059 and SP600125, respectively protected against 2-ClHDA-induced barrier dysfunction in vitro. The current data provide evidence that interference with the MPO pathway could protect against BBB dysfunction under (neuroinflammatory conditions.

  16. Effects of electromagnetic pulse exposure on gelatinase of blood-brain barrier in vitro.

    Science.gov (United States)

    Zhou, Yan; Qiu, Lian-Bo; An, Guang-Zhou; Zhou, Jia-Xing; Du, Le; Ma, Ya-Hong; Guo, Guo-Zhen; Ding, Gui-Rong

    2017-01-01

    The biological effects of electromagnetic pulse (EMP) on the brain have been focused on for years. It was reported that gelatinase played an important role in maintaining brain function through regulating permeability in the blood-brain barrier (BBB). To investigate the effects of EMP on gelatinase of BBB, an in vitro BBB model was established using primary cultured rat brain microvascular endothelial cells (BMVEC), astrocytes and half-contact culture of these cells in a transwell chamber. Cultured supernatant and cells were collected at different time points after exposure to EMP (peak intensity 400 kV/m, rise time 10 ns, pulse width 350 ns, 0.5 pps and 200 pulses). Protein levels of cellular gelatinase MMP-2 and MMP-9, and endogenous inhibitor TIMP-1 and TIMP-2 were detected by Western blot. The activity of gelatinase in culture supernatant was detected by gelatin zymography. It was found that compared with the sham-exposed group, the protein level of MMP-2 was significantly increased at 6 h (p < 0.05), and the protein level of its endogenous inhibitor TIMP-2 did not change after EMP exposure. In addition, the protein levels of MMP-9 and its endogenous inhibitor TIMP-1 did not change after EMP exposure. Gelatin zymography results showed that the activity of MMP-2 in the inner pool and the outer pool of the transwell chamber was significantly increased at 6 h after EMP exposure compared with that of the sham group. These results suggested that EMP exposure could affect the expression and activity of MMP-2 in the BBB model.

  17. Epigenetic regulation of the glucose transporter gene Slc2a1 by β-hydroxybutyrate underlies preferential glucose supply to the brain of fasted mice.

    Science.gov (United States)

    Tanegashima, Kosuke; Sato-Miyata, Yukiko; Funakoshi, Masabumi; Nishito, Yasumasa; Aigaki, Toshiro; Hara, Takahiko

    2017-01-01

    We carried out liquid chromatography-tandem mass spectrometry analysis of metabolites in mice. Those metabolome data showed that hepatic glucose content is reduced, but that brain glucose content is unaffected, during fasting, consistent with the priority given to brain glucose consumption during fasting. The molecular mechanisms for this preferential glucose supply to the brain are not fully understood. We also showed that the fasting-induced production of the ketone body β-hydroxybutyrate (β-OHB) enhances expression of the glucose transporter gene Slc2a1 (Glut1) via histone modification. Upon β-OHB treatment, Slc2a1 expression was up-regulated, with a concomitant increase in H3K9 acetylation at the critical cis-regulatory region of the Slc2a1 gene in brain microvascular endothelial cells and NB2a neuronal cells, shown by quantitative PCR analysis and chromatin immunoprecipitation assay. CRISPR/Cas9-mediated disruption of the Hdac2 gene increased Slc2a1 expression, suggesting that it is one of the responsible histone deacetylases (HDACs). These results confirm that β-OHB is a HDAC inhibitor and show that β-OHB plays an important role in fasting-induced epigenetic activation of a glucose transporter gene in the brain. © 2016 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

  18. Endothelial remodelling and intracellular calcium machinery.

    Science.gov (United States)

    Moccia, F; Tanzi, F; Munaron, L

    2014-05-01

    Rather being an inert barrier between vessel lumen and surrounding tissues, vascular endothelium plays a key role in the maintenance of cardiovascular homeostasis. The de-endothelialization of blood vessels is regarded as the early event that results in the onset of severe vascular disorders, including atherosclerosis, acute myocardial infarction, brain stroke, and aortic aneurysm. Restoration of the endothelial lining may be accomplished by the activation of neighbouring endothelial cells (ECs) freed by contact inhibition and by circulating endothelial progenitor cells (EPCs). Intracellular Ca(2+) signalling is essential to promote wound healing: however, the molecular underpinnings of the Ca(2+) response to injury are yet to be fully elucidated. Similarly, the components of the Ca(2+) toolkit that drive EPC incorporation into denuded vessels are far from being fully elucidated. The present review will survey the current knowledge on the role of Ca(2+) signalling in endothelial repair and in EPC activation. We propose that endothelial regeneration might be boosted by intraluminal release of specific Ca(2+) channel agonists or by gene transfer strategies aiming to enhance the expression of the most suitable Ca(2+) channels at the wound site. In this view, connexin (Cx) channels/hemichannels and store-operated Ca(2+) entry (SOCE) stand amid the most proper routes to therapeutically induce the regrowth of denuded vessels. Cx stimulation might trigger the proliferative and migratory behaviour of ECs facing the lesion site, whereas activation of SOCE is likely to favour EPC homing to the wounded vessel.

  19. Glutamate Transporters in the Blood-Brain Barrier

    DEFF Research Database (Denmark)

    Helms, Hans Christian Cederberg; Nielsen, Carsten Uhd; Waagepetersen, Helle S

    2017-01-01

    concentration of L-glutamate causes excitotoxicity. A tight control of the brain interstitial fluid L-glutamate levels is therefore imperative, in order to maintain optimal neurotransmission and to avoid such excitotoxicity. The blood-brain barrier, i.e., the endothelial lining of the brain capillaries...... cells. The mechanisms underlying transendothelial L-glutamate transport are however still not well understood. The present chapter summarizes the current knowledge on blood-brain barrier L-glutamate transporters and the suggested pathways for the brain-to-blood L-glutamate efflux......., regulates the exchange of nutrients, gases, and metabolic waste products between plasma and brain interstitial fluid. It has been suggested that brain capillary endothelial cells could play an important role in L-glutamate homeostasis by mediating brain-to-blood L-glutamate efflux. Both in vitro and in vivo...

  20. Effects of lead and mercury on histamine uptake by glial and endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Huszti, Z. [Semmelweis Univ. of Medicine, Dept. of Pharmacodynamics, Budapest (Hungary); Balogh, I. [Semmelweis Univ. of Medicine, Forensic Medicine, Budapest (Hungary)

    1995-06-01

    The effects of lead and mercury on [{sup 3}H]-histamine uptake by cultured astroglial and endothelial cells of rat brain were studied. Experimental data showed that both metal ions inhibited the uptake in both cell types of concentrations as low as 1-10 {mu}M. The effects were consistent with non/competitive inhibitions. With either lead or mercury exposure, the inhibition of the uptake was greater in astroglial than in cerebral endothelial cells. Contrary to the above finding, 100 {mu}M of mercuric chloride produced stimulation of histamine uptake and this stimulation was much more pronounced in cultured cerebral endothelial cells than in astroglial cells. Inhibition of [{sup 3}H]-histamine uptake by lead acetate and mercuric chloride was considered to be association with a loss of the transmembrane Na{sup +} and/or K{sup +} gradient while stimulation of the uptake by high concentration of mercury might be related to a direct effect on histamine transporter. It is note-worthy, that cultured astroglial cells, derived from neonatal rat brain, are much more sensitive to the toxic effects of these heavy metal ions than cultured endothelial cells derived from the brain capillaries often same species of animals. (au) 18 refs.

  1. Comparative study of four immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, and optimization of culture conditions, for an in vitro blood–brain barrier model for drug permeability studies

    OpenAIRE

    Eigenmann, Daniela E; Xue, Gongda; Kim, Kwang S; Moses, Ashlee V; Hamburger, Matthias; Oufir, Mouhssin

    2013-01-01

    Background: Reliable human in vitro blood brain barrier (BBB) models suitable for high throughput screening are urgently needed in early drug discovery and development for assessing the ability of promising bioactive compounds to overcome the BBB. To establish an improved human in vitro BBB model we compared four currently available and well characterized immortalized human brain capillary endothelial cell lines hCMEC/D3 hBMEC TY10 and BB19 with respect to barrier tightness and paracellular p...

  2. Boosting the signal: Endothelial inward rectifier K+ channels.

    Science.gov (United States)

    Jackson, William F

    2017-04-01

    Endothelial cells express a diverse array of ion channels including members of the strong inward rectifier family composed of K IR 2 subunits. These two-membrane spanning domain channels are modulated by their lipid environment, and exist in macromolecular signaling complexes with receptors, protein kinases and other ion channels. Inward rectifier K + channel (K IR ) currents display a region of negative slope conductance at membrane potentials positive to the K + equilibrium potential that allows outward current through the channels to be activated by membrane hyperpolarization, permitting K IR to amplify hyperpolarization induced by other K + channels and ion transporters. Increases in extracellular K + concentration activate K IR allowing them to sense extracellular K + concentration and transduce this change into membrane hyperpolarization. These properties position K IR to participate in the mechanism of action of hyperpolarizing vasodilators and contribute to cell-cell conduction of hyperpolarization along the wall of microvessels. The expression of K IR in capillaries in electrically active tissues may allow K IR to sense extracellular K + , contributing to functional hyperemia. Understanding the regulation of expression and function of microvascular endothelial K IR will improve our understanding of the control of blood flow in the microcirculation in health and disease and may provide new targets for the development of therapeutics in the future. © 2016 John Wiley & Sons Ltd.

  3. Vascular endothelial growth factor A protein level and gene expression in intracranial meningiomas with brain edema

    DEFF Research Database (Denmark)

    Nassehi, Damoun; Dyrbye, Henrik; Andresen, Morten

    2011-01-01

    Meningiomas are the second most common primary intracranial tumors in adults. Although meningiomas are mostly benign, more than 50% of patients with meningioma develop peritumoral brain edema (PTBE), which may be fatal because of increased intracranial pressure. Vascular endothelial growth factor....... Forty-three patients had primary, solitary, supratentorial meningiomas with PTBE. In these, correlations in PTBE, edema index, VEGF-A protein, VEGF gene expression, capillary length, and tumor water content were investigated. DNA-branched hybridization was used for measuring VEGF gene expression...... in tissue homogenates prepared from frozen tissue samples. The method for VEGF-A analysis resembled an ELISA assay, but was based on chemiluminescence. The edema index was positively correlated to VEGF-A protein (p = 0.014) and VEGF gene expression (p

  4. Effects of external radiation in a co-culture model of endothelial cells and adipose-derived stem cells

    International Nuclear Information System (INIS)

    Haubner, Frank; Leyh, Michaela; Ohmann, Elisabeth; Pohl, Fabian; Prantl, Lukas; Gassner, Holger G

    2013-01-01

    The inflammatory response clinically observed after radiation has been described to correlate with elevated expression of cytokines and adhesion molecules by endothelial cells. Therapeutic compensation for this microvascular compromise could be an important approach in the treatment of irradiated wounds. Clinical reports describe the potential of adipose-derived stem cells to enhance wound healing, but the underlying cellular mechanisms remain largely unclear. Human dermal microvascular endothelial cells (HDMEC) and human adipose-derived stem cells (ASC) were cultured in a co-culture setting and irradiated with sequential doses of 2 to 12 Gy. Cell count was determined 48 h after radiation using a semi-automated cell counting system. Levels of interleukin-6 (IL-6), basic fibroblast growth factor (FGF), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were determined in the supernatants using enzyme-linked immunosorbent assay (ELISA). Irradiated HDMEC and ASC as well as non-irradiated co-cultures, HDMEC or ASC respectively were used as controls. Cell count was significantly reduced in irradiated co-cultures of HDMEC and ASC compared to non-irradiated controls. Levels of IL-6, FGF, ICAM-1 and VCAM-1 in the supernatants of the co-cultures were significantly less affected by external radiation in comparison to HDMEC. The increased expression of cytokines and adhesion molecules by HDMEC after external radiation is mitigated in the co-culture setting with ASC. These in vitro changes seem to support the clinical observation that ASC may have a stabilizing effect when injected into irradiated wounds

  5. Active cooling of microvascular composites for battery packaging

    Science.gov (United States)

    Pety, Stephen J.; Chia, Patrick X. L.; Carrington, Stephen M.; White, Scott R.

    2017-10-01

    Batteries in electric vehicles (EVs) require a packaging system that provides both thermal regulation and crash protection. A novel packaging scheme is presented that uses active cooling of microvascular carbon fiber reinforced composites to accomplish this multifunctional objective. Microvascular carbon fiber/epoxy composite panels were fabricated and their cooling performance assessed over a range of thermal loads and experimental conditions. Tests were performed for different values of coolant flow rate, channel spacing, panel thermal conductivity, and applied heat flux. More efficient cooling occurs when the coolant flow rate is increased, channel spacing is reduced, and thermal conductivity of the host composite is increased. Computational fluid dynamics (CFD) simulations were also performed and correlate well with the experimental data. CFD simulations of a typical EV battery pack confirm that microvascular composite panels can adequately cool battery cells generating 500 W m-2 heat flux below 40 °C.

