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Sample records for brain endothelial cells

  1. Nanoparticle accumulation and transcytosis in brain endothelial cell layers

    NARCIS (Netherlands)

    Ye, Dong; Raghnaill, Michelle Nic; Bramini, Mattia; Mahon, Eugene; Åberg, Christoffer; Salvati, Anna; Dawson, Kenneth A

    2013-01-01

    The blood-brain barrier (BBB) is a selective barrier, which controls and limits access to the central nervous system (CNS). The selectivity of the BBB relies on specialized characteristics of the endothelial cells that line the microvasculature, including the expression of intercellular tight juncti

  2. Brain microvascular endothelial cell transplantation ameliorates ischemic white matter damage.

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    Puentes, Sandra; Kurachi, Masashi; Shibasaki, Koji; Naruse, Masae; Yoshimoto, Yuhei; Mikuni, Masahiko; Imai, Hideaki; Ishizaki, Yasuki

    2012-08-21

    Ischemic insults affecting the internal capsule result in sensory-motor disabilities which adversely affect the patient's life. Cerebral endothelial cells have been reported to exert a protective effect against brain damage, so the transplantation of healthy endothelial cells might have a beneficial effect on the outcome of ischemic brain damage. In this study, endothelin-1 (ET-1) was injected into the rat internal capsule to induce lacunar infarction. Seven days after ET-1 injection, microvascular endothelial cells (MVECs) were transplanted into the internal capsule. Meningeal cells or 0.2% bovine serum albumin-Hank's balanced salt solution were injected as controls. Two weeks later, the footprint test and histochemical analysis were performed. We found that MVEC transplantation improved the behavioral outcome based on recovery of hind-limb rotation angle (P<0.01) and induced remyelination (P<0.01) compared with the control groups. Also the inflammatory response was repressed by MVEC transplantation, judging from fewer ED-1-positive activated microglial cells in the MVEC-transplanted group than in the other groups. Elucidation of the mechanisms by which MVECs ameliorate ischemic damage of the white matter may provide important information for the development of effective therapies for white matter ischemia.

  3. Barrier Functionality of Porcine and Bovine Brain Capillary Endothelial Cells

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    Ailar Nakhlband

    2011-09-01

    Full Text Available Introduction: To date, isolated cell based blood-brain barrier (BBB models have been widely used for brain drug delivery and targeting, due to their relatively proper bioelectrical and permeability properties. However, primary cultures of brain capillary endothelial cells (BCECs isolated from different species vary in terms of bioelectrical and permeability properties. Methods: To pursue this, in the current investigation, primary porcine and bovine BCECs (PBCECs and BBCECs, respectively were isolated and used as an in vitro BBB model. The bioelectrical and permeability properties were assessed in BCECs co-cultured with C6 cells with/without hydrocortisone (550 nM. The bioelectrical properties were further validated by means of the permeability coefficients of transcellular and paracellular markers. Results: The primary PBCECs displayed significantly higher trans-endothelial electrical resistance (~900 W.cm2 than BBCECs (~700 W.cm2 - both co-cultured with C6 cells in presence of hydrocortisone. Permeability coefficients of propranolol/diazepam and mannitol/sucrose in PBCECs were ~21 and ~2 (×10-6 cm.sec-1, where these values for BBCECs were ~25 and ~5 (×10-6 cm.sec-1. Conclusion: Upon our bioelectrical and permeability findings, both models display discriminative barrier functionality but porcine BCECs seem to provide a better platform than bovine BCECs for drug screening and brain targeting.

  4. Lung endothelial cells strengthen, but brain endothelial cells weaken barrier properties of a human alveolar epithelium cell culture model.

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    Neuhaus, Winfried; Samwer, Fabian; Kunzmann, Steffen; Muellenbach, Ralf M; Wirth, Michael; Speer, Christian P; Roewer, Norbert; Förster, Carola Y

    2012-11-01

    The blood-air barrier in the lung consists of the alveolar epithelium, the underlying capillary endothelium, their basement membranes and the interstitial space between the cell layers. Little is known about the interactions between the alveolar and the blood compartment. The aim of the present study was to gain first insights into the possible interplay between these two neighbored cell layers. We established an in vitro Transwell model of the alveolar epithelium based on human cell line H441 and investigated the influence of conditioned medium obtained from human lung endothelial cell line HPMEC-ST1.6R on the barrier properties of the H441 layers. As control for tissue specificity H441 layers were exposed to conditioned medium from human brain endothelial cell line hCMEC/D3. Addition of dexamethasone was necessary to obtain stable H441 cell layers. Moreover, dexamethasone increased expression of cell type I markers (caveolin-1, RAGE) and cell type II marker SP-B, whereas decreased the transepithelial electrical resistance (TEER) in a concentration dependent manner. Soluble factors obtained from the lung endothelial cell line increased the barrier significantly proven by TEER values and fluorescein permeability on the functional level and by the differential expression of tight junctional proteins on the molecular level. In contrast to this, soluble factors derived from brain endothelial cells weakened the barrier significantly. In conclusion, soluble factors from lung endothelial cells can strengthen the alveolar epithelium barrier in vitro, which suggests communication between endothelial and epithelial cells regulating the integrity of the blood-air barrier.

  5. Rescue of Brain Function Using Tunneling Nanotubes Between Neural Stem Cells and Brain Microvascular Endothelial Cells.

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    Wang, Xiaoqing; Yu, Xiaowen; Xie, Chong; Tan, Zijian; Tian, Qi; Zhu, Desheng; Liu, Mingyuan; Guan, Yangtai

    2016-05-01

    Evidence indicates that neural stem cells (NSCs) can ameliorate cerebral ischemia in animal models. In this study, we investigated the mechanism underlying one of the neuroprotective effects of NSCs: tunneling nanotube (TNT) formation. We addressed whether the control of cell-to-cell communication processes between NSCs and brain microvascular endothelial cells (BMECs) and, particularly, the control of TNT formation could influence the rescue function of stem cells. In an attempt to mimic the cellular microenvironment in vitro, a co-culture system consisting of terminally differentiated BMECs from mice in a distressed state and NSCs was constructed. Additionally, engraftment experiments with infarcted mouse brains revealed that control of TNT formation influenced the effects of stem cell transplantation in vivo. In conclusion, our findings provide the first evidence that TNTs exist between NSCs and BMECs and that regulation of TNT formation alters cell function.

  6. Neutral amino acid transport across brain microvessel endothelial cell monolayers

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    Audus, K.L.; Borchardt, R.T.

    1986-03-01

    Brain microvessel endothelial cells (BMEC) which form the blood-brain barrier (BBB) possess an amino acid carrier specific for large neutral amino acids (LNAA). The carrier is important for facilitating the delivery of nutrient LNAA's and centrally acting drugs that are LNAA's, to the brain. Bovine BMEC's were isolated and grown up to complete monolayers on regenerated cellulose-membranes in primary culture. To study the transendothelial transport of leucine, the monolayers were placed in a side-by-side diffusion cell, and transport across the monolayers followed with (/sup 3/H)-leucine. The transendothelial transport of leucine in this in vitro model was determined to be bidirectional, and time-, temperature-, and concentration-dependent. The transport of leucine was saturable and the apparent K/sub m/ and V/sub max/, 0.18 mM and 6.3 nmol/mg/min, respectively. Other LNAA's, including the centrally acting drugs, ..cap alpha..-methyldopa, L-DOPA, ..cap alpha..-methyl-tyrosine, and baclofen, inhibited leucine transport. The leucine carrier was also found to be stereospecific and not sensitive to inhibitors of active transport. These results are consistent with previous in vitro and in vivo studies. Primary cultures of BMEC's appear to be a potentially important tool for investigating at the cellular level, the transport mechanisms of the BBB.

  7. Derivation of blood-brain barrier endothelial cells from human pluripotent stem cells.

    Science.gov (United States)

    Lippmann, Ethan S; Azarin, Samira M; Kay, Jennifer E; Nessler, Randy A; Wilson, Hannah K; Al-Ahmad, Abraham; Palecek, Sean P; Shusta, Eric V

    2012-08-01

    The blood-brain barrier (BBB) is crucial to the health of the brain and is often compromised in neurological disease. Moreover, because of its barrier properties, this endothelial interface restricts uptake of neurotherapeutics. Thus, a renewable source of human BBB endothelium could spur brain research and pharmaceutical development. Here we show that endothelial cells derived from human pluripotent stem cells (hPSCs) acquire BBB properties when co-differentiated with neural cells that provide relevant cues, including those involved in Wnt/β-catenin signaling. The resulting endothelial cells have many BBB attributes, including well-organized tight junctions, appropriate expression of nutrient transporters and polarized efflux transporter activity. Notably, they respond to astrocytes, acquiring substantial barrier properties as measured by transendothelial electrical resistance (1,450 ± 140 Ω cm2), and they possess molecular permeability that correlates well with in vivo rodent blood-brain transfer coefficients.

  8. Polylactic Acid Nanoparticles Targeted to Brain Microvascular Endothelial Cells

    Institute of Scientific and Technical Information of China (English)

    WANG Huafang; HU Yu; SUN Wangqiang; XIE Changsheng

    2005-01-01

    In this work, blank polylactic acid (PLA) nanoparticles with unstained surface were prepared by the nano-deposition method. On the basis of the preparation, the effect of surface modification on brain microvascular endothelial cells (BMECs) targeting was examined by in vivo experiments and fluorescence microscopy. The results showed that PLA nanoparticles are less toxic than PACA nanoparticles but their BMECs targeting is similar to PACA nanoparticles. The experiments suggest that drugs can be loaded onto the particles and become more stable through adsorption on the surface of PLA nanoparticles with high surface activity. The surface of PLA nanoparticles was obviously modified and the hydrophilicity was increased as well in the presence of non-ionic surfactants on PLA nanoparticles. As a targeting moiety, polysobate 80 (T-80) can facilitate BMECs targeting of PLA nanoparticles.

  9. Glioblastoma cell-secreted interleukin-8 induces brain endothelial cell permeability via CXCR2.

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    Julie Dwyer

    Full Text Available Glioblastoma constitutes the most aggressive and deadly of brain tumors. As yet, both conventional and molecular-based therapies have met with limited success in treatment of this cancer. Among other explanations, the heterogeneity of glioblastoma and the associated microenvironment contribute to its development, as well as resistance and recurrence in response to treatments. Increased vascularity suggests that tumor angiogenesis plays an important role in glioblastoma progression. However, the molecular crosstalk between endothelial and glioblastoma cells requires further investigation. To examine the effects of glioblastoma-derived signals on endothelial homeostasis, glioblastoma cell secretions were collected and used to treat brain endothelial cells. Here, we present evidence that the glioblastoma secretome provides pro-angiogenic signals sufficient to disrupt VE-cadherin-mediated cell-cell junctions and promote endothelial permeability in brain microvascular endothelial cells. An unbiased angiogenesis-specific antibody array screen identified the chemokine, interleukin-8, which was further demonstrated to function as a key factor involved in glioblastoma-induced permeability, mediated through its receptor CXCR2 on brain endothelia. This underappreciated interface between glioblastoma cells and associated endothelium may inspire the development of novel therapeutic strategies to induce tumor regression by preventing vascular permeability and inhibiting angiogenesis.

  10. Immortalized endothelial cell lines for in vitro blood-brain barrier models: A systematic review.

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    Rahman, Nurul Adhwa; Rasil, Alifah Nur'ain Haji Mat; Meyding-Lamade, Uta; Craemer, Eva Maria; Diah, Suwarni; Tuah, Ani Afiqah; Muharram, Siti Hanna

    2016-07-01

    Endothelial cells play the most important role in construction of the blood-brain barrier. Many studies have opted to use commercially available, easily transfected or immortalized endothelial cell lines as in vitro blood-brain barrier models. Numerous endothelial cell lines are available, but we do not currently have strong evidence for which cell lines are optimal for establishment of such models. This review aimed to investigate the application of immortalized endothelial cell lines as in vitro blood-brain barrier models. The databases used for this review were PubMed, OVID MEDLINE, ProQuest, ScienceDirect, and SpringerLink. A narrative systematic review was conducted and identified 155 studies. As a result, 36 immortalized endothelial cell lines of human, mouse, rat, porcine and bovine origins were found for the establishment of in vitro blood-brain barrier and brain endothelium models. This review provides a summary of immortalized endothelial cell lines as a guideline for future studies and improvements in the establishment of in vitro blood-brain barrier models. It is important to establish a good and reproducible model that has the potential for multiple applications, in particular a model of such a complex compartment such as the blood-brain barrier.

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

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    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.

  12. Changes in the permeability of blood brain barrier and endothelial cell damage after cerebral ischemia

    Institute of Scientific and Technical Information of China (English)

    Ke Liu; Jiansheng Li

    2006-01-01

    OBJECTIVE: To investigate the effect of endothelial cells on the permeability of blood brain barrier (BBB) after brain injury and its effect mechanism.DATA SOURCES: We searched for the articles of permeability of BBB and endothelial cell injury after brain ischemia, which were published between January 1982 and December 2005, with the key words of "cerebral ischemia damage,blood brain barrier ( BBB),permeability,effect of endothelial cell (EC) and its variation mechanism"in English.STUDY SELECTION: The materials were primarily selected. The articles related to the changes in the permeability of BBB and the effect of endothelial cells as well as the change mechanism after cerebral ischemia damage were chosen. Repetitive studies or review articles were excluded.DATA EXTRACTION: Totally 55 related articles were collected, and 35 were excluded due to repetitive or review articles, finally 20 articles were involved.DATA SYNTHESIS: The content or viewpoints of involved literatures were analyzed. Cerebral ischemia had damage for endothelial cells, such as the inflow of a lot of Ca2+, the production of nitrogen monoxide and oxygen free radical, and aggravated destruction of BBB. After acceptors of inflammatory mediators on cerebrovascular endothelial cell membrane, such as histamine, bradykinin , 5-hydroxytryptamine and so on are activated, endothelial cells shrink and the permeability of BBB increases. Its mechanism involves in the inflow of extracellular Ca2+and the release of intracellular Ca2+ in the cells. Glycocalyx molecule on the surface of endothelial cell, having structural polytropy, is the determinative factor of the permeability of BBB. VEGF, intensively increasing the vasopermeability and mainly effecting on postcapillary vein and veinlet, is the strongest known blood vessel permeation reagent. Its chronic overexpression in the brain can lead the destruction of BBB.CONCLUSION: The injury of endothelial cell participants in the pathological mechanism of BBB

  13. Brain endothelial cells increase the proliferation of Plasmodium falciparum through production of soluble factors.

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    Khaw, L T; Ball, H J; Mitchell, A J; Grau, G E; Stocker, R; Golenser, J; Hunt, N H

    2014-10-01

    We here describe the novel finding that brain endothelial cells in vitro can stimulate the growth of Plasmodium falciparum through the production of low molecular weight growth factors. By using a conditioned medium approach, we show that the brain endothelial cells continued to release these factors over time. If this mirrors the in vivo situation, these growth factors potentially would provide an advantage, in terms of enhanced growth, for sequestered parasitised red blood cells in the brain microvasculature. We observed this phenomenon with brain endothelial cells from several sources as well as a second P. falciparum strain. The characteristics of the growth factors included: heat stable, and in part chloroform soluble. Future efforts should be directed at identifying these growth factors, since blocking their production or actions might be of benefit for reducing parasite load and, hence, malaria pathology.

  14. Endothelial cells of the blood-brain barrier: a target for glucocorticoids and estrogens?

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    Dietrich, Jean-Bernard

    2004-01-01

    Adhesion molecules are involved in the leukocyte recruitment of leukocytes at the blood-brain barrier. For this reason, it is important to understand how the regulation of their gene expression controls lymphocyte adhesion to endothelial cells in microvessels. Indeed, due to their specificity and diversity, adhesion molecules involved in extravasation play an essential role in the recruitment of activated leukocytes and activation of inflammation. Multiple sclerosis results from a chronic inflammation of the CNS which is mediated by infiltration of inflammatory cells from the immune system. Administration of glucocorticoids is a routine method to control multiple sclerosis since naturally derived or synthetic glucocorticoids are potent immunosuppressive and anti-inflammatory agents. Glucocorticoids also have beneficial effects in stabilizing the blood-brain barrier, as steroid hormones regulate the expression of adhesion molecule genes in endothelial cells. Other hormones such as estrogens modulate many endothelial cell biological activities, among them adhesion to leukocytes. They regulate expression of adhesion molecules genes on endothelial cells and are useful for the treatment of experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis. The effects of glucocorticoids and estrogens on the expression of adhesion molecules on endothelial cells, including microvascular endothelial cells of the blood-brain barrier, are reviewed in this paper, as well as the involvement of these hormones in the therapy of experimental autoimmune encephalomyelitis and multiple sclerosis.

  15. Gene delivery of therapeutic polypeptides into brain capillary endothelial cells for protein secretion

    DEFF Research Database (Denmark)

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

    has been to investigate the usage of BCEC as factories for recombinant protein production. A non-viral gene carrier was prepared from pullulan-spermine conjugated with plasmid DNA (Thomsen et al., 2011). In vitro transfection of Rat Brain Endothelial Cells (RBE4) and Human Brain Microvascular...... Endothelial cells (HBMECs) were conducted with three plasmids bearing cDNA encoding human BDNF, EPO or the FGL peptide. Results revealed a high expression of BDNF, EPO and FGL transcripts in transfected cells compared to the non-transfected cells, which strongly suggest that transfection were successful...