  6. Contrast ultrasound targeted treatment of gliomas in mice via drug-bearing nanoparticle delivery and microvascular ablation.

    Science.gov (United States)

    Burke, Caitlin W; Price, Richard J

    2010-12-15

    We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the microvasculature are controlled by varying ultrasound pulsing parameters. Specifically, we are testing whether such approaches may be used to treat malignant brain tumors through drug delivery and microvascular ablation. Preliminary studies have been performed to determine whether targeted drug-bearing nanoparticle delivery can be facilitated by the ultrasound mediated destruction of "composite" delivery agents comprised of 100nm poly(lactide-co-glycolide) (PLAGA) nanoparticles that are adhered to albumin shelled microbubbles. We denote these agents as microbubble-nanoparticle composite agents (MNCAs). When targeted to subcutaneous C6 gliomas with ultrasound, we observed an immediate 4.6-fold increase in nanoparticle delivery in MNCA treated tumors over tumors treated with microbubbles co-administered with nanoparticles and a 8.5 fold increase over non-treated tumors. Furthermore, in many cancer applications, we believe it may be desirable to perform targeted drug delivery in conjunction with ablation of the tumor microcirculation, which will lead to tumor hypoxia and apoptosis. To this end, we have tested the efficacy of non-theramal cavitation-induced microvascular ablation, showing that this approach elicits tumor perfusion reduction, apoptosis, significant growth inhibition, and necrosis. Taken together, these results indicate that our ultrasound-targeted approach has the potential to increase therapeutic efficiency by creating tumor necrosis through microvascular ablation and/or simultaneously enhancing the drug payload in gliomas.

  7. Potential roles of cell-derived microparticles in ischemic brain disease.

    Science.gov (United States)

    Horstman, Lawrence L; Jy, Wenche; Bidot, Carlos J; Nordberg, Mary L; Minagar, Alireza; Alexander, J Steven; Kelley, Roger E; Ahn, Yeon S

    2009-10-01

    The objective of this study is to review the role of cell-derived microparticles in ischemic cerebrovascular diseases. An extensive PubMed search of literature pertaining to this study was performed in April 2009 using specific keyword search terms related to cell-derived microparticles and ischemic stroke. Some references are not cited here as it is not possible to be all inclusive or due to space limitation. Cell-derived microparticles are small membranous vesicles released from the plasma membranes of platelets, leukocytes, red cells and endothelial cells in response to diverse biochemical agents or mechanical stresses. They are the main carriers of circulating tissue factor, the principal initiator of intravascular thrombosis, and are implicated in a variety of thrombotic and inflammatory disorders. This review outlines evidence suggesting that cell-derived microparticles are involved predominantly with microvascular, as opposed to macrovascular, thrombosis. More specifically, cell-derived microparticles may substantially contribute to ischemic brain disease in several settings, as well as to neuroinflammatory conditions. If further work confirms this hypothesis, novel therapeutic strategies for minimizing cell-derived microparticles-mediated ischemia are available or can be developed, as discussed.

  8. Microvascular imaging: techniques and opportunities for clinical physiological measurements

    International Nuclear Information System (INIS)

    Allen, John; Howell, Kevin

    2014-01-01

    The microvasculature presents a particular challenge in physiological measurement because the vessel structure is spatially inhomogeneous and perfusion can exhibit high variability over time. This review describes, with a clinical focus, the wide variety of methods now available for imaging of the microvasculature and their key applications. Laser Doppler perfusion imaging and laser speckle contrast imaging are established, commercially-available techniques for determining microvascular perfusion, with proven clinical utility for applications such as burn-depth assessment. Nailfold capillaroscopy is also commercially available, with significant published literature that supports its use for detecting microangiopathy secondary to specific connective tissue diseases in patients with Raynaud's phenomenon. Infrared thermography measures skin temperature and not perfusion directly, and it has only gained acceptance for some surgical and peripheral microvascular applications. Other emerging technologies including imaging photoplethysmography, optical coherence tomography, photoacoustic tomography, hyperspectral imaging, and tissue viability imaging are also described to show their potential as techniques that could become established tools for clinical microvascular assessment. Growing interest in the microcirculation has helped drive the rapid development in perfusion imaging of the microvessels, bringing exciting opportunities in microvascular research. (topical review)

  9. Alda-1 Protects Against Acrolein-Induced Acute Lung Injury and Endothelial Barrier Dysfunction.

    Science.gov (United States)

    Lu, Qing; Mundy, Miles; Chambers, Eboni; Lange, Thilo; Newton, Julie; Borgas, Diana; Yao, Hongwei; Choudhary, Gaurav; Basak, Rajshekhar; Oldham, Mahogany; Rounds, Sharon

    2017-12-01

    Inhalation of acrolein, a highly reactive aldehyde, causes lung edema. The underlying mechanism is poorly understood and there is no effective treatment. In this study, we demonstrated that acrolein not only dose-dependently induced lung edema but also promoted LPS-induced acute lung injury. Importantly, acrolein-induced lung injury was prevented and rescued by Alda-1, an activator of mitochondrial aldehyde dehydrogenase 2. Acrolein also dose-dependently increased monolayer permeability, disrupted adherens junctions and focal adhesion complexes, and caused intercellular gap formation in primary cultured lung microvascular endothelial cells (LMVECs). These effects were attenuated by Alda-1 and the antioxidant N-acetylcysteine, but not by the NADPH inhibitor apocynin. Furthermore, acrolein inhibited AMP-activated protein kinase (AMPK) and increased mitochondrial reactive oxygen species levels in LMVECs-effects that were associated with impaired mitochondrial respiration. AMPK total protein levels were also reduced in lung tissue of mice and LMVECs exposed to acrolein. Activation of AMPK with 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside blunted an acrolein-induced increase in endothelial monolayer permeability, but not mitochondrial oxidative stress or inhibition of mitochondrial respiration. Our results suggest that acrolein-induced mitochondrial dysfunction may not contribute to endothelial barrier dysfunction. We speculate that detoxification of acrolein by Alda-1 and activation of AMPK may be novel approaches to prevent and treat acrolein-associated acute lung injury, which may occur after smoke inhalation.

  10. [Mesh structure of two-dimensional tumor microvascular architecture phenotype heterogeneity in non-small cell lung cancer].

    Science.gov (United States)

    Xiong, Zeng; Zhou, Hui; Liu, Jin-Kang; Hu, Cheng-Ping; Zhou, Mo-Ling; Xia, Yu; Zhou, Jian-Hua

    2009-11-01

    To investigate the structural characteristics and clinical significance of two-dimensional tumor microvascular architecture phenotype (2D-TMAP) in non-small cell lung cancer (NSCLC). Thirty surgical specimens of NSCLC were collected. The sections of the tumor tissues corresponding to the slice of CT perfusion imaging were selected to construct the 2D-TMAP expression. Spearman correlation analysis was used to examine the relation between the 2D-TMAP expression and the clinicopathological features of NSCLC. A heterogeneity was noted in the 2D-TMAP expression of NSCLC. The microvascular density (MVD) in the area surrounding the tumor was higher than that in the central area, but the difference was not statistically significant. The density of the microvessels without intact lumen was significantly greater in the surrounding area than in the central area (P=0.030). The total MVD was not correlated to tumor differentiation (r=0.042, P=0.831). The density of the microvessels without intact lumen in the surrounding area was positively correlated to degree of tumor differentiation and lymph node metastasis (r=0.528 and 0.533, P=0.041 and 0.028, respectively), and also to the expressions of vascular endothelial growth factor (VEGF), ephrinB2, EphB4, and proliferating cell nuclear antigen (PCNA) (r=0.504, 0.549, 0.549, and 0.370; P=0.005, 0.002, 0.002, and 0.048, respectively). The degree of tumor differentiation was positively correlated to PCNA and VEGF expression (r=0.604 and 0.370, P=0.001 and 0.048, respectively), but inversely to the integrity of microvascular basement membrane (r=-0.531, P=0.033). The 2D-TMAP suggests the overall state of the micro-environment for tumor growth. The 2D-TMAP of NSCLC regulates angiogenesis and tumor cell proliferation through a mesh-like structure, and better understanding of the characteristics and possible mechanism of 2D-TMAP expression can be of great clinical importance.

  11. Pilot study on microvascular anastomosis: performance and future educational prospects.

    Science.gov (United States)

    Berretti, G; Colletti, G; Parrinello, G; Iavarone, A; Vannucchi, P; Deganello, A

    2017-11-30

    The introduction of microvascular free flaps has revolutionised modern reconstructive surgery. Unfortunately, access to training opportunities at standardised training courses is limited and expensive. We designed a pilot study on microvascular anastomoses with the aim of verifying if a short course, easily reproducible, could transmit microvascular skills to participants; if the chosen pre-test was predictive of final performance; and if age could influence the outcome. A total of 30 participants (10 students, 10 residents and 10 surgeons) without any previous microvascular experience were instructed and tested during a single 3 to 5 hour course. The two microanastomoses evaluated were the first ever performed by each participant. More than the half of the cohort was able to produce both patent microanastomoses in less than 2 hours; two-thirds of the attempted microanastomoses were patent. The pretest predicted decent scores from poor performances with a sensitivity of 61.5%, specificity of 100%, positive predictive value of 100% and negative predictive value of 40%. Students and residents obtained significantly higher scores than surgeons. Since our course model is short, cost-effective and highly reproducible, it could be introduced and implemented anywhere as an educational prospect for preselecting young residents showing talent and natural predisposition and having ambitions towards microvascular reconstructive surgery. © Copyright by Società Italiana di Otorinolaringologia e Chirurgia Cervico-Facciale.

  12. Carotid Artery Intima-Media Thickness and Cutaneous Microvascular Function are Associated With Vitamin C Levels in Young Patients With Type 1 Diabetes

    DEFF Research Database (Denmark)

    Odermarsky, Michal; Lykkesfeldt, Jens; Liuba, Petru

    2008-01-01

    Background: Vascular endothelial dysfunction and accelerated thickening of arterial intima contribute to increased cardiovascular morbidity in type 1 diabetes. Although vitamin C has important antioxidant functions, and increased oxidative stress is a central mechanism of vascular abnormalities......, lower plasma levels of vitamin C appears to predispose to more pronounced adverse changes in both microcirculation and peripheral arteries. Further studies are needed to investigate whether dietary supplementation with vitamin C could retard the development of microvasculopathy and atherosclerosis...... in diabetes, the relationship between these two in young patients with this disease has not been yet investigated. Methods: Carotid artery intima-media thickness (cIMT) and cutaneous microvascular reactivity to endothelium-dependent (acetylcholine, ACH) and independent (sodium nitroprusside, SNP) were...

  13. Synthesis and deposition of basement membrane proteins by primary brain capillary endothelial cells in a murine model of the blood-brain barrier.

    Science.gov (United States)

    Thomsen, Maj Schneider; Birkelund, Svend; Burkhart, Annette; Stensballe, Allan; Moos, Torben

    2017-03-01

    The brain vascular basement membrane is important for both blood-brain barrier (BBB) development, stability, and barrier integrity and the contribution hereto from brain capillary endothelial cells (BCECs), pericytes, and astrocytes of the BBB is probably significant. The aim of this study was to analyse four different in vitro models of the murine BBB for expression and possible secretion of major basement membrane proteins from murine BCECs (mBCECs). mBCECs, pericytes and glial cells (mainly astrocytes and microglia) were prepared from brains of C57BL/6 mice. The mBCECs were grown as monoculture, in co-culture with pericytes or mixed glial cells, or as a triple-culture with both pericytes and mixed glial cells. The integrity of the BBB models was validated by measures of transendothelial electrical resistance (TEER) and passive permeability to mannitol. The expression of basement membrane proteins was analysed using RT-qPCR, mass spectrometry and immunocytochemistry. Co-culturing mBCECs with pericytes, mixed glial cells, or both significantly increased the TEER compared to the monoculture, and a low passive permeability was correlated with high TEER. The mBCECs expressed all major basement membrane proteins such as laminin-411, laminin-511, collagen [α1(IV)] 2 α2(IV), agrin, perlecan, and nidogen 1 and 2 in vitro. Increased expression of the laminin α5 subunit correlated with the addition of BBB-inducing factors (hydrocortisone, Ro 20-1724, and pCPT-cAMP), whereas increased expression of collagen IV α1 primarily correlated with increased levels of cAMP. In conclusion, BCECs cultured in vitro coherently form a BBB and express basement membrane proteins as a feature of maturation. Cover Image for this issue: doi: 10.1111/jnc.13789. © 2016 International Society for Neurochemistry.