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

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    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

  17. Lipopolysaccharide-induced apoptosis in transformed bovine brain endothelial cells and human dermal microvessel endothelial cells: the role of JNK.

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    Karahashi, Hisae; Michelsen, Kathrin S; Arditi, Moshe

    2009-06-01

    Stimulation of transformed bovine brain endothelial cells (TBBEC) with LPS leads to apoptosis while human microvessel endothelial cells (HMEC) need the presence of cycloheximide (CHX) with LPS to induce apoptosis. To investigate the molecular mechanism of LPS-induced apoptosis in HMEC or TBBEC, we analyzed the involvement of MAPK and PI3K in TBBEC and HMEC. LPS-induced apoptosis in TBBEC was hallmarked by the activation of caspase 3, caspase 6, and caspase 8 after the stimulation of LPS, followed by poly(ADP-ribose) polymerase cleavage and lactate dehydrogenase release. We also observed DNA cleavage determined by TUNEL staining in TBBEC treated with LPS. Herbimycin A, a tyrosine kinase inhibitor, and SP600125, a JNK inhibitor, suppressed the activation of caspases and lactate dehydrogenase release. Moreover, a PI3K inhibitor (LY294002) suppressed activation of caspases and combined treatment with both SP600125 and LY294002 completely inhibited the activation of caspases. These results suggest that the JNK signaling pathway through the tyrosine kinase and PI3K pathways is involved in the induction of apoptosis in LPS-treated TBBEC. On the other hand, we observed sustained JNK activation in HMEC treated with LPS and CHX, and neither ERK1/2 nor AKT were activated. The addition of SP600125 suppressed phosphorylation of JNK and the activation of caspase 3 in HMEC treated with LPS and CHX. These results suggest that JNK plays an important role in the induction of apoptosis in endothelial cells.

  18. Influence of curvature on the morphology of brain microvascular endothelial cells

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    Ye, Mao; Yang, Zhen; Wong, Andrew; Searson, Peter; Searson Group Team

    2013-03-01

    There are hundreds or thousands of endothelial cells around the perimeter of a single artery or vein, and hence an individual cell experiences little curvature. In contrast, a single endothelial cell may wrap around itself to form the lumen of a brain capillary. Curvature plays a key role in many biological, chemical and physical processes, however, its role in dictating the morphology and polarization of brain capillary endothelial cells has not been investigated. We hypothesize that curvature and shear flow play a key role in determining the structure and function of the blood-brain barrier (BBB). We have developed the ``rod'' assay to study the influence of curvature on the morphology of confluent monolayers of endothelial cells. In this assay cells are plated onto glass rods pulled down to the desired diameter in the range from 5 - 500 μm and coated with collagen. We show that curvature has a significant influence on the morphology of endothelial cells and may have an important role in blood-brain barrier function.

  19. Human Brain Microvascular Endothelial Cells Derived from the BC1 iPS Cell Line Exhibit a Blood-Brain Barrier Phenotype

    OpenAIRE

    Katt, Moriah E.; Xu, Zinnia S.; Gerecht, Sharon; Searson, Peter C.

    2016-01-01

    The endothelial cells that form capillaries in the brain are highly specialized, with tight junctions that minimize paracellular transport and an array of broad-spectrum efflux pumps that make drug delivery to the brain extremely challenging. One of the major limitations in blood-brain barrier research and the development of drugs to treat central nervous system diseases is the lack of appropriate cell lines. Recent reports indicate that the derivation of human brain microvascular endothelial...

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

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    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.

  1. 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

    In vitro models of the blood-brain barrier are useful tools to study blood-brain barrier function as well as drug permeation from the systemic circulation to the brain parenchyma. However, a large number of the available in vitro models fail to reflect the tightness of the in vivo blood-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....

  2. Transport and regulation mechanism of the colloidal gold liposomes in the brain microvascular endothelial cells

    Institute of Scientific and Technical Information of China (English)

    WANG Lipeng; CHANG Yanzhong

    2015-01-01

    Objective:Blood-brain barrier is the key barrier of brain in the innate immune. It can prevent the harmful substances from the blood into the brain. In order to keep the brain in a relatively stable environment and maintain the normal function of the nervous system, it can also pump harmful substances or excess substances outside the brain selectively. Among them, brain microvascular endothelial cell tissue is a key part in the blood-brain barrier's function. The number of the patients with central nervous system ( CNS) diseases increased year by year. The therapeutic drug is usually inhibited by the blood-brain barrier and is difficult to work. Therefore, how to modify the drug and to make it easier to cross the blood brain barrier is the key point to cure CNS. At present, more than 95% research focus only on how nano drugs can enter the cell, the way and efficiency to enter the cell and the research of effect of nano drug etc. For the process of drug carrier in endocytosis, intracellular transport and release and regulation of research are rarely reported. Clathrin and P-glycoprotein are related protein in endo-cytosis and exocytosis with nano drug. Clathrin is located on the plasma membrane. It participates in endocytosis of some nutrients, and maybe the entry into the cell of some drugs. P-glycoprotein is located in the membrane of cer-ebral capillary endothelial cells. It can efflux drugs relying on ATP. Although there is a certain understanding of the cell in the inner swallow and efflux. But the process of the liposome drug is not clear. To solve the above prob-lems, using colloidal gold liposome nano materials to trace liposome's transport and regulation mechanism in brain microvascular endothelial cells, and study endocytosis, release, distribution and regulation mechanism of nano lipo-somes in brain microvascular. The solution of this problem can guide to construct reasonable drug carrier, and look forward to clarifing the molecular basis and mechanism of

  3. Pentosan polysulfate protects brain endothelial cells against bacterial lipopolysaccharide-induced damages.

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    Veszelka, Szilvia; Pásztói, Mária; Farkas, Attila E; Krizbai, István; Ngo, Thi Khue Dung; Niwa, Masami; Abrahám, Csongor S; Deli, Mária A

    2007-01-01

    Peripheral inflammation can aggravate local brain inflammation and neuronal death. The blood-brain barrier (BBB) is a key player in the event. On a relevant in vitro model of primary rat brain endothelial cells co-cultured with primary rat astroglia cells lipopolysaccharide (LPS)-induced changes in several BBB functions have been investigated. LPS-treatment resulted in a dose- and time-dependent decrease in the integrity of endothelial monolayers: transendothelial electrical resistance dropped, while flux of permeability markers fluorescein and albumin significantly increased. Immunostaining for junctional proteins ZO-1, claudin-5 and beta-catenin was significantly weaker in LPS-treated endothelial cells than in control monolayers. LPS also reduced the intensity and changed the pattern of ZO-1 immunostaining in freshly isolated rat brain microvessels. The activity of P-glycoprotein, an important efflux pump at the BBB, was also inhibited by LPS. At the same time production of reactive oxygen species and nitric oxide was increased in brain endothelial cells treated with LPS. Pentosan polysulfate, a polyanionic polysaccharide could reduce the deleterious effects of LPS on BBB permeability, and P-glycoprotein activity. LPS-stimulated increase in the production of reactive oxygen species and nitric oxide was also decreased by pentosan treatment. The protective effect of pentosan for brain endothelium can be of therapeutical significance in bacterial infections affecting the BBB.

  4. The Wnt/planar cell polarity signaling pathway contributes to the integrity of tight junctions in brain endothelial cells

    OpenAIRE

    2013-01-01

    Wnt morphogens released by neural precursor cells were recently reported to control blood–brain barrier (BBB) formation during development. Indeed, in mouse brain endothelial cells, activation of the Wnt/β-catenin signaling pathway, also known as the canonical Wnt pathway, was shown to stabilize endothelial tight junctions (TJs) through transcriptional regulation of the expression of TJ proteins. Because Wnt proteins activate several distinct β-catenin-dependent and independent signaling path...

  5. Pericyte abundance affects sucrose permeability in cultures of rat brain microvascular endothelial cells.

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    Parkinson, Fiona E; Hacking, Cindy

    2005-07-05

    The blood-brain barrier is a physical and metabolic barrier that restricts diffusion of blood-borne substances into brain. In vitro models of the blood-brain barrier are used to characterize this structure, examine mechanisms of damage and repair and measure permeability of test substances. The core component of in vitro models of the blood-brain barrier is brain microvascular endothelial cells. We cultured rat brain microvascular endothelial cells (RBMEC) from isolated rat cortex microvessels. After 2-14 days in vitro (DIV), immunohistochemistry of these cells showed strong labeling for zona occludens 1 (ZO-1), a tight junction protein expressed in endothelial cells. Pericytes were also present in these cultures, as determined by expression of alpha-actin. The present study was performed to test different cell isolation methods and to compare the resulting cell cultures for abundance of pericytes and for blood-brain barrier function, as assessed by 14C-sucrose flux. Two purification strategies were used. First, microvessels were preabsorbed onto uncoated plastic for 4 h, then unattached microvessels were transferred to coated culture ware. Second, microvessels were incubated with an antibody to platelet-endothelial cell adhesion molecule 1 (PECAM-1; CD31) precoupled to magnetic beads, and a magnetic separation procedure was performed. Our results indicate that immunopurification, but not preadsorption, was an effective method to purify microvessels and reduce pericyte abundance in the resulting cultures. This purification significantly reduced 14C-sucrose fluxes across cell monolayers. These data indicate that pericytes can interfere with the development of blood-brain barrier properties in in vitro models that utilize primary cultures of RBMECs.

  6. Magnetic particle spectroscopy allows precise quantification of nanoparticles after passage through human brain microvascular endothelial cells

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    Gräfe, C.; Slabu, I.; Wiekhorst, F.; Bergemann, C.; von Eggeling, F.; Hochhaus, A.; Trahms, L.; Clement, J. H.

    2016-06-01

    Crossing the blood-brain barrier is an urgent requirement for the treatment of brain disorders. Superparamagnetic iron oxide nanoparticles (SPIONs) are a promising tool as carriers for therapeutics because of their physical properties, biocompatibility, and their biodegradability. In order to investigate the interaction of nanoparticles with endothelial cell layers in detail, in vitro systems are of great importance. Human brain microvascular endothelial cells are a well-suited blood-brain barrier model. Apart from generating optimal conditions for the barrier-forming cell units, the accurate detection and quantification of SPIONs is a major challenge. For that purpose we use magnetic particle spectroscopy to sensitively and directly quantify the SPION-specific iron content. We could show that SPION concentration depends on incubation time, nanoparticle concentration and location. This model system allows for further investigations on particle uptake and transport at cellular barriers with regard to parameters including particles’ shape, material, size, and coating.

  7. 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

    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

  8. 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

  9. Human brain microvascular endothelial cells resist elongation due to shear stress.

    Science.gov (United States)

    Reinitz, Adam; DeStefano, Jackson; Ye, Mao; Wong, Andrew D; Searson, Peter C

    2015-05-01

    Endothelial cells in straight sections of vessels are known to elongate and align in the direction of flow. This phenotype has been replicated in confluent monolayers of bovine aortic endothelial cells and human umbilical vein endothelial cells (HUVECs) in cell culture under physiological shear stress. Here we report on the morphological response of human brain microvascular endothelial cells (HBMECs) in confluent monolayers in response to shear stress. Using a microfluidic platform we image confluent monolayers of HBMECs and HUVECs under shear stresses up to 16 dyne cm(-2). From live-cell imaging we quantitatively analyze the cell morphology and cell speed as a function of time. We show that HBMECs do not undergo a classical transition from cobblestone to spindle-like morphology in response to shear stress. We further show that under shear stress, actin fibers are randomly oriented in the cells indicating that there is no cytoskeletal remodeling. These results suggest that HBMECs are programmed to resist elongation and alignment under shear stress, a phenotype that may be associated with the unique properties of the blood-brain barrier.

  10. Transcriptional Profile of HIV-induced Nuclear Translocation of Amyloid β in Brain Endothelial Cells

    Science.gov (United States)

    András, Ibolya E.; Rampersaud, Evadnie; Eum, Sung Yong; Toborek, Michal

    2015-01-01

    Background and Aims Increased amyloid deposition in HIV-infected brains may contribute to the pathogenesis of neurocognitive dysfunction in infected patients. We have previously shown that exposure to HIV results in enhanced amyloid β (Aβ) levels in human brain microvascular endothelial cells, suggesting that brain endothelial cells contribute to accumulation of Aβ in HIV-infected brains. Importantly, Aβ not only accumulates in the cytoplasm of HIV-exposed cells but also enters the nuclei of brain endothelial cells. Methods cDNA microarray analysis was performed in order to examine changes in the transcriptional profile associated with Aβ nuclear entry in the presence of HIV-1. Results Gene network analysis indicated that inhibition of nuclear entry of Aβ resulted in enrichment in gene sets involved in apoptosis and survival, endoplasmic reticulum stress response, immune response, cell cycle, DNA damage, oxidative stress, cytoskeleton remodeling and transforming growth factor b (TGFβ) receptor signaling. Conclusions The obtained data indicate that HIV-induced Aβ nuclear uptake affects several cellular stress-related pathways relevant for HIV-induced Aβ pathology. PMID:25446617

  11. Gene delivery into primary brain capillary endothelial cells for protein secretion

    DEFF Research Database (Denmark)

    Larsen, Annette Burkhart; Thomsen, Louiza Bohn; Lichota, Jacek;

    model was established by co-culturing primary BCECs together with primary astrocytes, both of which were isolated from rats. This was done in order to study the possibility of using gene transfection in an environment closer to the in-vivo BBB situation. The in-vitro BBB barrier model showed trans...... for recombinant protein production. We have previously shown that it is possible to transfect human microvascular endothelial cells (HBMEC) and rat brain endothelial (RBE4) cells with genetic material encoding the human growth hormone (GH1) (Thomsen et al 2011). In the present study, however, an in-vitro BBB...

  12. Brain microvascular endothelial cell association and distribution of a 5 nm ceria engineered nanomaterial

    Directory of Open Access Journals (Sweden)

    Dan M

    2012-07-01

    with the capillary fraction. Electron microscopy showed the ceria ENM located on the endothelial cell luminal surface.Conclusion: Ceria ENM association with brain capillary endothelial cells saturated between 20 and 60 seconds and ceria ENM brain uptake was not diffusion-mediated. During the 120-second ceria ENM perfusion, ceria ENM predominately associated with the surface of the brain capillary cells, providing the opportunity for its cell uptake or redistribution back into circulating blood.Keywords: ceria engineered nanomaterial, brain microvascular endothelial cell association, in situ brain perfusion, capillary depletion

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

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    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

  14. Galectin-1 suppresses methamphetamine induced neuroinflammation in human brain microvascular endothelial cells: Neuroprotective role in maintaining blood brain barrier integrity.

    Science.gov (United States)

    Parikh, Neil U; Aalinkeel, R; Reynolds, J L; Nair, B B; Sykes, D E; Mammen, M J; Schwartz, S A; Mahajan, S D

    2015-10-22

    Methamphetamine (Meth) abuse can lead to the breakdown of the blood-brain barrier (BBB) integrity leading to compromised CNS function. The role of Galectins in the angiogenesis process in tumor-associated endothelial cells (EC) is well established; however no data are available on the expression of Galectins in normal human brain microvascular endothelial cells and their potential role in maintaining BBB integrity. We evaluated the basal gene/protein expression levels of Galectin-1, -3 and -9 in normal primary human brain microvascular endothelial cells (BMVEC) that constitute the BBB and examined whether Meth altered Galectin expression in these cells, and if Galectin-1 treatment impacted the integrity of an in-vitro BBB. Our results showed that BMVEC expressed significantly higher levels of Galectin-1 as compared to Galectin-3 and -9. Meth treatment increased Galectin-1 expression in BMVEC. Meth induced decrease in TJ proteins ZO-1, Claudin-3 and adhesion molecule ICAM-1 was reversed by Galectin-1. Our data suggests that Galectin-1 is involved in BBB remodeling and can increase levels of TJ proteins ZO-1 and Claudin-3 and adhesion molecule ICAM-1 which helps maintain BBB tightness thus playing a neuroprotective role. Galectin-1 is thus an important regulator of immune balance from neurodegeneration to neuroprotection, which makes it an important therapeutic agent/target in the treatment of drug addiction and other neurological conditions.

  15. Modulation of cellular adhesion in bovine brain microvessel endothelial cells by a decapeptide.

    Science.gov (United States)

    Pal, D; Audus, K L; Siahaan, T J

    1997-01-30

    The importance of cell adhesion molecules in maintaining the cellular integrity of the endothelial layer is well recognized, yet their exact participation in regulating the blood-brain barrier (BBB) is poorly understood. Both Ca(2+)-dependent and Ca(2+)-independent cell adhesion molecules are found in endothelial cells. In this study, we used immunofluorescence, ELISA, Western blot and cell adhesion assay to identify a Ca(2+)-dependent cell adhesion molecule, E-cadherin, in bovine brain microvessel endothelial cells (BBMECs). Monoclonal anti-E-cadherin antibody specifically interacted with cultured BBMECs and decorated the cellular junctions with a series of punctate fluorescence spots as seen by indirect immunofluorescence using a confocal microscope. The intensity of these fluorescence spots increased after brief treatment with hIFN-gamma or CPT-cAMP. In the cellular extract of BBMECs, a 120 kDa protein was immunoprecipitated with anti-E-cadherin antibody. BBMECs did not react with anti-N-cadherin antibody, but recognized the FITC-labeled LRAHAVDVNG-NH2, a decapeptide generated from the EC-1 domain of N-cadherin, which decorated the lateral margins of the cells with fluorescence spots. A concentration-dependent binding of this decapeptide was also observed in the flow cytometry assay. BBMECs dissociated with trypsin plus Ca2+ were able to reaggregate only in the presence of Ca2+. However, such cell-cell aggregations of BBMECs were prevented by the presence of either anti-E-cadherin antibody or the decapeptide in the assay medium. These results confirm that BBMECs possess a distinct Ca(2+)-dependent cell adhesion mechanism that can be modulated by the decapeptide. This modulation of cell-cell adhesion in BBMECs by the decapeptide is thought-provoking for creating channels for paracellular drug delivery across the BBB.