  14. Fate of cerium dioxide nanoparticles in endothelial cells: exocytosis

    Energy Technology Data Exchange (ETDEWEB)

    Strobel, Claudia, E-mail: Claudia.Strobel@med.uni-jena.de [Jena University Hospital – Friedrich Schiller University Jena, Department of Experimental Radiology, Institute of Diagnostic and Interventional Radiology (Germany); Oehring, Hartmut [Jena University Hospital – Friedrich Schiller University Jena, Institute of Anatomy II (Germany); Herrmann, Rudolf [University of Augsburg, Department of Physics (Germany); Förster, Martin [Jena University Hospital – Friedrich Schiller University Jena, Department of Internal Medicine I, Division of Pulmonary Medicine and Allergy/Immunology (Germany); Reller, Armin [University of Augsburg, Department of Physics (Germany); Hilger, Ingrid, E-mail: ingrid.hilger@med.uni-jena.de [Jena University Hospital – Friedrich Schiller University Jena, Department of Experimental Radiology, Institute of Diagnostic and Interventional Radiology (Germany)

    2015-05-15

    Although cytotoxicity and endocytosis of nanoparticles have been the subject of numerous studies, investigations regarding exocytosis as an important mechanism to reduce intracellular nanoparticle accumulation are rather rare and there is a distinct lack of knowledge. The current study investigated the behavior of human microvascular endothelial cells to exocytose cerium dioxide (CeO{sub 2}) nanoparticles (18.8 nm) by utilization of specific inhibitors [brefeldin A; nocodazole; methyl-β-cyclodextrin (MβcD)] and different analytical methods (flow cytometry, transmission electron microscopy, inductively coupled plasma mass spectrometry). Overall, it was found that endothelial cells were able to release CeO{sub 2} nanoparticles via exocytosis after the migration of nanoparticle containing endosomes toward the plasma membrane. The exocytosis process occurred mainly by fusion of vesicular membranes with plasma membrane resulting in the discharge of vesicular content to extracellular environment. Nevertheless, it seems to be likely that nanoparticles present in the cytosol could leave the cells in a direct manner. MβcD treatment led to the strongest inhibition of the nanoparticle exocytosis indicating a significant role of the plasma membrane cholesterol content in the exocytosis process. Brefeldin A (inhibitor of Golgi-to-cell-surface-transport) caused a higher inhibitory effect on exocytosis than nocodazole (inhibitor of microtubules). Thus, the transfer from distal Golgi compartments to the cell surface influenced the exocytosis process of the CeO{sub 2} nanoparticles more than the microtubule-associated transport. In conclusion, endothelial cells, which came in contact with nanoparticles, e.g., after intravenously applied nano-based drugs, can regulate their intracellular nanoparticle amount, which is necessary to avoid adverse nanoparticle effects on cells.

  15. Characterization of the L-glutamate clearance pathways across the blood-brain barrier and the effect of astrocytes in an in vitro blood-brain barrier model

    DEFF Research Database (Denmark)

    Helms, Hans CC; Aldana, Blanca I; Groth, Simon

    2017-01-01

    The aim was to characterize the clearance pathways for L-glutamate from the brain interstitial fluid across the blood-brain barrier using a primary in vitro bovine endothelial/rat astrocyte co-culture. Transporter profiling was performed using uptake studies of radiolabeled L-glutamate with co...... brain to blood via the concerted action of abluminal and luminal transport proteins, but the total brain clearance is highly dependent on metabolism in astrocytes and endothelial cells followed by transport of metabolites....

  16. Alpha-, gamma- and delta-tocopherols reduce inflammatory angiogenesis in human microvascular endothelial cells.

    Science.gov (United States)

    Wells, Shannon R; Jennings, Merilyn H; Rome, Courtney; Hadjivassiliou, Vicky; Papas, Konstantinos A; Alexander, Jonathon S

    2010-07-01

    Vitamin E, a micronutrient (comprising alpha-, beta-, gamma- and delta-tocopherols, alpha-, beta-, gamma- and delta-tocotrienols), has documented antioxidant and non-antioxidant effects, some of which inhibit inflammation and angiogenesis. We compared the abilities of alpha-, gamma- and delta-tocopherols to regulate human blood cytotoxicity (BEC) and lymphatic endothelial cytotoxicity (LEC), proliferation, invasiveness, permeability, capillary formation and suppression of TNF-alpha-induced VCAM-1 as in vitro models of inflammatory angiogenesis. alpha-, gamma- and delta-tocopherols were not toxic to either cell type up to 40 microM. In BEC, confluent cell density was decreased by all concentrations of delta- and gamma-tocopherol (10-40 microM) but not by alpha-tocopherol. LEC showed no change in cell density in response to tocopherols. delta-Tocopherol (40 microM), but not other isomers, decreased BEC invasiveness. In LEC, all doses of gamma-tocopherol, as well as the highest dose of alpha-tocopherol (40 microM), decreased cell invasiveness. delta-Tocopherol had no effect on LEC invasiveness at any molarity. delta-Tocopherol dose dependently increased cell permeability at 48 h in BEC and LEC; alpha- and gamma-tocopherols showed slight effects. Capillary tube formation was decreased by high dose (40 microM) concentrations of alpha-, gamma- and delta-tocopherol, but showed no effects with smaller doses (10-20 microM) in BEC. gamma-Tocopherol (10-20 microM) and alpha-tocopherol (10 microM), but not delta-tocopherol, increased LEC capillary tube formation. Lastly, in BEC, alpha-, gamma- and delta-tocopherol each dose-dependently reduced TNF-alpha-induced expression of VCAM-1. In LEC, there was no significant change to TNF-alpha-induced VCAM-1 expression with any concentration of alpha-, gamma- or delta-tocopherol. These data demonstrate that physiological levels (0-40 microM) of alpha-, gamma- and delta-tocopherols are nontoxic and dietary tocopherols, especially delta

  17. Optimization of the Ultrasound-Induced Blood-Brain Barrier Opening

    OpenAIRE

    Konofagou, Elisa E.

    2012-01-01

    Current treatments of neurological and neurodegenerative diseases are limited due to the lack of a truly non-invasive, transient, and regionally selective brain drug delivery method. The brain is particularly difficult to deliver drugs to because of the blood-brain barrier (BBB). The impermeability of the BBB is due to the tight junctions connecting adjacent endothelial cells and highly regulatory transport systems of the endothelial cell membranes. The main function of the BBB is ion and vol...

  18. Development of the blood-brain barrier: a historical point of view.

    Science.gov (United States)

    Ribatti, Domenico; Nico, Beatrice; Crivellato, Enrico; Artico, Marco

    2006-01-01

    Although there has been considerable controversy since the observation by Ehrlich more than 100 years ago that the brain did not take up dyes from the vascular system, the concept of an endothelial blood-brain barrier (BBB) was confirmed by the unequivocal demonstration that the passage of molecules from blood to brain and vice versa was prevented by endothelial tight junctions (TJs). There are three major functions implicated in the term "BBB": protection of the brain from the blood milieu, selective transport, and metabolism or modification of blood- or brain-borne substances. The BBB phenotype develops under the influence of associated brain cells, especially astrocytic glia, and consists of complex TJs and a number of specific transport and enzyme systems that regulate molecular traffic across the endothelial cells. The development of the BBB is a complex process that leads to endothelial cells with unique permeability characteristics due to high electrical resistance and the expression of specific transporters and metabolic pathways. This review article summarizes the historical background underlying our current knowledge of the cellular and molecular mechanisms involved in the development and maintenance of the BBB. (c) 2006 Wiley-Liss, Inc.

  19. Drug-induced trafficking of p-glycoprotein in human brain capillary endothelial cells as demonstrated by exposure to mitomycin C.

    Science.gov (United States)

    Noack, Andreas; Noack, Sandra; Hoffmann, Andrea; Maalouf, Katia; Buettner, Manuela; Couraud, Pierre-Olivier; Romero, Ignacio A; Weksler, Babette; Alms, Dana; Römermann, Kerstin; Naim, Hassan Y; Löscher, Wolfgang

    2014-01-01

    P-glycoprotein (Pgp; ABCB1/MDR1) is a major efflux transporter at the blood-brain barrier (BBB), restricting the penetration of various compounds. In other tissues, trafficking of Pgp from subcellular stores to the cell surface has been demonstrated and may constitute a rapid way of the cell to respond to toxic compounds by functional membrane insertion of the transporter. It is not known whether drug-induced Pgp trafficking also occurs in brain capillary endothelial cells that form the BBB. In this study, trafficking of Pgp was investigated in human brain capillary endothelial cells (hCMEC/D3) that were stably transfected with a doxycycline-inducible MDR1-EGFP fusion plasmid. In the presence of doxycycline, these cells exhibited a 15-fold increase in Pgp-EGFP fusion protein expression, which was associated with an increased efflux of the Pgp substrate rhodamine 123 (Rho123). The chemotherapeutic agent mitomycin C (MMC) was used to study drug-induced trafficking of Pgp. Confocal fluorescence microscopy of single hCMEC/D3-MDR1-EGFP cells revealed that Pgp redistribution from intracellular pools to the cell surface occurred within 2 h of MMC exposure. Pgp-EGFP exhibited a punctuate pattern at the cell surface compatible with concentrated regions of the fusion protein in membrane microdomains, i.e., lipid rafts, which was confirmed by Western blot analysis of biotinylated cell surface proteins in Lubrol-resistant membranes. MMC exposure also increased the functionality of Pgp as assessed in three functional assays with Pgp substrates (Rho123, eFluxx-ID Gold, calcein-AM). However, this increase occurred with some delay after the increased Pgp expression and coincided with the release of Pgp from the Lubrol-resistant membrane complexes. Disrupting rafts by depleting the membrane of cholesterol increased the functionality of Pgp. Our data present the first direct evidence of drug-induced Pgp trafficking at the human BBB and indicate that Pgp has to be released from lipid

  20. Drug-Induced Trafficking of P-Glycoprotein in Human Brain Capillary Endothelial Cells as Demonstrated by Exposure to Mitomycin C

    Science.gov (United States)

    Noack, Andreas; Noack, Sandra; Hoffmann, Andrea; Maalouf, Katia; Buettner, Manuela; Couraud, Pierre-Olivier; Romero, Ignacio A.; Weksler, Babette; Alms, Dana; Römermann, Kerstin; Naim, Hassan Y.; Löscher, Wolfgang

    2014-01-01

    P-glycoprotein (Pgp; ABCB1/MDR1) is a major efflux transporter at the blood-brain barrier (BBB), restricting the penetration of various compounds. In other tissues, trafficking of Pgp from subcellular stores to the cell surface has been demonstrated and may constitute a rapid way of the cell to respond to toxic compounds by functional membrane insertion of the transporter. It is not known whether drug-induced Pgp trafficking also occurs in brain capillary endothelial cells that form the BBB. In this study, trafficking of Pgp was investigated in human brain capillary endothelial cells (hCMEC/D3) that were stably transfected with a doxycycline-inducible MDR1-EGFP fusion plasmid. In the presence of doxycycline, these cells exhibited a 15-fold increase in Pgp-EGFP fusion protein expression, which was associated with an increased efflux of the Pgp substrate rhodamine 123 (Rho123). The chemotherapeutic agent mitomycin C (MMC) was used to study drug-induced trafficking of Pgp. Confocal fluorescence microscopy of single hCMEC/D3-MDR1-EGFP cells revealed that Pgp redistribution from intracellular pools to the cell surface occurred within 2 h of MMC exposure. Pgp-EGFP exhibited a punctuate pattern at the cell surface compatible with concentrated regions of the fusion protein in membrane microdomains, i.e., lipid rafts, which was confirmed by Western blot analysis of biotinylated cell surface proteins in Lubrol-resistant membranes. MMC exposure also increased the functionality of Pgp as assessed in three functional assays with Pgp substrates (Rho123, eFluxx-ID Gold, calcein-AM). However, this increase occurred with some delay after the increased Pgp expression and coincided with the release of Pgp from the Lubrol-resistant membrane complexes. Disrupting rafts by depleting the membrane of cholesterol increased the functionality of Pgp. Our data present the first direct evidence of drug-induced Pgp trafficking at the human BBB and indicate that Pgp has to be released from lipid

  1. Insulin-resistant glucose metabolism in patients with microvascular angina--syndrome X

    DEFF Research Database (Denmark)