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

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    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.

  17. Circulating endothelial progenitor cells in traumatic brain injury: an emerging therapeutic target?

    Institute of Scientific and Technical Information of China (English)

    WEI Hui-jie; JIANG Rong-cai; LIU Li; ZHANG Jian-ning

    2010-01-01

    Traumatic brain injury (TBI) is a major cause ofmortality and morbidity in the world. Recent clinical investigations and basic researches suggest that strategies to improve angiogenesis following TBI may provide promising opportunities to improve clinical outcomes and brain functional recovery. More and more evidences show that circulating endothelial progenitor cells (EPCs), which have been identified in the peripheral blood, may play an important role in the pathologic and physiological angiogenesis in adults. Moreover, impressive data demonstrate that EPCs are mobilized from bone marrow to blood circulation in response to traumatic or inflammatory stimulations.In this review, we discussed the role of EPCs in the repair of brain injury and the possible therapeutic implication for functional recovery of TBl in the future.

  18. Endocytosis and intracellular processing of platelet microparticles by brain endothelial cells.

    Science.gov (United States)

    Faille, Dorothée; El-Assaad, Fatima; Mitchell, Andrew J; Alessi, Marie-Christine; Chimini, Giovanna; Fusai, Thierry; Grau, Georges E; Combes, Valéry

    2012-08-01

    Platelet-derived microparticles (PMP) bind and modify the phenotype of many cell types including endothelial cells. Recently, we showed that PMP were internalized by human brain endothelial cells (HBEC). Here we intend to better characterize the internalization mechanisms of PMP and their intracellular fate. Confocal microscopy analysis of PKH67-labelled PMP distribution in HBEC showed PMP in early endosome antigen 1 positive endosomes and in LysoTracker-labelled lysosomes, confirming a role for endocytosis in PMP internalization. No fusion of calcein-loaded PMP with HBEC membranes was observed. Quantification of PMP endocytosis using flow cytometry revealed that it was partially inhibited by trypsin digestion of PMP surface proteins and by extracellular Ca(2+) chelation by EDTA, suggesting a partial role for receptor-mediated endocytosis in PMP uptake. This endocytosis was independent of endothelial receptors such as intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 and was not increased by tumour necrosis factor stimulation of HBEC. Platelet-derived microparticle internalization was dramatically increased in the presence of decomplemented serum, suggesting a role for PMP opsonin-dependent phagocytosis. Platelet-derived microparticle uptake was greatly diminished by treatment of HBEC with cytochalasin D, an inhibitor of microfilament formation required for both phagocytosis and macropinocytosis, with methyl-β-cyclodextrin that depletes membrane cholesterol needed for macropinocytosis and with amiloride that inhibits the Na(+)/H(+) exchanger involved in macropinocytosis. In conclusion, PMP are taken up by active endocytosis in HBEC, involving mechanisms consistent with both phagocytosis and macropinocytosis. These findings identify new processes by which PMP could modify endothelial cell phenotype and functions.

  19. 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 ...

  20. The effect of beta-turn structure on the permeation of peptides across monolayers of bovine brain microvessel endothelial cells

    DEFF Research Database (Denmark)

    Sorensen, M; Steenberg, B; Knipp, G T;

    1997-01-01

    PURPOSE: To investigate the effects of the beta-turn structure of a peptide on its permeation via the paracellular and transcellular routes across cultured bovine brain microvessel endothelial cell (BBMEC) monolayers, an in vitro model of the blood-brain barrier (BBB). METHODS: The effective...

  1. 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

    of the proteins. Morphological examination of the protein expression was determined using immunofluorescence detecting FLAG. Additionally, the transfection efficiency were determined by Flow cytometry. Perspective: Our study opens for knowledge on how non-viral gene therapy to BCECs can lead to protein secretion......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....... 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...

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

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    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

  3. Human brain endothelial cells endeavor to immunoregulate CD8 T cells via PD-1 ligand expression in multiple sclerosis

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    Pittet Camille L

    2011-11-01

    Full Text Available Abstract Background Multiple sclerosis (MS, an inflammatory disease of the central nervous system (CNS, is characterized by blood-brain barrier (BBB disruption and massive infiltration of activated immune cells. Engagement of programmed cell death-1 (PD-1 expressed on activated T cells with its ligands (PD-L1 and PD-L2 suppresses T cell responses. We recently demonstrated in MS lesions elevated PD-L1 expression by glial cells and absence of PD-1 on many infiltrating CD8 T cells. We have now investigated whether human brain endothelial cells (HBECs, which maintain the BBB, can express PD-L1 or PD-L2 and thereby modulate T cells. Methods We used primary cultures of HBECs isolated from non-tumoral CNS tissue either under basal or inflamed conditions. We assessed the expression of PD-L1 and PD-L2 using qPCR and flow cytometry. Human CD8 T cells were isolated from peripheral blood of healthy donors and co-cultured with HBECs. Following co-culture with HBECs, proliferation and cytokine production by human CD8 T cells were measured by flow cytometry whereas transmigration was determined using a well established in vitro model of the BBB. The functional impact of PD-L1 and PD-L2 provided by HBECs was determined using blocking antibodies. We performed immunohistochemistry for the detection of PD-L1 or PD-L2 concurrently with caveolin-1 (a cell specific marker for endothelial cells on post-mortem human brain tissues obtained from MS patients and normal controls. Results Under basal culture conditions, PD-L2 is expressed on HBECs, whilst PD-L1 is not detected. Both ligands are up-regulated under inflammatory conditions. Blocking PD-L1 and PD-L2 leads to increased transmigration and enhanced responses by human CD8 T cells in co-culture assays. Similarly, PD-L1 and PD-L2 blockade significantly increases CD4 T cell transmigration. Brain endothelium in normal tissues and MS lesions does not express detectable PD-L1; in contrast, all blood vessels in normal

  4. Modulation of P-glycoprotein function by amlodipine derivatives in brain microvessel endothelial cells of rats

    Institute of Scientific and Technical Information of China (English)

    Bian-sheng JI; Ling HE; Guo-qing LIU

    2005-01-01

    Aim: To investigate whether the amlodipine derivatives, CJX1 and CJX2, have a modulative effect on P-glycoprotein (P-gp) function in rat brain microvessel endothelial cells (RBMEC). Methods: Isolated RBMEC were cultured in DMEM/ F1 2 (1:1) medium. The amount of intracellular rhodamine (Rh 123) was determined, using a fluorescence spectrophotometer, to evaluate the function of P-gp. Results:The accumulation of Rh123 in RBMEC was potentiated in a concentrationdependent manner after incubation with CJX1 and CJX2 at 1, 2.5, 5, and 10μmol/L (P<0.01), but no accumulation of Rh123 was observed in human umbilical vein endothelial cells after incubation with CJX1 and CJX2 10 μmol/L (P>0.05). Accumulation of intracellular Rh123 was increased and efflux of intracellular Rh123 was decreased in a time-dependent manner from 0-100 min after CJX1 and CXJ2 at 10 μmol/L treatment. The inhibitory effect of CJX1 and CJX2 on P-gp function was reversible and remained even at 120 min after removal of CJX1 and CJX2 at 2.5 μmol/L from the medium. Conclusion: CJX1 and CJX2 exhibited a potent effect in the inhibition of P-gp function in vitro.

  5. Mononuclear phagocyte intercellular crosstalk facilitates transmission of cell-targeted nanoformulated antiretroviral drugs to human brain endothelial cells

    Directory of Open Access Journals (Sweden)

    Kanmogne GD

    2012-05-01

    Full Text Available Georgette D Kanmogne1, Sangya Singh1, Upal Roy1, Xinming Liu1, JoEllyn McMillan1, Santhi Gorantla1, Shantanu Balkundi1, Nathan Smith1, Yazen Alnouti2, Nagsen Gautam2, You Zhou3, Larisa Poluektova1, Alexander Kabanov2, Tatiana Bronich2, Howard E Gendelman11Departments of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, 2Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE; 3Center for Biotechnology, University of Nebraska-Lincoln, Lincoln, NE, USAAbstract: Despite the successes of antiretroviral therapy (ART, HIV-associated neurocognitive disorders remain prevalent in infected people. This is due, in part, to incomplete ART penetration across the blood–brain barrier (BBB and lymph nodes and to the establishment of viral sanctuaries within the central nervous system. In efforts to improve ART delivery, our laboratories developed a macrophage-carriage system for nanoformulated crystalline ART (nanoART (atazanavir, ritonavir, indinavir, and efavirenz. We demonstrate that nanoART transfer from mononuclear phagocytes (MP to human brain microvascular endothelial cells (HBMEC can be realized through cell-to-cell contacts, which can facilitate drug passage across the BBB. Coculturing of donor MP containing nanoART with recipient HBMEC facilitates intercellular particle transfer. NanoART uptake was observed in up to 52% of HBMEC with limited cytotoxicity. Folate coating of nanoART increased MP to HBMEC particle transfer by up to 77%. To translate the cell assays into relevant animal models of disease, ritonavir and atazanavir nanoformulations were injected into HIV-1-infected NOD/scid-γcnull mice reconstituted with human peripheral blood lymphocytes. Atazanavir and ritonavir levels in brains of mice treated with folate-coated nanoART were three- to four-fold higher than in mice treated with noncoated particles. This was associated with decreased viral load in the spleen and

  6. Ephrin-A3 and ephrin-A4 contribute to microglia-induced angiogenesis in brain endothelial cells.

    Science.gov (United States)

    Li, Ying; Liu, Dong-Xin; Li, Mei-Yang; Qin, Xiao-Xue; Fang, Wen-Gang; Zhao, Wei-Dong; Chen, Yu-Hua

    2014-10-01

    The association of microglia with brain vasculature during development and the reduced brain vascular complexity in microglia-deficient mice suggest the role of microglia in cerebrovascular angiogenesis. However, the underlying molecular mechanism remains unclear. Here, using an in vitro angiogenesis model, we found the culture supernatant of BV2 microglial cells significantly enhanced capillary-like tube formation and migration of brain microvascular endothelial cells (BMECs). The expression of angiogenic factors, ephrin-A3 and ephrin-A4, were specifically upregulated in BMECs exposed to BV2-derived culture supernatant. Knockdown of ephrin-A3 and ephrin-A4 in BMECs by siRNA significantly attenuated the enhanced angiogenesis and migration of BMECs induced by BV2 supernatant. Our further results indicated that the ability of BV2 supernatant to promote endothelial angiogenesis was caused by the soluble tumor necrosis factor α (TNF-α) released from BV2 microglial cells. Moreover, the upregulations of ephrin-A3 and ephrin-A4 in BMECs in response to BV2 supernatant were effectively abolished by neutralization antibody against TNF-α and TNF receptor 1, respectively. The present study provides evidence that microglia upregulates endothelial ephrin-A3 and ephrin-A4 to facilitate in vitro angiogenesis of brain endothelial cells, which is mediated by microglia-released TNF-α.

  7. The surface-anchored NanA protein promotes pneumococcal brain endothelial cell invasion.

    Science.gov (United States)

    Uchiyama, Satoshi; Carlin, Aaron F; Khosravi, Arya; Weiman, Shannon; Banerjee, Anirban; Quach, Darin; Hightower, George; Mitchell, Tim J; Doran, Kelly S; Nizet, Victor

    2009-08-31

    In humans, Streptococcus pneumoniae (SPN) is the leading cause of bacterial meningitis, a disease with high attributable mortality and frequent permanent neurological sequelae. The molecular mechanisms underlying the central nervous system tropism of SPN are incompletely understood, but include a primary interaction of the pathogen with the blood-brain barrier (BBB) endothelium. All SPN strains possess a gene encoding the surface-anchored sialidase (neuraminidase) NanA, which cleaves sialic acid on host cells and proteins. Here, we use an isogenic SPN NanA-deficient mutant and heterologous expression of the protein to show that NanA is both necessary and sufficient to promote SPN adherence to and invasion of human brain microvascular endothelial cells (hBMECs). NanA-mediated hBMEC invasion depends only partially on sialidase activity, whereas the N-terminal lectinlike domain of the protein plays a critical role. NanA promotes SPN-BBB interaction in a murine infection model, identifying the protein as proximal mediator of CNS entry by the pathogen.

  8. Differential roles for endothelial ICAM-1, ICAM-2, and VCAM-1 in shear-resistant T cell arrest, polarization, and directed crawling on blood-brain barrier endothelium.

    Science.gov (United States)

    Steiner, Oliver; Coisne, Caroline; Cecchelli, Roméo; Boscacci, Rémy; Deutsch, Urban; Engelhardt, Britta; Lyck, Ruth

    2010-10-15

    Endothelial ICAM-1 and ICAM-2 were shown to be essential for T cell diapedesis across the blood-brain barrier (BBB) in vitro under static conditions. Crawling of T cells prior to diapedesis was only recently revealed to occur preferentially against the direction of blood flow on the endothelial surface of inflamed brain microvessels in vivo. Using live cell-imaging techniques, we prove that Th1 memory/effector T cells predominantly crawl against the direction of flow on the surface of BBB endothelium in vitro. Analysis of T cell interaction with wild-type, ICAM-1-deficient, ICAM-2-deficient, or ICAM-1 and ICAM-2 double-deficient primary mouse brain microvascular endothelial cells under physiological flow conditions allowed us to dissect the individual contributions of endothelial ICAM-1, ICAM-2, and VCAM-1 to shear-resistant T cell arrest, polarization, and crawling. Although T cell arrest was mediated by endothelial ICAM-1 and VCAM-1, T cell polarization and crawling were mediated by endothelial ICAM-1 and ICAM-2 but not by endothelial VCAM-1. Therefore, our data delineate a sequential involvement of endothelial ICAM-1 and VCAM-1 in mediating shear-resistant T cell arrest, followed by endothelial ICAM-1 and ICAM-2 in mediating T cell crawling to sites permissive for diapedesis across BBB endothelium.

  9. Effects of Neospora caninum infection on brain microvascular endothelial cells bioenergetics

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    Elsheikha Hany M

    2013-01-01

    Full Text Available Abstract Background The brain is the most commonly affected organ during Neospora caninum infection but the mechanisms utilized by this protozoan parasite for traversal of the blood–brain barrier (BBB are not yet understood. Herein, we investigate the cellular pathogenicity of N. caninum infection on bioenergetics of human brain microvascular endothelial cells (HBMECs, a fundamental component of the BBB. Methods We tracked the growth kinetics of N. caninum in HBMECs. Focusing on cell bioenergetics, oxygen consumption rate (OCR was determined using Clark electrode system and mitochondrial membrane potential (ΔΨm was evaluated using DePsipher staining by fluorescence microscopy in the presence and absence of infection. Results HBMECs provided a receptive environment for parasite proliferation. N. caninum tachyzoites were able to invade and replicate within HBMECs without significantly altering cell proliferation rate, as measured with the MTT assay, up to 24 hr post infection (pi. The oxygen consumption rate (OCR was significantly inhibited (p 6 cell min-1 and from −0.29±0.09 to −0.16±0.1 nmol 106 cell min-1 for uninfected HBMECs and free N. Caninum tachyzoites, respectively]. After normalization for DNA content the basal OCR did not differ between two host cell types: HBMECs and K562. The OCR of HBMECs was significantly elevated 24 hr pi in the absence of substrate, in 10 mM glucose and in the presence of a tetramethyl-p-phenylenediamine (TMPD/ascorbate redox shuttle. Although quantitatively similar results were observed for uninfected K562 cells, there was no effect on their OCR 24 hr pi with N. caninum under any of the above substrate conditions. 6mM azide abolished OCR in all situations. Mitochondrial staining with DePsipher indicated no change in their membrane potential (Δψm up to 24 hr pi. Conclusions N. caninum is able to grow in HBMECs without markedly disrupting their normal proliferation or mitochondrial integrity. However

  10. NADPH OXIDASE AND LIPID RAFT-ASSOCIATED REDOX SIGNALING ARE REQUIRED FOR PCB153-INDUCED UPREGULATION OF CELL ADHESION MOLECULES IN HUMAN BRAIN ENDOTHELIAL CELLS

    Science.gov (United States)

    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. PMID:19632255

  11. P. falciparum isolate-specific distinct patterns of induced apoptosis in pulmonary and brain endothelial cells.

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    Nadine N'Dilimabaka

    Full Text Available The factors implicated in the transition from uncomplicated to severe clinical malaria such as pulmonary oedema and cerebral malaria remain unclear. It is known that alterations in vascular integrity due to endothelial cell (EC activation and death occur during severe malaria. In this study, we assessed the ability of different P. falciparum clinical isolates to induce apoptosis in ECs derived from human lung and brain. We observed that induction of EC apoptosis was sensitive to the environmental pH and required direct contact between the parasite and the cell, though it was not correlated to the ability of the parasite to cytoadhere. Moreover, the extent of induced apoptosis in the two EC types varied with the isolate. Analysis of parasite genes transcript led us to propose that the activation of different pathways, such as Plasmodium apoptosis-linked pathogenicity factors (PALPF, PALPF-2, PALPF-5 and PF11_0521, could be implied in EC death. These observations provide an experimental framework to decipher the molecular mechanism implicated in the genesis of severe malaria.