    Vestergaard, H; Skøtt, P; Steffensen, R

    1995-01-01

    Studies in patients with microvascular angina (MA) or the cardiologic syndrome X have shown a hyperinsulinemic response to an oral glucose challenge, suggesting insulin resistance and a role for increased serum insulin in coronary microvascular dysfunction. The aim of the present study was to exa......Studies in patients with microvascular angina (MA) or the cardiologic syndrome X have shown a hyperinsulinemic response to an oral glucose challenge, suggesting insulin resistance and a role for increased serum insulin in coronary microvascular dysfunction. The aim of the present study...... was to examine whether patients with MA are insulin-resistant. Nine patients with MA and seven control subjects were studied. All were sedentary and glucose-tolerant. Coronary arteriography was normal in all participants, and exercise-induced coronary ischemia was demonstrated in all MA patients. A euglycemic...... metabolism (8.4 +/- 0.9 v 12.5 +/- 1.3 mg.kg FFM-1.min-1, P

  2. Radionuclide assessment of pulmonary microvascular permeability

    Energy Technology Data Exchange (ETDEWEB)

    Groeneveld, A.B.J. [Medical Intensive Care Unit, Department of Internal Medicine, Free University Hospital, De Boelelaan 1117, 1081 HV Amsterdam (Netherlands)

    1997-04-01

    The literature has been reviewed to evaluate the technique and clinical value of radionuclide measurements of microvascular permeability and oedema formation in the lungs. Methodology, modelling and interpretation vary widely among studies. Nevertheless, most studies agree on the fact that the measurement of permeability via pulmonary radioactivity measurements of intravenously injected radiolabelled proteins versus that in the blood pool, the so-called pulmonary protein transport rate (PTR), can assist the clinician in discriminating between permeability oedema of the lungs associated with the adult respiratory distress syndrome (ARDS) and oedema caused by an increased filtration pressure, for instance in the course of cardiac disease, i.e. pressure-induced pulmonary oedema. Some of the techniques used to measure PTR are also able to detect subclinical forms of lung microvascular injury not yet complicated by permeability oedema. This may occur after cardiopulmonary bypass and major vascular surgery, for instance. By paralleling the clinical severity and course of the ARDS, the PTR method may also serve as a tool to evaluate new therapies for the syndrome. Taken together, the currently available radionuclide methods, which are applicable at the bedside in the intensive care unit, may provide a gold standard for detecting minor and major forms of acute microvascular lung injury, and for evaluating the severity, course and response to treatment. (orig.). With 2 tabs.

  3. Profile of Microvascular Disease in Type 2 Diabetes in a Tertiary ...

    African Journals Online (AJOL)

    Background: Diabetes mellitus (DM) is a metabolic disorder complicated by microvascular and macrovascular diseases. The clinical profile of these complications has not been adequately studied in many tertiary health care centers in India. Aim: The authors studied the clinical profile of microvascular diabetes ...

  4. Effect of rosuvastatin on fasting and postprandial endothelial biomarker levels and microvascular reactivity in patients with type 2 diabetes and dyslipidemia: a preliminary report.

    Science.gov (United States)

    Kim, Kyoung Min; Jung, Kyong Yeun; Yun, Han Mi; Lee, Seo Young; Oh, Tae Jung; Jang, Hak Chul; Lim, Soo

    2017-11-09

    The cardiovascular benefits of statins have been proven, but their effect on circulation in small vessels has not been examined fully. We investigated the effect of 20 mg rosuvastatin on biomarkers, including paraoxonase-1 (PON-1) and asymmetric dimethylarginine (ADMA), and on microvascular reactivity. We enrolled 20 dyslipidemic patients with type 2 diabetes and 20 age- and body mass index (BMI)-matched healthy controls. Rosuvastatin (20 mg/day) was given to the patient group for 12 weeks. Biochemical parameters, including PON-1 and ADMA, were compared between the patient and control groups, and before and after rosuvastatin treatment in the patient group. Fasting and 2 h postprandial levels of PON-1 and ADMA after mixed-meal challenge were also compared. Microvascular reactivity in a peripheral artery was examined using laser Doppler flowmetry. The respective mean ± standard deviation of age and BMI were 50.1 ± 3.8 year and 25.8 ± 3.7 kg/m 2 in the patients and 50.2 ± 3.2 year and 25.4 ± 3.4 kg/m 2 in the controls. The patient group had worse profiles of cardiometabolic biomarkers, including PON-1 and ADMA, than the controls. In the patients treated with 20 mg rosuvastatin, low-density lipoprotein (LDL)-cholesterol decreased from 147.2 ± 26.5 to 68.3 ± 24.5 mg/dL and high-density lipoprotein (HDL)-cholesterol increased from 42.4 ± 5.2 to 44.7 ± 6.2 mg/dL (both P fasting and 2 h postprandial levels of PON-1 increased and those of ADMA decreased after treatment with rosuvastatin for 12 weeks. The changes in postprandial levels of both biomarkers were greater than those after fasting. Microcirculation assessed as reactive hyperemia in the patients after an ischemic challenge increased significantly from 335.3 ± 123.4 to 402.7 ± 133.4% after rosuvastatin treatment. The postprandial changes in the biomarkers were significantly associated with improvement of microvascular reactivity. Rosuvastatin treatment for 12

  5. Urinary Leukotriene E4 Is Associated with Renal Function but Not with Endothelial Function in Type 2 Diabetes

    Directory of Open Access Journals (Sweden)

    Arnar Rafnsson

    2013-01-01

    Full Text Available Leukotrienes are inflammatory and vasoactive mediators implicated in endothelium-dependent relaxations and atherosclerosis. Urinary leukotriene E4 (U-LTE4 is a validated disease marker of asthma and increases also in diabetes and acute coronary syndromes. The aim of the present study was to evaluate the association of U-LTE4 and CRP with endothelial function. Urine samples were obtained from 30 subjects (80% males; median age 65 with type 2 diabetes of at least two years duration and a median glomerular filtration rate (eGFR of 71 (14–129 mL/min. Reactive hyperemia index (RHI was used as a measure of microvascular endothelial function, whereas macrovascular endothelial function was determined be means of flow-mediated dilatation of the brachial artery (FMD. Decreased renal function was associated with lower concentrations of U-LTE4. In addition, U-LTE4 was correlated with serum creatinine (R=−0.572; P=0.001 and eGFR (R=0.517; P=0.0036. A stepwise multiple linear regression analysis identified eGFR as an independent predictor of U-LTE4 concentrations. In conclusion, the present results did not establish an association of U-LTE4 with endothelial dysfunction. However, eGFR was an independent predictor of U-LTE4, but not CRP, in this cohort, suggesting that GFR should be considered in biomarker studies of U-LTE4.

  6. Engineering Microvascularized 3D Tissue Using Alginate-Chitosan Microcapsules

    OpenAIRE

    Zhang, Wujie; Choi, Jung K.; He, Xiaoming

    2017-01-01

    Construction of vascularized tissues is one of the major challenges of tissue engineering. The goal of this study was to engineer 3D microvascular tissues by incorporating the HUVEC-CS cells with a collagen/alginate-chitosan (AC) microcapsule scaffold. In the presence of AC microcapsules, a 3D vascular-like network was clearly observable. The results indicated the importance of AC microcapsules in engineering microvascular tissues -- providing support and guiding alignment of HUVEC-CS cells. ...

  7. Opening of brain blood barrier induced by red light and central analgesic improvement of cobra neurotoxin.

    Science.gov (United States)

    Ye, Yong; Li, Yue; Fang, Fei

    2014-05-05

    Cobra neurotoxin (NT) has central analgesic effects, but it is difficult to pass through brain blood barrier (BBB). A novel method of red light induction is designed to help NT across BBB, which is based on photosensitizer activation by red light to generate reactive oxygen species (ROS) to open BBB. The effects were evaluated on cell models and animals in vivo with illumination by semiconductor laser at 670nm on photosensitizer pheophorbide isolated from silkworm excrement. Brain microvascular endothelial cells and astrocytes were co-cultured to build up BBB cell model. The radioactivity of (125)I-NT was measured in cells and tissues for NT permeation. Three ways of cranial irradiation, nasal cavity and intravascular irradiation were tested with combined injection of (125)I-NT 20μg/kg and pheophorbide 100μg/kg to rats, and organs of rats were separated and determined the radioactivity. Paw pressure test in rats, hot plate and writhing test in mice were applied to appraise the analgesic effects. NT across BBB cell model increased with time of illumination, and reached stable level after 60min. So did ROS in cells. NT mainly distributed in liver and kidney of rats, significantly increased in brain after illumination, and improved analgesic effects. Excitation of pheophorbide at red light produces ROS to open BBB, help NT enter brain, and enhance its central action. This research provides a new method for drug across BBB to improve its central role. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Integration of Self-Assembled Microvascular Networks with Microfabricated PEG-Based Hydrogels.

    Science.gov (United States)

    Cuchiara, Michael P; Gould, Daniel J; McHale, Melissa K; Dickinson, Mary E; West, Jennifer L

    2012-11-07

    Despite tremendous efforts, tissue engineered constructs are restricted to thin, simple tissues sustained only by diffusion. The most significant barrier in tissue engineering is insufficient vascularization to deliver nutrients and metabolites during development in vitro and to facilitate rapid vascular integration in vivo. Tissue engineered constructs can be greatly improved by developing perfusable microvascular networks in vitro in order to provide transport that mimics native vascular organization and function. Here a microfluidic hydrogel is integrated with a self-assembling pro-vasculogenic co-culture in a strategy to perfuse microvascular networks in vitro. This approach allows for control over microvascular network self-assembly and employs an anastomotic interface for integration of self-assembled micro-vascular networks with fabricated microchannels. As a result, transport within the system shifts from simple diffusion to vessel supported convective transport and extra-vessel diffusion, thus improving overall mass transport properties. This work impacts the development of perfusable prevascularized tissues in vitro and ultimately tissue engineering applications in vivo.

  9. Therapeutic Effects of PPARα on Neuronal Death and Microvascular Impairment

    Directory of Open Access Journals (Sweden)

    Elizabeth P. Moran

    2015-01-01

    Full Text Available Peroxisome-proliferator activated receptor-alpha (PPARα is a broadly expressed nuclear hormone receptor and is a transcription factor for diverse target genes possessing a PPAR response element (PPRE in the promoter region. The PPRE is highly conserved, and PPARs thus regulate transcription of an extensive array of target genes involved in energy metabolism, vascular function, oxidative stress, inflammation, and many other biological processes. PPARα has potent protective effects against neuronal cell death and microvascular impairment, which have been attributed in part to its antioxidant and anti-inflammatory properties. Here we discuss PPARα’s effects in neurodegenerative and microvascular diseases and also recent clinical findings that identified therapeutic effects of a PPARα agonist in diabetic microvascular complications.

  10. Cutaneous microvascular perfusion responses to insulin iontophoresis are differentially affected by insulin resistance after spinal cord injury.

    Science.gov (United States)

    La Fountaine, Michael F; Cirnigliaro, Christopher M; Azarelo, Frank; Hobson, Joshua C; Tascione, Oriana; Swonger, Kirsten N; Dyson-Hudson, Trevor; Bauman, William A

    2017-09-01

    What is the central question of this study? What impact does insulin resistance have on cutaneous perfusion responses to insulin iontophoresis in vascular beds with markedly reduced or functionally ablated sympathetic nervous system vasomotor function resulting from spinal cord injury? What is the main finding and its importance? Persons with spinal cord injury have sublesional microvascular endothelial dysfunction, as indicated by a blunted cutaneous perfusion response to acetylcholine iontophoresis, and the presence of insulin resistance has a further confounding effect on endothelium-mediated changes to cutaneous perfusion in the lower extremities. Endothelium-mediated mechanisms that regulate skin blood flow might play an integral role in optimizing skin perfusion in vascular beds with sympathetic nervous system vasomotor impairment, such as in spinal cord injury (SCI). Insulin is a vasoactive hormone and second messenger of nitric oxide that facilitates endothelium-mediated dilatation. The effects of insulin resistance (IR) on sublesional cutaneous perfusion responses to insulin provocation have yet to be described in persons with SCI. Persons with SCI and an able-bodied (AB) cohort were divided into subgroups based upon fasting plasma insulin concentration cut-offs for IR (≥13.13 mIU ml -1 ) or insulin sensitivity (IS; insulin, acetylcholine or placebo iontophoresis in the lower extremities; BPU responses were log 10 transformed to facilitate comparisons, and the net insulin response (NetIns) BPU response was calculated (insulin minus placebo BPU response). The NetIns was significantly greater in both IS groups compared with their corresponding IR group. The acetylcholine-mediated BPU responses in the SCI subgroups were significantly lower than those in the ABIS group. The proportional BPU responses of NetIns to acetylcholine in the IS cohorts (i.e. ABIS and SCIS) were significantly greater (P < 0.05) than that of each IR subgroup. The presence of IR

  11. Correlation of vascular endothelial growth factor and microvascular damage in diabetic retinopathy%糖尿病性视网膜病变患者 VEGF 与微血管损伤的相关性

    Institute of Scientific and Technical Information of China (English)