  12. 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.

  13. Human Brain Microvascular Endothelial Cells Derived from the BC1 iPS Cell Line Exhibit a Blood-Brain Barrier Phenotype.

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    Katt, Moriah E; Xu, Zinnia S; Gerecht, Sharon; Searson, Peter C

    2016-01-01

    The endothelial cells that form capillaries in the brain are highly specialized, with tight junctions that minimize paracellular transport and an array of broad-spectrum efflux pumps that make drug delivery to the brain extremely challenging. One of the major limitations in blood-brain barrier research and the development of drugs to treat central nervous system diseases is the lack of appropriate cell lines. Recent reports indicate that the derivation of human brain microvascular endothelial cells (hBMECs) from human induced pluripotent stem cells (iPSCs) may provide a solution to this problem. Here we demonstrate the derivation of hBMECs extended to two new human iPSC lines: BC1 and GFP-labeled BC1. These hBMECs highly express adherens and tight junction proteins VE-cadherin, ZO-1, occludin, and claudin-5. The addition of retinoic acid upregulates VE-cadherin expression, and results in a significant increase in transendothelial electrical resistance to physiological values. The permeabilities of tacrine, rhodamine 123, and Lucifer yellow are similar to values obtained for MDCK cells. The efflux ratio for rhodamine 123 across hBMECs is in the range 2-4 indicating polarization of efflux transporters. Using the rod assay to assess cell organization in small vessels and capillaries, we show that hBMECs resist elongation with decreasing diameter but show progressive axial alignment. The derivation of hBMECs with a blood-brain barrier phenotype from the BC1 cell line highlights that the protocol is robust. The expression of GFP in hBMECs derived from the BC1-GFP cell line provides an important new resource for BBB research.

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

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    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.

  15. Brain Endothelial Cells Produce Amyloid β from Amyloid Precursor Protein 770 and Preferentially Secrete the O-Glycosylated Form*

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    Kitazume, Shinobu; Tachida, Yuriko; Kato, Masaki; Yamaguchi, Yoshiki; Honda, Takashi; Hashimoto, Yasuhiro; Wada, Yoshinao; Saito, Takashi; Iwata, Nobuhisa; Saido, Takaomi; Taniguchi, Naoyuki

    2010-01-01

    Deposition of amyloid β (Aβ) in the brain is closely associated with Alzheimer disease (AD). Aβ is generated from amyloid precursor protein (APP) by the actions of β- and γ-secretases. In addition to Aβ deposition in the brain parenchyma, deposition of Aβ in cerebral vessel walls, termed cerebral amyloid angiopathy, is observed in more than 80% of AD individuals. The mechanism for how Aβ accumulates in blood vessels remains largely unknown. In the present study, we show that brain endothelial cells expressed APP770, a differently spliced APP mRNA isoform from neuronal APP695, and produced Aβ40 and Aβ42. Furthermore, we found that the endothelial APP770 had sialylated core 1 type O-glycans. Interestingly, Ο-glycosylated APP770 was preferentially processed by both α- and β-cleavage and secreted into the media, suggesting that O-glycosylation and APP processing involved related pathways. By immunostaining human brain sections with an anti-APP770 antibody, we found that APP770 was expressed in vascular endothelial cells. Because we were able to detect O-glycosylated sAPP770β in human cerebrospinal fluid, this unique soluble APP770β has the potential to serve as a marker for cortical dementias such as AD and vascular dementia. PMID:20952385

  16. Effects of flow on LOX-1 and oxidized low-density lipoprotein interactions in brain endothelial cell cultures.

    Science.gov (United States)

    Mao, Xiaoou; Xie, Lin; Greenberg, David A

    2015-12-01

    Fluid shear stress and uptake of oxidized low-density lipoprotein (ox-LDL) into the vessel wall both contribute to atherosclerosis, but the relationship between shear stress and ox-LDL uptake is unclear. We examined the effects of flow, induced by orbital rotation of bEnd.3 brain endothelial cell cultures for 1 wk, on ox-LDL receptor (LOX-1) protein expression, ox-LDL uptake and ox-LDL toxicity. Orbitally rotated cultures showed no changes in LOX-1 protein expression, ox-LDL uptake or ox-LDL toxicity, compared to stationary cultures. Flow alone does not modify ox-LDL/LOX-1 signaling in bEnd.3 brain endothelial cells in vitro, suggesting that susceptibility of atheroprone vascular sites to lipid accumulation is not due solely to effects of altered flow on endothelium.

  17. Cocaine inhibits store-operated Ca2+ entry in brain microvascular endothelial cells: critical role for sigma-1 receptors.

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    Brailoiu, G Cristina; Deliu, Elena; Console-Bram, Linda M; Soboloff, Jonathan; Abood, Mary E; Unterwald, Ellen M; Brailoiu, Eugen

    2016-01-01

    Sigma-1 receptor (Sig-1R) is an intracellular chaperone protein with many ligands, located at the endoplasmic reticulum (ER). Binding of cocaine to Sig-1R has previously been found to modulate endothelial functions. In the present study, we show that cocaine dramatically inhibits store-operated Ca(2+) entry (SOCE), a Ca(2+) influx mechanism promoted by depletion of intracellular Ca(2+) stores, in rat brain microvascular endothelial cells (RBMVEC). Using either Sig-1R shRNA or pharmacological inhibition with the unrelated Sig-1R antagonists BD-1063 and NE-100, we show that cocaine-induced SOCE inhibition is dependent on Sig-1R. In addition to revealing new insight into fundamental mechanisms of cocaine-induced changes in endothelial function, these studies indicate an unprecedented role for Sig-1R as a SOCE inhibitor.

  18. Protease activated receptor signaling is required for African trypanosome traversal of human brain microvascular endothelial cells.

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    Dennis J Grab

    Full Text Available BACKGROUND: Using human brain microvascular endothelial cells (HBMECs as an in vitro model for how African trypanosomes cross the human blood-brain barrier (BBB we recently reported that the parasites cross the BBB by generating calcium activation signals in HBMECs through the activity of parasite cysteine proteases, particularly cathepsin L (brucipain. In the current study, we examined the possible role of a class of protease stimulated HBMEC G protein coupled receptors (GPCRs known as protease activated receptors (PARs that might be implicated in calcium signaling by African trypanosomes. METHODOLOGY/PRINCIPAL FINDINGS: Using RNA interference (RNAi we found that in vitro PAR-2 gene (F2RL1 expression in HBMEC monolayers could be reduced by over 95%. We also found that the ability of Trypanosoma brucei rhodesiense to cross F2RL1-silenced HBMEC monolayers was reduced (39%-49% and that HBMECs silenced for F2RL1 maintained control levels of barrier function in the presence of the parasite. Consistent with the role of PAR-2, we found that HBMEC barrier function was also maintained after blockade of Galpha(q with Pasteurella multocida toxin (PMT. PAR-2 signaling has been shown in other systems to have neuroinflammatory and neuroprotective roles and our data implicate a role for proteases (i.e. brucipain and PAR-2 in African trypanosome/HBMEC interactions. Using gene-profiling methods to interrogate candidate HBMEC pathways specifically triggered by brucipain, several pathways that potentially link some pathophysiologic processes associated with CNS HAT were identified. CONCLUSIONS/SIGNIFICANCE: Together, the data support a role, in part, for GPCRs as molecular targets for parasite proteases that lead to the activation of Galpha(q-mediated calcium signaling. The consequence of these events is predicted to be increased permeability of the BBB to parasite transmigration and the initiation of neuroinflammation, events precursory to CNS disease.

  19. Endomorphins exit the brain by a saturable efflux system at the basolateral surface of cerebral endothelial cells.

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    Somogyvari-Vigh, Aniko; Kastin, Abba J; Liao, Jie; Zadina, James E; Pan, Weihong

    2004-05-01

    Endomorphin-1 (EM-1) and endomorphin-2 (EM-2) are two highly selective mu-opiate receptor agonists. We recently demonstrated that EM-1 and EM-2 have a saturable transport system from brain-to-blood in vivo. Since the endothelial cells are the main component of the non-fenestrated microvessels of the blood-brain barrier (BBB), we examined whether these endogenous tetrapeptides have a saturable transport system in cultured cerebral endothelial cells. EM-1 and EM-2 binding and transport were studied in a transwell system in which primary mouse endothelial cells were co-cultured with rat glioma cells. We found that binding of both endomorphins was greater on the basolateral than the apical cell surface. Flux of EM-1 and EM-2 occurred predominantly in the basolateral to apical direction, each showing self-inhibition with an excess of the respective endomorphin. Transport was not influenced by the addition of the P-glycoprotein inhibitor, cyclosporin A. Neither the mu-opiate receptor agonist DAMGO nor the delta-opiate receptor agonist DPDPE had any effect on the transport. Thus, the results show that a saturable transport system for EM-1 and EM-2 occurs at the level of endothelial cells of the BBB, and unlike beta-endorphin and morphine, P-glycoprotein is not needed for the brain-to-blood transport. Cross-inhibition of the transport of each endomorphin by the other suggests a shared transport system that is different from mu- or delta-opiate receptors. As endormorphins are mainly produced in the CNS, the presence of the efflux system at the BBB could play an important role in pain modulation and neuroendocrine control.