    温积权; 汪怿; 杨杰; 吴若欣

    2015-01-01

    ?AIM:To explore the correlation of vascular endothelial growth factor ( VEGF ) level and microvascular damage degree in patients with proliferative diabetic retinopathy ( DR) . ?METHODS:Seventy-one patients with diabetes were analyzed retrospectively, and divided into 3 groups according to the degree of DR:the pure diabetes group ( n=31), the pure DR group (n=22) and the proliferative DR group ( n=18 ) .The incidence of microvascular damage was compared;At the same time, fasting venous blood of patients was extracted, and the VEGF levels were detected with ELISA kits, the endothelial cells ( ECs ) , endothelial progenitor cells ( EPCs ) and circulating progenitor cells ( CPCs ) counts were detected with flow cytometry. ?RESULTS:The incidence of diabetic nephropathy and diabetic neuropathy was significantly different in three groups, proliferative DR group was higher than pure DR group and pure diabetes group, the difference was statistically significant (P ?CONCLUSION:VEGF has important significance in the clinical diagnosis and medical treatment of diabetic retinopathy, especially proliferative diabetic retinopathy.%目的:探讨糖尿病视网膜病变( diabetic retinopathy, DR)患者血管内皮生长因子( vascular endothelial growth factor, VEGF)水平与微血管损伤程度的相关性。  方法:回顾性分析本院收治的糖尿病患者71例,根据有无DR及病变程度分为三组:单纯糖尿病组( n=31)、单纯型DR组( n=22)、增殖型DR组( n=18),比较各组微血管病变发生率。同时,取患者空腹肘静脉血,采用ELISA试剂盒测定血清VEGF水平,采用流式细胞仪检测内皮细胞( ECs)、内皮祖细胞( EPCs)、循环祖细胞( CPCs)计数。  结果:各组糖尿病肾病和糖尿病神经病变发生率有明显差异,增殖型DR组高于单纯型DR组和单纯糖尿病组,差异有统计学意义( P<0.05

  12. Down-regulation of selected Blood-brain Barrier Specific Genes from Capillaries to Bovine In Vitro Models

    DEFF Research Database (Denmark)

    Goldeman, Charlotte; Saaby, Lasse; Brodin, Birger

    Cultures of primary bovine brain endothelial cells (BECs) grown, often together with astrocytes, on permeable supports in two-compartment culture systems are commonly used as an in vitro model of the blood-brain barrier (BBB). While trans-endothelial electrical resistance, restriction...... the in vivo gene expression of brain capillary endothelial cells. Primary bovine endothelial cells and rat astrocytes were cultured in different culture configurations and the mRNA expression of selected genes (vWF, Glut-1, P-gp, claudin-1,-5, occludin, JAM-1, LAT-1, SLC16A1, MRP-1,-4, BCRP, ZO-1, AP, TPA...

  13. Novel brain arteriovenous malformation mouse models for type 1 hereditary hemorrhagic telangiectasia.

    Directory of Open Access Journals (Sweden)

    Eun-Jung Choi

    Full Text Available Endoglin (ENG is a causative gene of type 1 hereditary hemorrhagic telangiectasia (HHT1. HHT1 patients have a higher prevalence of brain arteriovenous malformation (AVM than the general population and patients with other HHT subtypes. The pathogenesis of brain AVM in HHT1 patients is currently unknown and no specific medical therapy is available to treat patients. Proper animal models are crucial for identifying the underlying mechanisms for brain AVM development and for testing new therapies. However, creating HHT1 brain AVM models has been quite challenging because of difficulties related to deleting Eng-floxed sequence in Eng(2fl/2fl mice. To create an HHT1 brain AVM mouse model, we used several Cre transgenic mouse lines to delete Eng in different cell-types in Eng(2fl/2fl mice: R26CreER (all cell types after tamoxifen treatment, SM22α-Cre (smooth muscle and endothelial cell and LysM-Cre (lysozyme M-positive macrophage. An adeno-associated viral vector expressing vascular endothelial growth factor (AAV-VEGF was injected into the brain to induce focal angiogenesis. We found that SM22α-Cre-mediated Eng deletion in the embryo caused AVMs in the postnatal brain, spinal cord, and intestines. Induction of Eng deletion in adult mice using R26CreER plus local VEGF stimulation induced the brain AVM phenotype. In both models, Eng-null endothelial cells were detected in the brain AVM lesions, and formed mosaicism with wildtype endothelial cells. However, LysM-Cre-mediated Eng deletion in the embryo did not cause AVM in the postnatal brain even after VEGF stimulation. In this study, we report two novel HHT1 brain AVM models that mimic many phenotypes of human brain AVM and can thus be used for studying brain AVM pathogenesis and testing new therapies. Further, our data indicate that macrophage Eng deletion is insufficient and that endothelial Eng homozygous deletion is required for HHT1 brain AVM development.

  14. Restoration of Impaired Metabolic Energy Balance (ATP Pool and Tube Formation Potential of Endothelial Cells under “high glucose”, Diabetic Conditions by the Bioinorganic Polymer Polyphosphate

    Directory of Open Access Journals (Sweden)

    Xiaohong Wang

    2017-11-01

    Full Text Available Micro-vascularization is a fast, energy-dependent process that is compromised by elevated glucose concentrations such as in diabetes mellitus disease. Here, we studied the effect of the physiological bioinorganic polymer, polyphosphate (polyP, on the reduced ATP content and impaired function of endothelial cells cultivated under “high glucose” (35 mM diabetes mellitus conditions concentrations. This high-energy biopolymer has been shown to provide a source of metabolic energy, stored in its phosphoanhydride bonds. We show that exposure of human umbilical vein endothelial cells (HUVEC cells to “high glucose” levels results in reduced cell viability, increased apoptotic cell death, and a decline in intracellular ATP level. As a consequence, the ability of HUVEC cells to form tube-like structures in the in vitro cell tube formation assay was almost completely abolished under “high glucose” conditions. Those cells were grown onto a physiological collagen scaffold (collagen/basement membrane extract. We demonstrate that these adverse effects of increased glucose levels can be reversed by administration of polyP to almost normal values. Using Na-polyP, complexed in a stoichiometric (molar ratio to Ca2+ ions and in the physiological concentration range between 30 and 300 µM, an almost complete restoration of the reduced ATP pool of cells exposed to “high glucose” was found, as well as a normalization of the number of apoptotic cells and energy-dependent tube formation. It is concluded that the adverse effects on endothelial cells caused by the metabolic energy imbalance at elevated glucose concentrations can be counterbalanced by polyP, potentially opening new strategies for treatment of the micro-vascular complications in diabetic patients.

  15. Differential regulation of TRPV1 channels by H2O2: implications for diabetic microvascular dysfunction

    Science.gov (United States)

    DelloStritto, Daniel J.; Connell, Patrick J.; Dick, Gregory M.; Fancher, Ibra S.; Klarich, Brittany; Fahmy, Joseph N.; Kang, Patrick T.; Chen, Yeong-Renn; Damron, Derek S.; Thodeti, Charles K.

    2016-01-01

    We demonstrated previously that TRPV1-dependent coupling of coronary blood flow (CBF) to metabolism is disrupted in diabetes. A critical amount of H2O2 contributes to CBF regulation; however, excessive H2O2 impairs responses. We sought to determine the extent to which differential regulation of TRPV1 by H2O2 modulates CBF and vascular reactivity in diabetes. We used contrast echocardiography to study TRPV1 knockout (V1KO), db/db diabetic, and wild type C57BKS/J (WT) mice. H2O2 dose-dependently increased CBF in WT mice, a response blocked by the TRPV1 antagonist SB366791. H2O2-induced vasodilation was significantly inhibited in db/db and V1KO mice. H2O2 caused robust SB366791-sensitive dilation in WT coronary microvessels; however, this response was attenuated in vessels from db/db and V1KO mice, suggesting H2O2-induced vasodilation occurs, in part, via TRPV1. Acute H2O2 exposure potentiated capsaicin-induced CBF responses and capsaicin-mediated vasodilation in WT mice, whereas prolonged luminal H2O2 exposure blunted capsaicin-induced vasodilation. Electrophysiology studies re-confirms acute H2O2 exposure activated TRPV1 in HEK293A and bovine aortic endothelial cells while establishing that H2O2 potentiate capsaicin-activated TRPV1 currents, whereas prolonged H2O2 exposure attenuated TRPV1 currents. Verification of H2O2-mediated activation of intrinsic TRPV1 specific currents were found in isolated mouse coronary endothelial cells from WT mice and decreased in endothelial cells from V1KO mice. These data suggest prolonged H2O2 exposure impairs TRPV1-dependent coronary vascular signaling. This may contribute to microvascular dysfunction and tissue perfusion deficits characteristic of diabetes. PMID:26907473

  16. Aryl hydrocarbon receptor is necessary to protect fetal human pulmonary microvascular endothelial cells against hyperoxic injury: Mechanistic roles of antioxidant enzymes and RelB

    International Nuclear Information System (INIS)

    Zhang, Shaojie; Patel, Ananddeep; Chu, Chun; Jiang, Weiwu; Wang, Lihua; Welty, Stephen E.; Moorthy, Bhagavatula; Shivanna, Binoy

    2015-01-01

    Hyperoxia contributes to the development of bronchopulmonary dysplasia (BPD) in premature infants. Activation of the aryl hydrocarbon receptor (AhR) protects adult and newborn mice against hyperoxic lung injury by mediating increases in the expression of phase I (cytochrome P450 (CYP) 1A) and phase II (NADP(H) quinone oxidoreductase (NQO1)) antioxidant enzymes (AOE). AhR positively regulates the expression of RelB, a component of the nuclear factor-kappaB (NF-κB) protein that contributes to anti-inflammatory processes in adult animals. Whether AhR regulates the expression of AOE and RelB, and protects fetal primary human lung cells against hyperoxic injury is unknown. Therefore, we tested the hypothesis that AhR-deficient fetal human pulmonary microvascular endothelial cells (HPMEC) will have decreased RelB activation and AOE, which will in turn predispose them to increased oxidative stress, inflammation, and cell death compared to AhR-sufficient HPMEC upon exposure to hyperoxia. AhR-deficient HPMEC showed increased hyperoxia-induced reactive oxygen species (ROS) generation, cleavage of poly(ADP-ribose) polymerase (PARP), and cell death compared to AhR-sufficient HPMEC. Additionally, AhR-deficient cell culture supernatants displayed increased macrophage inflammatory protein 1α and 1β, indicating a heightened inflammatory state. Interestingly, loss of AhR was associated with a significantly attenuated CYP1A1, NQO1, superoxide dismutase 1(SOD1), and nuclear RelB protein expression. These findings support the hypothesis that decreased RelB activation and AOE in AhR-deficient cells is associated with increased hyperoxic injury compared to AhR-sufficient cells. - Highlights: • AhR deficiency potentiates oxygen toxicity in human fetal lung cells. • Deficient AhR signaling increases hyperoxia-induced cell death. • AhR deficiency increases hyperoxia-induced ROS generation and inflammation. • Anti-oxidant enzyme levels are attenuated in AhR-deficient lung cells

  17. Aryl hydrocarbon receptor is necessary to protect fetal human pulmonary microvascular endothelial cells against hyperoxic injury: Mechanistic roles of antioxidant enzymes and RelB

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Shaojie; Patel, Ananddeep; Chu, Chun; Jiang, Weiwu; Wang, Lihua; Welty, Stephen E.; Moorthy, Bhagavatula; Shivanna, Binoy, E-mail: shivanna@bcm.edu

    2015-07-15

    Hyperoxia contributes to the development of bronchopulmonary dysplasia (BPD) in premature infants. Activation of the aryl hydrocarbon receptor (AhR) protects adult and newborn mice against hyperoxic lung injury by mediating increases in the expression of phase I (cytochrome P450 (CYP) 1A) and phase II (NADP(H) quinone oxidoreductase (NQO1)) antioxidant enzymes (AOE). AhR positively regulates the expression of RelB, a component of the nuclear factor-kappaB (NF-κB) protein that contributes to anti-inflammatory processes in adult animals. Whether AhR regulates the expression of AOE and RelB, and protects fetal primary human lung cells against hyperoxic injury is unknown. Therefore, we tested the hypothesis that AhR-deficient fetal human pulmonary microvascular endothelial cells (HPMEC) will have decreased RelB activation and AOE, which will in turn predispose them to increased oxidative stress, inflammation, and cell death compared to AhR-sufficient HPMEC upon exposure to hyperoxia. AhR-deficient HPMEC showed increased hyperoxia-induced reactive oxygen species (ROS) generation, cleavage of poly(ADP-ribose) polymerase (PARP), and cell death compared to AhR-sufficient HPMEC. Additionally, AhR-deficient cell culture supernatants displayed increased macrophage inflammatory protein 1α and 1β, indicating a heightened inflammatory state. Interestingly, loss of AhR was associated with a significantly attenuated CYP1A1, NQO1, superoxide dismutase 1(SOD1), and nuclear RelB protein expression. These findings support the hypothesis that decreased RelB activation and AOE in AhR-deficient cells is associated with increased hyperoxic injury compared to AhR-sufficient cells. - Highlights: • AhR deficiency potentiates oxygen toxicity in human fetal lung cells. • Deficient AhR signaling increases hyperoxia-induced cell death. • AhR deficiency increases hyperoxia-induced ROS generation and inflammation. • Anti-oxidant enzyme levels are attenuated in AhR-deficient lung cells

  18. Activation of RhoA, but Not Rac1, Mediates Early Stages of S1P-Induced Endothelial Barrier Enhancement.

    Science.gov (United States)

    Zhang, Xun E; Adderley, Shaquria P; Breslin, Jerome W

    2016-01-01

    Compromised endothelial barrier function is a hallmark of inflammation. Rho family GTPases are critical in regulating endothelial barrier function, yet their precise roles, particularly in sphingosine-1-phosphate (S1P)-induced endothelial barrier enhancement, remain elusive. Confluent cultures of human umbilical vein endothelial cells (HUVEC) or human dermal microvascular endothelial cells (HDMEC) were used to model the endothelial barrier. Barrier function was assessed by determining the transendothelial electrical resistance (TER) using an electrical cell-substrate impedance sensor (ECIS). The roles of Rac1 and RhoA were tested in S1P-induced barrier enhancement. The results show that pharmacologic inhibition of Rac1 with Z62954982 failed to block S1P-induced barrier enhancement. Likewise, expression of a dominant negative form of Rac1, or knockdown of native Rac1 with siRNA, failed to block S1P-induced elevations in TER. In contrast, blockade of RhoA with the combination of the inhibitors Rhosin and Y16 significantly reduced S1P-induced increases in TER. Assessment of RhoA activation in real time using a fluorescence resonance energy transfer (FRET) biosensor showed that S1P increased RhoA activation primarily at the edges of cells, near junctions. This was complemented by myosin light chain-2 phosphorylation at cell edges, and increased F-actin and vinculin near intercellular junctions, which could all be blocked with pharmacologic inhibition of RhoA. The results suggest that S1P causes activation of RhoA at the cell periphery, stimulating local activation of the actin cytoskeleton and focal adhesions, and resulting in endothelial barrier enhancement. S1P-induced Rac1 activation, however, does not appear to have a significant role in this process.

  19. Activation of RhoA, but Not Rac1, Mediates Early Stages of S1P-Induced Endothelial Barrier Enhancement.

    Directory of Open Access Journals (Sweden)

    Xun E Zhang

    Full Text Available Compromised endothelial barrier function is a hallmark of inflammation. Rho family GTPases are critical in regulating endothelial barrier function, yet their precise roles, particularly in sphingosine-1-phosphate (S1P-induced endothelial barrier enhancement, remain elusive. Confluent cultures of human umbilical vein endothelial cells (HUVEC or human dermal microvascular endothelial cells (HDMEC were used to model the endothelial barrier. Barrier function was assessed by determining the transendothelial electrical resistance (TER using an electrical cell-substrate impedance sensor (ECIS. The roles of Rac1 and RhoA were tested in S1P-induced barrier enhancement. The results show that pharmacologic inhibition of Rac1 with Z62954982 failed to block S1P-induced barrier enhancement. Likewise, expression of a dominant negative form of Rac1, or knockdown of native Rac1 with siRNA, failed to block S1P-induced elevations in TER. In contrast, blockade of RhoA with the combination of the inhibitors Rhosin and Y16 significantly reduced S1P-induced increases in TER. Assessment of RhoA activation in real time using a fluorescence resonance energy transfer (FRET biosensor showed that S1P increased RhoA activation primarily at the edges of cells, near junctions. This was complemented by myosin light chain-2 phosphorylation at cell edges, and increased F-actin and vinculin near intercellular junctions, which could all be blocked with pharmacologic inhibition of RhoA. The results suggest that S1P causes activation of RhoA at the cell periphery, stimulating local activation of the actin cytoskeleton and focal adhesions, and resulting in endothelial barrier enhancement. S1P-induced Rac1 activation, however, does not appear to have a significant role in this process.

  20. eEF-2 Phosphorylation Down-Regulates P-Glycoprotein Over-Expression in Rat Brain Microvessel Endothelial Cells.

    Directory of Open Access Journals (Sweden)

    Xing Hua Tang

    Full Text Available We investigated whether glutamate, NMDA receptors, and eukaryote elongation factor-2 kinase (eEF-2K/eEF-2 regulate P-glycoprotein expression, and the effects of the eEF-2K inhibitor NH125 on the expression of P-glycoprotein in rat brain microvessel endothelial cells (RBMECs.Cortex was obtained from newborn Wistar rat brains. After surface vessels and meninges were removed, the pellet containing microvessels was resuspended and incubated at 37°C in culture medium. Cell viability was assessed by the MTT assay. RBMECs were identified by immunohistochemistry with anti-vWF. P-glycoprotein, phospho-eEF-2, and eEF-2 expression were determined by western blot analysis. Mdr1a gene expression was analyzed by RT-PCR.Mdr1a mRNA, P-glycoprotein and phospho-eEF-2 expression increased in L-glutamate stimulated RBMECs. P-glycoprotein and phospho-eEF-2 expression were down-regulated after NH125 treatment in L-glutamate stimulated RBMECs.eEF-2K/eEF-2 should have played an important role in the regulation of P-glycoprotein expression in RBMECs. eEF-2K inhibitor NH125 could serve as an efficacious anti-multidrug resistant agent.

  1. HLA class I antibodies trigger increased adherence of monocytes to endothelial cells by eliciting an increase in endothelial P-selectin and, depending on subclass, by engaging FcγRs.

    Science.gov (United States)

    Valenzuela, Nicole M; Mulder, Arend; Reed, Elaine F

    2013-06-15

    Ab-mediated rejection (AMR) of solid organ transplants is characterized by intragraft macrophages. It is incompletely understood how donor-specific Ab binding to graft endothelium promotes monocyte adhesion, and what, if any, contribution is made by the Fc region of the Ab. We investigated the mechanisms underlying monocyte recruitment by HLA class I (HLA I) Ab-activated endothelium. We used a panel of murine mAbs of different subclasses to crosslink HLA I on human aortic, venous, and microvascular endothelial cells and measured the binding of human monocytic cell lines and peripheral blood monocytes. Both anti-HLA I murine (m)IgG1 and mIgG2a induced endothelial P-selectin, which was required for monocyte adhesion to endothelium irrespective of subclass. mIgG2a but not mIgG1 could bind human FcγRs. Accordingly, HLA I mIgG2a but not mIgG1 treatment of endothelial cells significantly augmented recruitment, predominantly through FcγRI, and, to a lesser extent, FcγRIIa. Moreover, HLA I mIgG2a promoted firm adhesion of monocytes to ICAM-1 through Mac-1, which may explain the prominence of monocytes during AMR. We confirmed these observations using human HLA allele-specific mAbs and IgG purified from transplant patient sera. HLA I Abs universally elicit endothelial exocytosis leading to monocyte adherence, implying that P-selectin is a putative therapeutic target to prevent macrophage infiltration during AMR. Importantly, the subclass of donor-specific Ab may influence its pathogenesis. These results imply that human IgG1 and human IgG3 should have a greater capacity to trigger monocyte infiltration into the graft than IgG2 or IgG4 due to enhancement by FcγR interactions.

  2. Gap filling of 3-D microvascular networks by tensor voting.

    Science.gov (United States)

    Risser, L; Plouraboue, F; Descombes, X

    2008-05-01

    We present a new algorithm which merges discontinuities in 3-D images of tubular structures presenting undesirable gaps. The application of the proposed method is mainly associated to large 3-D images of microvascular networks. In order to recover the real network topology, we need to fill the gaps between the closest discontinuous vessels. The algorithm presented in this paper aims at achieving this goal. This algorithm is based on the skeletonization of the segmented network followed by a tensor voting method. It permits to merge the most common kinds of discontinuities found in microvascular networks. It is robust, easy to use, and relatively fast. The microvascular network images were obtained using synchrotron tomography imaging at the European Synchrotron Radiation Facility. These images exhibit samples of intracortical networks. Representative results are illustrated.

  3. Activation of endothelial cells after exposure to ambient ultrafine particles: The role of NADPH oxidase

    International Nuclear Information System (INIS)

    Mo Yiqun; Wan Rong; Chien Sufan; Tollerud, David J.; Zhang Qunwei

    2009-01-01

    Several studies have shown that ultrafine particles (UFPs) may pass from the lungs to the circulation because of their very small diameter, and induce lung oxidative stress with a resultant increase in lung epithelial permeability. The direct effects of UFPs on vascular endothelium remain unknown. We hypothesized that exposure to UFPs leads to endothelial cell O 2 ·- generation via NADPH oxidase and results in activation of endothelial cells. Our results showed that UFPs, at a non-toxic dose, induced reactive oxygen species (ROS) generation in mouse pulmonary microvascular endothelial cells (MPMVEC) that was inhibited by pre-treatment with the ROS scavengers or inhibitors, but not with the mitochondrial inhibitor, rotenone. UFP-induced ROS generation in MPMVEC was abolished by p67 phox siRNA transfection and UFPs did not cause ROS generation in MPMVEC isolated from gp91 phox knock-out mice. UFP-induced ROS generation in endothelial cells was also determined in vivo by using a perfused lung model with imaging. Moreover, Western blot and immunofluorescence staining results showed that MPMVEC treated with UFPs resulted in the translocation of cytosolic proteins of NADPH oxidase, p47 phox , p67 phox and rac 1, to the plasma membrane. These results demonstrate that NADPH oxidase in the pulmonary endothelium is involved in ROS generation following exposure to UFPs. To investigate the activation of endothelial cells by UFP-induced oxidative stress, we determined the activation of the mitogen-activated protein kinases (MAPKs) in MPMVEC. Our results showed that exposure of MPMVEC to UFPs caused increased phosphorylation of p38 and ERK1/2 MAPKs that was blocked by pre-treatment with DPI or p67 phox siRNA. Exposure of MPMVEC obtained from gp91 phox knock-out mice to UFPs did not cause increased phosphorylation of p38 and ERK1/2 MAPKs. These findings confirm that UFPs can cause endothelial cells to generate ROS directly via activation of NADPH oxidase. UFP-induced ROS lead to

  4. Analysis of circulating hem-endothelial marker RNA levels in preterm infants

    Directory of Open Access Journals (Sweden)

    Kuint Jacob

    2009-06-01

    Full Text Available Abstract Background Circulating endothelial cells may serve as novel markers of angiogenesis. These include a subset of hem-endothelial progenitor cells that play a vital role in vascular growth and repair. The presence and clinical implications of circulating RNA levels as an expression for hematopoietic and endothelial-specific markers have not been previously evaluated in preterm infants. This study aims to determine circulating RNA levels of hem-endothelial marker genes in peripheral blood of preterm infants and begin to correlate these findings with prenatal complications. Methods Peripheral blood samples from seventeen preterm neonates were analyzed at three consecutive post-delivery time points (day 3–5, 10–15 and 30. Using quantitative reverse transcription-polymerase chain reaction we studied the expression patterns of previously established hem-endothelial-specific progenitor-associated genes (AC133, Tie-2, Flk-1 (VEGFR2 and Scl/Tal1 in association with characteristics of prematurity and preterm morbidity. Results Circulating Tie-2 and SCL/Tal1 RNA levels displayed an inverse correlation to gestational age (GA. We observed significantly elevated Tie-2 levels in preterm infants born to mothers with amnionitis, and in infants with sustained brain echogenicity on brain sonography. Other markers showed similar expression patterns yet we could not demonstrate statistically significant correlations. Conclusion These preliminary findings suggest that circulating RNA levels especially Tie2 and SCL decline with maturation and might relate to some preterm complication. Further prospective follow up of larger cohorts are required to establish this association.

  5. Endothelial RIG-I activation impairs endothelial function

    International Nuclear Information System (INIS)

    Asdonk, Tobias; Motz, Inga; Werner, Nikos; Coch, Christoph; Barchet, Winfried; Hartmann, Gunther; Nickenig, Georg; Zimmer, Sebastian

    2012-01-01

    Highlights: ► RIG-I activation impairs endothelial function in vivo. ► RIG-I activation alters HCAEC biology in vitro. ► EPC function is affected by RIG-I stimulation in vitro. -- Abstract: Background: Endothelial dysfunction is a crucial part of the chronic inflammatory atherosclerotic process and is mediated by innate and acquired immune mechanisms. Recent studies suggest that pattern recognition receptors (PRR) specialized in immunorecognition of nucleic acids may play an important role in endothelial biology in a proatherogenic manner. Here, we analyzed the impact of endothelial retinoic acid inducible gene I (RIG-I) activation upon vascular endothelial biology. Methods and results: Wild type mice were injected intravenously with 32.5 μg of the RIG-ligand 3pRNA (RNA with triphosphate at the 5′end) or polyA control every other day for 7 days. In 3pRNA-treated mice, endothelium-depended vasodilation was significantly impaired, vascular oxidative stress significantly increased and circulating endothelial microparticle (EMP) numbers significantly elevated compared to controls. To gain further insight in RIG-I dependent endothelial biology, cultured human coronary endothelial cells (HCAEC) and endothelial progenitor cells (EPC) were stimulated in vitro with 3pRNA. Both cells types express RIG-I and react with receptor upregulation upon stimulation. Reactive oxygen species (ROS) formation is enhanced in both cell types, whereas apoptosis and proliferation is not significantly affected in HCAEC. Importantly, HCAEC release significant amounts of proinflammatory cytokines in response to RIG-I stimulation. Conclusion: This study shows that activation of the cytoplasmatic nucleic acid receptor RIG-I leads to endothelial dysfunction. RIG-I induced endothelial damage could therefore be an important pathway in atherogenesis.

  6. Increased toll-like receptor 4 in cerebral endothelial cells contributes to the astrocyte swelling and brain edema in acute hepatic encephalopathy.

    Science.gov (United States)

    Jayakumar, Arumugam R; Tong, Xiao Y; Curtis, Kevin M; Ruiz-Cordero, Roberto; Abreu, Maria T; Norenberg, Michael D

    2014-03-01

    Astrocyte swelling and the subsequent increase in intracranial pressure and brain herniation are major clinical consequences in patients with acute hepatic encephalopathy. We recently reported that conditioned media from brain endothelial cells (ECs) exposed to ammonia, a mixture of cytokines (CKs) or lipopolysaccharide (LPS), when added to astrocytes caused cell swelling. In this study, we investigated the possibility that ammonia and inflammatory agents activate the toll-like receptor 4 (TLR4) in ECs, resulting in the release of factors that ultimately cause astrocyte swelling. We found a significant increase in TLR4 protein expression when ECs were exposed to ammonia, CKs or LPS alone, while exposure of ECs to a combination of these agents potentiate such effects. In addition, astrocytes exposed to conditioned media from TLR4-silenced ECs that were treated with ammonia, CKs or LPS, resulted in a significant reduction in astrocyte swelling. TLR4 protein up-regulation was also detected in rat brain ECs after treatment with the liver toxin thioacetamide, and that thioacetamide-treated TLR4 knock-out mice exhibited a reduction in brain edema. These studies strongly suggest that ECs significantly contribute to the astrocyte swelling/brain edema in acute hepatic encephalopathy, likely as a consequence of increased TLR4 protein expression by blood-borne noxious agents. © 2013 International Society for Neurochemistry.

  7. Sphingosine 1 Phosphate at the Blood Brain Barrier: Can the Modulation of S1P Receptor 1 Influence the Response of Endothelial Cells and Astrocytes to Inflammatory Stimuli?

    Directory of Open Access Journals (Sweden)

    Simona F Spampinato

    Full Text Available The ability of the Blood Brain Barrier (BBB to maintain proper barrier functions, keeping an optimal environment for central nervous system (CNS activity and regulating leukocytes' access, can be affected in CNS diseases. Endothelial cells and astrocytes are the principal BBB cellular constituents and their interaction is essential to maintain its function. Both endothelial cells and astrocytes express the receptors for the bioactive sphingolipid S1P. Fingolimod, an immune modulatory drug whose structure is similar to S1P, has been approved for treatment in multiple sclerosis (MS: fingolimod reduces the rate of MS relapses by preventing leukocyte egress from the lymph nodes. Here, we examined the ability of S1P and fingolimod to act on the BBB, using an in vitro co-culture model that allowed us to investigate the effects of S1P on endothelial cells, astrocytes, and interactions between the two. Acting selectively on endothelial cells, S1P receptor signaling reduced cell death induced by inflammatory cytokines. When acting on astrocytes, fingolimod treatment induced the release of a factor, granulocyte macrophage colony-stimulating factor (GM-CSF that reduced the effects of cytokines on endothelium. In an in vitro BBB model incorporating shear stress, S1P receptor modulation reduced leukocyte migration across the endothelial barrier, indicating a novel mechanism that might contribute to fingolimod efficacy in MS treatment.

  8. Regulatory mechanisms for iron transport across the blood-brain barrier.

    Science.gov (United States)

    Duck, Kari A; Simpson, Ian A; Connor, James R

    2017-12-09

    Many critical metabolic functions in the brain require adequate and timely delivery of iron. However, most studies when considering brain iron uptake have ignored the iron requirements of the endothelial cells that form the blood-brain barrier (BBB). Moreover, current models of BBB iron transport do not address regional regulation of brain iron uptake or how neurons, when adapting to metabolic demands, can acquire more iron. In this study, we demonstrate that both iron-poor transferrin (apo-Tf) and the iron chelator, deferoxamine, stimulate release of iron from iron-loaded endothelial cells in an in vitro BBB model. The role of the endosomal divalent metal transporter 1 (DMT1) in BBB iron acquisition and transport has been questioned. Here, we show that inhibition of DMT1 alters the transport of iron and Tf across the endothelial cells. These data support an endosome-mediated model of Tf-bound iron uptake into the brain and identifies mechanisms for local regional regulation of brain iron uptake. Moreover, our data provide an explanation for the disparity in the ratio of Tf to iron transport into the brain that has confounded the field. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Studies of pathological dynamics after microvascular injury using nonlinear optical methods

    Science.gov (United States)

    Rosidi, Nathanael L.

    Microvascular lesions are a common feature in the aging brain and clinical evidence has correlated microvascular pathology with the development of neurodegenerative diseases such as Alzheimer's disease and dementia. Traditional animal models that replicate hemorrhagic and ischemic lesions in the brain typically affect large regions in the cortex and do not reproduce the small-scale lesions linked to neurodegeneration that likely stem from injuries to single microvessels. Due in part to this lack of small-scale injury animal models, there remains an incomplete understanding of the cellular and pathophysiological dynamics following small-scale vascular lesions, making progress on therapeutic strategies difficult. We used tightly focused femtosecond laser pulses to injure single penetrating arterioles (PA) (i.e., arterioles that plunge into the brain) in the cortex of live anesthetized rodents and used two-photon excited fluorescence (2PEF) imaging to quantify blood flow changes and to determine the time course of pathological consequences in the brain after injury. We find that after ischemic occlusion of a PA, nearby pial and penetrating arterioles do not actively compensate for the reduction of blood flow observed near the occluded blood vessel. We find that capillaries connected downstream to the clotted vessel dilate but other capillaries in the vicinity do not, suggesting that any compensatory signal that results in a physiological response travels vascularly. We ruptured individual PAs to induce microhemorrhages that resulted in extravasation of blood into the parenchyma. We find that tissue compression due to the hematoma does not collapse capillaries and cause acute ischemia. 2PEF imaging of mice expressing yellow fluorescent protein (YFP) in a subset of cortical neurons revealed no dendrite degeneration out to seven days after microhemorrhage. However, we did observe an inflammatory response by microglia/macrophages as quickly as 1.5-hrs after

  10. CD36 and Fyn kinase mediate malaria-induced lung endothelial barrier dysfunction in mice infected with Plasmodium berghei.

    Directory of Open Access Journals (Sweden)

    Ifeanyi U Anidi

    Full Text Available Severe malaria can trigger acute lung injury characterized by pulmonary edema resulting from increased endothelial permeability. However, the mechanism through which lung fluid conductance is altered during malaria remains unclear. To define the role that the scavenger receptor CD36 may play in mediating this response, C57BL/6J (WT and CD36-/- mice were infected with P. berghei ANKA and monitored for changes in pulmonary endothelial barrier function employing an isolated perfused lung system. WT lungs demonstrated a >10-fold increase in two measures of paracellular fluid conductance and a decrease in the albumin reflection coefficient (σalb compared to control lungs indicating a loss of barrier function. In contrast, malaria-infected CD36-/- mice had near normal fluid conductance but a similar reduction in σalb. In WT mice, lung sequestered iRBCs demonstrated production of reactive oxygen species (ROS. To determine whether knockout of CD36 could protect against ROS-induced endothelial barrier dysfunction, mouse lung microvascular endothelial monolayers (MLMVEC from WT and CD36-/- mice were exposed to H2O2. Unlike WT monolayers, which showed dose-dependent decreases in transendothelial electrical resistance (TER from H2O2 indicating loss of barrier function, CD36-/- MLMVEC demonstrated dose-dependent increases in TER. The differences between responses in WT and CD36-/- endothelial cells correlated with important differences in the intracellular compartmentalization of the CD36-associated Fyn kinase. Malaria infection increased total lung Fyn levels in CD36-/- lungs compared to WT, but this increase was due to elevated production of the inactive form of Fyn further suggesting a dysregulation of Fyn-mediated signaling. The importance of Fyn in CD36-dependent endothelial signaling was confirmed using in vitro Fyn knockdown as well as Fyn-/- mice, which were also protected from H2O2- and malaria-induced lung endothelial leak, respectively. Our

  11. Perioperative antibiotics in the setting of microvascular free tissue transfer: current practices

    NARCIS (Netherlands)

    Reiffel, Alyssa J.; Kamdar, Mehul R.; Kadouch, Daniel J. M.; Rohde, Christine H.; Spector, Jason A.

    2010-01-01

    Microvascular free tissue transfer is a ubiquitous and routine method of restoring anatomic defects. There is a paucity of data regarding the role of perioperative antibiotics in free tissue transfer. We designed a survey to explore usage patterns among microvascular surgeons and thereby define a

  12. Effects of fisetin on hyperhomocysteinemia-induced experimental endothelial dysfunction and vascular dementia.

    Science.gov (United States)

    Hemanth Kumar, Boyina; Arun Reddy, Ravula; Mahesh Kumar, Jerald; Dinesh Kumar, B; Diwan, Prakash V

    2017-01-01

    This study was designed to investigate the effects of fisetin (FST) on hyperhomocysteinemia (HHcy)-induced experimental endothelial dysfunction (ED) and vascular dementia (VaD) in rats. Wistar rats were randomly divided into 8 groups: control, vehicle control, l-methionine, FST (5, 10, and 25 mg/kg, p.o.), FST-per se (25 mg/kg, p.o.), and donepezil (0.1 mg/kg, p.o.). l-Methionine administration (1.7 g/kg, p.o.) for 32 days induced HHcy. ED and VaD induced by HHcy were determined by vascular reactivity measurements, behavioral analysis using Morris water maze and Y-maze, along with a biochemical and histological evaluation of thoracic aorta and brain tissues. Administration of l-methionine developed behavioral deficits; triggered brain lipid peroxidation (LPO); compromised brain acetylcholinesterase activity (AChE); and reduced the levels of brain superoxide dismutase (SOD), brain catalase (CAT), brain reduced glutathione (GSH), and serum nitrite; and increased serum homocysteine and cholesterol levels. These effects were accompanied by decreased vascular NO bioavailability, marked intimal thickening of the aorta, and multiple necrotic foci in brain cortex. HHcy-induced alterations in the activities of SOD, CAT, GSH, AChE, LPO, behavioral deficits, ED, and histological aberrations were significantly attenuated by treatment with fisetin in a dose-dependent manner. Collectively, our results indicate that fisetin exerts endothelial and neuroprotective effects against HHcy-induced ED and VaD.

  13. Interactions of the gasotransmitters contribute to microvascular tone (dysregulation in the preterm neonate.

    Directory of Open Access Journals (Sweden)

    Rebecca M Dyson

    Full Text Available Hydrogen sulphide (H2S, nitric oxide (NO, and carbon monoxide (CO are involved in transitional microvascular tone dysregulation in the preterm infant; however there is conflicting evidence on the interaction of these gasotransmitters, and their overall contribution to the microcirculation in newborns is not known. The aim of this study was to measure the levels of all 3 gasotransmitters, characterise their interrelationships and elucidate their combined effects on microvascular blood flow.90 preterm neonates were studied at 24h postnatal age. Microvascular studies were performed by laser Doppler. Arterial COHb levels (a measure of CO were determined through co-oximetry. NO was measured as nitrate and nitrite in urine. H2S was measured as thiosulphate by liquid chromatography. Relationships between levels of the gasotransmitters and microvascular blood flow were assessed through partial correlation controlling for the influence of gestational age. Structural equation modelling was used to examine the combination of these effects on microvascular blood flow and derive a theoretical model of their interactions.No relationship was observed between NO and CO (p = 0.18, r = 0.18. A positive relationship between NO and H2S (p = 0.008, r = 0.28 and an inverse relationship between CO and H2S (p = 0.01, r = -0.33 exists. Structural equation modelling was used to examine the combination of these effects on microvascular blood flow. The model with the best fit is presented.The relationships between NO and H2S, and CO and H2S may be of importance in the preterm newborn, particularly as NO levels in males are associated with higher H2S levels and higher microvascular blood flow and CO in females appears to convey protection against vascular dysregulation. Here we present a theoretical model of these interactions and their overall effects on microvascular flow in the preterm newborn, upon which future mechanistic studies may be based.

  14. Engineering Microvascularized 3D Tissue Using Alginate-Chitosan Microcapsules.

    Science.gov (United States)

    Zhang, Wujie; Choi, Jung K; He, Xiaoming

    2017-02-01

    Construction of vascularized tissues is one of the major challenges of tissue engineering. The goal of this study was to engineer 3D microvascular tissues by incorporating the HUVEC-CS cells with a collagen/alginate-chitosan (AC) microcapsule scaffold. In the presence of AC microcapsules, a 3D vascular-like network was clearly observable. The results indicated the importance of AC microcapsules in engineering microvascular tissues -- providing support and guiding alignment of HUVEC-CS cells. This approach provides an alternative and promising method for constructing vascularized tissues.

  15. Endothelial RIG-I activation impairs endothelial function

    Energy Technology Data Exchange (ETDEWEB)

    Asdonk, Tobias, E-mail: tobias.asdonk@ukb.uni-bonn.de [Department of Medicine/Cardiology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn (Germany); Motz, Inga; Werner, Nikos [Department of Medicine/Cardiology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn (Germany); Coch, Christoph; Barchet, Winfried; Hartmann, Gunther [Institute for Clinical Chemistry and Clinical Pharmacology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn (Germany); Nickenig, Georg; Zimmer, Sebastian [Department of Medicine/Cardiology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn (Germany)

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer RIG-I activation impairs endothelial function in vivo. Black-Right-Pointing-Pointer RIG-I activation alters HCAEC biology in vitro. Black-Right-Pointing-Pointer EPC function is affected by RIG-I stimulation in vitro. -- Abstract: Background: Endothelial dysfunction is a crucial part of the chronic inflammatory atherosclerotic process and is mediated by innate and acquired immune mechanisms. Recent studies suggest that pattern recognition receptors (PRR) specialized in immunorecognition of nucleic acids may play an important role in endothelial biology in a proatherogenic manner. Here, we analyzed the impact of endothelial retinoic acid inducible gene I (RIG-I) activation upon vascular endothelial biology. Methods and results: Wild type mice were injected intravenously with 32.5 {mu}g of the RIG-ligand 3pRNA (RNA with triphosphate at the 5 Prime end) or polyA control every other day for 7 days. In 3pRNA-treated mice, endothelium-depended vasodilation was significantly impaired, vascular oxidative stress significantly increased and circulating endothelial microparticle (EMP) numbers significantly elevated compared to controls. To gain further insight in RIG-I dependent endothelial biology, cultured human coronary endothelial cells (HCAEC) and endothelial progenitor cells (EPC) were stimulated in vitro with 3pRNA. Both cells types express RIG-I and react with receptor upregulation upon stimulation. Reactive oxygen species (ROS) formation is enhanced in both cell types, whereas apoptosis and proliferation is not significantly affected in HCAEC. Importantly, HCAEC release significant amounts of proinflammatory cytokines in response to RIG-I stimulation. Conclusion: This study shows that activation of the cytoplasmatic nucleic acid receptor RIG-I leads to endothelial dysfunction. RIG-I induced endothelial damage could therefore be an important pathway in atherogenesis.

  16. HLA class I antibodies trigger increased adherence of monocytes to endothelial cells by eliciting an increase in endothelial P-selectin and, depending on subclass, by engaging FcγRs1

    Science.gov (United States)

    Valenzuela, Nicole M; Mulder, Arend; Reed, Elaine F

    2013-01-01

    Antibody-mediated rejection of solid organ transplants is characterized by intragraft macrophages. It is incompletely understood how donor specific antibody binding to graft endothelium promotes monocyte adhesion, and what, if any, contribution is made by the Fc region of the antibody. We investigated the mechanisms underlying monocyte recruitment by HLA class I antibody-activated endothelium. We used a panel of murine monoclonal antibodies of different subclasses to crosslink HLA I on human aortic, venous and microvascular endothelial cells, and measured the binding of human monocytic cell lines and peripheral blood monocytes. Both anti-HLA I murine IgG1 and mIgG2a induced endothelial P-selectin, which was required for monocyte adhesion to endothelium irrespective of subclass. Mouse IgG2a but not mIgG1 could bind human FcγRs. Accordingly, HLA I mIgG2a but not mIgG1 treatment of endothelial cells significantly augmented recruitment, predominantly through FcγRI, and, to a lesser extent, FcγRIIa. Moreover, HLA I mIgG2a promoted firm adhesion of monocytes to ICAM-1 through Mac-1, which may explain the prominence of monocytes during antibody mediated rejection. We confirmed these observations using human HLA allele specific monoclonal antibodies and IgG purified from transplant patient sera. HLA I antibodies universally elicit endothelial exocytosis leading to monocyte adherence, implying that P-selectin is a putative therapeutic target to prevent macrophage infiltration during antibody-mediated rejection. Importantly, the subclass of donor specific antibody may influence its pathogenesis. These results imply that hIgG1 and hIgG3 should have a greater capacity to trigger monocyte infiltration into the graft than IgG2 or IgG4 due to enhancement by FcγR interactions. PMID:23690477

  17. Restraint stress-induced morphological changes at the blood-brain barrier in adult rats

    Directory of Open Access Journals (Sweden)

    Petra eSántha

    2016-01-01

    Full Text Available Stress is well known to contribute to the development of both neurological and psychiatric diseases. While the role of the blood-brain barrier is increasingly recognised in the development of neurodegenerative disorders, such as Alzheimer’s disease, dysfunction of the blood-brain barrier has been linked to stress-related psychiatric diseases only recently. In the present study the effects of restraint stress with different duration (1, 3 and 21 days were investigated on the morphology of the blood-brain barrier in male adult Wistar rats. Frontal cortex and hippocampus sections were immunostained for markers of brain endothelial cells (claudin-5, occludin and glucose transporter-1 and astroglia (GFAP. Staining pattern and intensity were visualized by confocal microscopy and evaluated by several types of image analysis. The ultrastructure of brain capillaries was investigated by electron microscopy. Morphological changes and intensity alterations in brain endothelial tight junction proteins claudin-5 and occludin were induced by stress. Following restraint stress significant increases in the fluorescence intensity of glucose transporter-1 were detected in brain endothelial cells in the frontal cortex and hippocampus. Significant reductions in GFAP fluorescence intensity were observed in the frontal cortex in all stress groups. As observed by electron microscopy, one-day acute stress induced morphological changes indicating damage in capillary endothelial cells in both brain regions. After 21 days of stress thicker and irregular capillary basal membranes in the hippocampus and edema in astrocytes in both regions were seen. These findings indicate that stress exerts time-dependent changes in the staining pattern of tight junction proteins occludin, claudin-5 and glucose transporter-1 at the level of brain capillaries and in the ultrastructure of brain endothelial cells and astroglial endfeet, which may contribute to neurodegenerative processes

  18. Bipolar disorder and related mood states are not associated with endothelial function of small arteries in adults without heart disease.

    Science.gov (United States)

    Tong, Brian; Abosi, Oluchi; Schmitz, Samantha; Myers, Janie; Pierce, Gary L; Fiedorowicz, Jess G

    Individuals with bipolar disorder are at increased risk for adverse cardiovascular disease (CVD) events. This study aimed to assess endothelial function and wave reflection, a risk factor for CVD, as measured by finger plethysmography in bipolar disorder to investigate whether CVD risk was higher in bipolar disorder and altered during acute mood episodes. We hypothesized that EndoPAT would detect a lower reactive hyperemia index (RHI) and higher augmentation index (AIX) in individuals with bipolar disorder compared with controls. Second, we predicted lower RHI and higher AIX during acute mood episodes. Reactive hyperemia index and augmentation index, measures of microvascular endothelial function and arterial pressure wave reflection respectively, were assessed using the EndoPAT 2000 device in a sample of 56 participants with a DSM-IV diagnosis of bipolar I disorder with 82 measures spanning different mood states (mania, depression, euthymia) and cross-sectionally in 26 healthy controls. RHI and AIX were not different between adults with and without bipolar disorder (mean age 40.3 vs. 41.2years; RHI: 2.04±0.67 vs. 2.05±0.51; AIX@75 (AIX adjusted for heart rate of 75): 1.4±19.7 vs. 0.8±22.4). When modeled in linear mixed models with a random intercept (to account for repeated observations of persons with bipolar disorder) and adjusting for age and sex, there were no significant differences between those with bipolar disorder and controls (p=0.89 for RHI; p=0.85 for AIX@75). Microvascular endothelial function and wave reflection estimated by finger plethysmography were unable to detect differences between adults with and without bipolar disorder or changes with mood states. Future research is necessary to identify more proximal and sensitive, yet relevant, biomarkers of abnormal mood-related influences on CVD risk or must target higher risk samples. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. The Deletion of Endothelial Sodium Channel α (αENaC Impairs Endothelium-Dependent Vasodilation and Endothelial Barrier Integrity in Endotoxemia in Vivo

    Directory of Open Access Journals (Sweden)

    Magdalena Sternak

    2018-04-01

    Full Text Available The role of epithelial sodium channel (ENaC activity in the regulation of endothelial function is not clear. Here, we analyze the role of ENaC in the regulation of endothelium-dependent vasodilation and endothelial permeability in vivo in mice with conditional αENaC subunit gene inactivation in the endothelium (endo-αENaCKO mice using unique MRI-based analysis of acetylcholine-, flow-mediated dilation and vascular permeability. Mice were challenged or not with lipopolysaccharide (LPS, from Salmonella typhosa, 10 mg/kg, i.p.. In addition, changes in vascular permeability in ex vivo organs were analyzed by Evans Blue assay, while changes in vascular permeability in perfused mesenteric artery were determined by a FITC-dextran-based assay. In basal conditions, Ach-induced response was completely lost, flow-induced vasodilation was inhibited approximately by half but endothelial permeability was not changed in endo-αENaCKO vs. control mice. In LPS-treated mice, both Ach- and flow-induced vasodilation was more severely impaired in endo-αENaCKO vs. control mice. There was also a dramatic increase in permeability in lungs, brain and isolated vessels as evidenced by in vivo and ex vivo analysis in endotoxemic endo-αENaCKO vs. control mice. The impaired endothelial function in endotoxemia in endo-αENaCKO was associated with a decrease of lectin and CD31 endothelial staining in the lung as compared with control mice. In conclusion, the activity of endothelial ENaC in vivo contributes to endothelial-dependent vasodilation in the physiological conditions and the preservation of endothelial barrier integrity in endotoxemia.

  20. Intercellular adhesion molecule-1 blockade attenuates inflammatory response and improves microvascular perfusion in rat pancreas grafts.

    Science.gov (United States)

    Preissler, Gerhard; Eichhorn, Martin; Waldner, Helmut; Winter, Hauke; Kleespies, Axel; Massberg, Steffen

    2012-10-01

    After pancreas transplantation (PTx), early capillary malperfusion and leukocyte recruitment indicate the manifestation of severe ischemia/reperfusion injury (IRI). Oscillatory blood-flow redistribution (intermittent capillary perfusion, IP), leading to an overall decrease in erythrocyte flux, precedes complete microvascular perfusion failure with persistent blood flow cessation. We addressed the role of intercellular adhesion molecule-1 (ICAM-1) for leukocyte-endothelial interactions (LEIs) after PTx and evaluated the contribution of IP and malperfusion. Pancreas transplantation was performed in rats after 18-hour preservation, receiving either isotype-matched IgG or monoclonal anti-ICAM-1 antibodies (10 mg/kg intravenously) once before reperfusion. Leukocyte-endothelial interaction, IP, erythrocyte flux, and functional capillary density, respectively, were examined in vivo during 2-hour reperfusion. Nontransplanted animals served as controls. Tissue samples were analyzed by histomorphometry. In grafts of IgG-treated animals, IP was encountered already at an early stage after reperfusion and steadily increased over 2 hours, whereas erythrocyte flux declined continuously. In contrast, inhibition of ICAM-1 significantly improved erythrocyte flux and delayed IP appearance by 2 hours. Further, anti-ICAM-1 significantly reduced LEI and leukocyte tissue infiltration when compared to IgG; edema development was less pronounced in response to anti-ICAM-1 monoclonal antibody. Intercellular adhesion molecule-1 blockade significantly attenuates IRI via immediate reduction of LEI and concomitant improvement of capillary perfusion patterns, emphasizing its central role during IRI in PTx.