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

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    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.

  1. Transient oxygen-glucose deprivation sensitizes brain capillary endothelial cells to rtPA at 4h of reoxygenation.

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    Kuntz, Mélanie; Mysiorek, Caroline; Pétrault, Olivier; Boucau, Marie-Christine; Aijjou, Rachid; Uzbekov, Rustem; Bérézowski, Vincent

    2014-01-01

    Thrombolysis treatment of acute ischemic stroke is limited by the pro-edematous and hemorrhagic effects exerted by reperfusion, which disrupts the blood-brain barrier (BBB) capillary endothelium in the infarct core. Most studies of the ischemic BBB overlook the complexity of the penumbral area, where the affected brain cells are still viable following deprivation. Our present objective was to examine in vitro the kinetic impact of reoxygenation on the integrity of ischemic BBB cells after oxygen-glucose deprivation. Through the use of a co-culture of brain capillary endothelial cells and glial cells, we first showed that the transendothelial permeability increase induced by deprivation can occur with both preserved cell viability and interendothelial tight junction network. The subtle and heterogeneous alteration of the tight junctions was observable only through electron microscopy. A complete permeability recovery was then found after reoxygenation, when Vimentin and Actin networks were reordered. However, still sparse ultrastructural alterations of tight junctions suggested an acquired vulnerability. Endothelial cells were then exposed to recombinant tissue-type plasminogen activator (rtPA) to define a temporal profile for the toxic effect of this thrombolytic on transendothelial permeability. Interestingly, the reoxygenated BBB broke down with aggravated tight junction disruption when exposed to rtPA only at 4h after reoxygenation. Moreover, this breakdown was enhanced by 50% when ischemic glial cells were present during the first hours of reoxygenation. Our results suggest that post-stroke reoxygenation enables retrieval of the barrier function of brain capillary endothelium when in a non-necrotic environment, but may sensitize it to rtPA at the 4-hour time point, when both endothelial breakdown mechanisms and glial secretions could be identified and targeted in a therapeutical perspective.

  2. Live-cell imaging to detect phosphatidylserine externalization in brain endothelial cells exposed to ionizing radiation: implications for the treatment of brain arteriovenous malformations.

    Science.gov (United States)

    Zhao, Zhenjun; Johnson, Michael S; Chen, Biyi; Grace, Michael; Ukath, Jaysree; Lee, Vivienne S; McRobb, Lucinda S; Sedger, Lisa M; Stoodley, Marcus A

    2016-06-01

    OBJECT Stereotactic radiosurgery (SRS) is an established intervention for brain arteriovenous malformations (AVMs). The processes of AVM vessel occlusion after SRS are poorly understood. To improve SRS efficacy, it is important to understand the cellular response of blood vessels to radiation. The molecular changes on the surface of AVM endothelial cells after irradiation may also be used for vascular targeting. This study investigates radiation-induced externalization of phosphatidylserine (PS) on endothelial cells using live-cell imaging. METHODS An immortalized cell line generated from mouse brain endothelium, bEnd.3 cells, was cultured and irradiated at different radiation doses using a linear accelerator. PS externalization in the cells was subsequently visualized using polarity-sensitive indicator of viability and apoptosis (pSIVA)-IANBD, a polarity-sensitive probe. Live-cell imaging was used to monitor PS externalization in real time. The effects of radiation on the cell cycle of bEnd.3 cells were also examined by flow cytometry. RESULTS Ionizing radiation effects are dose dependent. Reduction in the cell proliferation rate was observed after exposure to 5 Gy radiation, whereas higher radiation doses (15 Gy and 25 Gy) totally inhibited proliferation. In comparison with cells treated with sham radiation, the irradiated cells showed distinct pseudopodial elongation with little or no spreading of the cell body. The percentages of pSIVA-positive cells were significantly higher (p = 0.04) 24 hours after treatment in the cultures that received 25- and 15-Gy doses of radiation. This effect was sustained until the end of the experiment (3 days). Radiation at 5 Gy did not induce significant PS externalization compared with the sham-radiation controls at any time points (p > 0.15). Flow cytometric analysis data indicate that irradiation induced growth arrest of bEnd.3 cells, with cells accumulating in the G2 phase of the cell cycle. CONCLUSIONS Ionizing radiation

  3. Effect of curcumin on the adhesion of platelets to brain microvascular endothelial cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Li ZHANG; Zhen-lun GU; Zheng-hong QIN; Zhong-qin LIANG

    2008-01-01

    Aim: To determine whether curcumin prevents the adhesion of platelets to brain microvascular endothelial cells (BMECs) cultured in vitro. Methods: [3H]Ad-chine-labeled platelets were incubated with BMECs to investigate the role of curcumin in the adhesion of platelets to BMECs. The number of platelets adher-ing to the BMECs monolayer was determined by liquid scintillation spectroscopy. The thrombin-induced expression of platelets P-selectin, glycoprotein Ⅱb (GPⅡb), and glycoprotein Ⅲa (GPⅢa) on the cell surface, was measured by flow cytometry. P-selectin mRNA levels of BMECs were determined by RT-PCR. The TNF-α-induced expressions of P-selectin and E-selectin on the surface of BMECs were determined by Western blotting. Results: The adhesion between thrombin-acti-vated platelets and normal BMECs, and that of TNF-α-activated BMECs and normal platelets were significantly increased, and this increase could be inhibited by curcumin (30-240 μmol/L) in a concentration-dependant manner. The platelets activated with thrombin and BMECs stimulated by TNF-α demonstrated an upregulated expressions of P-selectin and E-selectin, and this increase, when pretreated with curcumin for 30 min, could be restrained dose dependently. Curcumin also inhibited the increase of the GPⅡb/GPⅢa expression of thrombin-activated platelets in a concentration-dependent manner. Conclusion: Curcumin can inhibit the platelets to BMECs. This effect may be related to the decreased expressions of P-selectin, E-selectin, and GPⅡb/GPⅢa on platelets and BMECs.

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

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    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.

  5. Activation of sonic hedgehog signaling attenuates oxidized low-density lipoprotein-stimulated brain microvascular endothelial cells dysfunction in vitro.

    Science.gov (United States)

    Jiang, Xiu-Long; Chen, Ting; Zhang, Xu

    2015-01-01

    The study was performed to investigate the role of sonic hedgehog (SHH) in the oxidized low-density lipoprotein (oxLDL)-induced blood-brain barrier (BBB) disruption. The primary mouse brain microvascular endothelial cells (MBMECs) were exposed to oxLDL. The results indicated that treatment of MBMECs with oxLDL decreased the cell viability, and oxidative stress was involved in oxLDL-induce MBMECs dysfunction with increasing intracellular ROS and MDA formation as well as decreasing NO release and eNOS mRNA expression. In addition, SHH signaling components, such as SHH, Smo and Gli1, mRNA and protein levels were significantly decreased after incubation with increasing concentrations of oxLDL. Treatment with oxLDL alone or SHH loss-of-function significantly increased the permeability of MBMECs, and overexpression of SHH attenuated oxLDL-induced elevation of permeability in MBMECs. Furthermore, SHH gain-of-function could reverse oxLDL-induced apoptosis through inhibition caspase3 and caspase8 levels in MBMECs. Taken together, these results demonstrated that the suppression of SHH in MBMECs might contribute to the oxLDL-induced disruption of endothelial barrier. However, the overexpression of SHH could reverse oxLDL-induced endothelial cells dysfunction in vitro.

  6. B7-H1 shapes T-cell–mediated brain endothelial cell dysfunction and regional encephalitogenicity in spontaneous CNS autoimmunity

    Science.gov (United States)

    Klotz, Luisa; Kuzmanov, Ivan; Hucke, Stephanie; Gross, Catharina C.; Posevitz, Vilmos; Dreykluft, Angela; Schulte-Mecklenbeck, Andreas; Janoschka, Claudia; Lindner, Maren; Herold, Martin; Schwab, Nicholas; Ludwig-Portugall, Isis; Kurts, Christian; Meuth, Sven G.; Kuhlmann, Tanja; Wiendl, Heinz

    2016-01-01

    Molecular mechanisms that determine lesion localization or phenotype variation in multiple sclerosis are mostly unidentified. Although transmigration of activated encephalitogenic T cells across the blood–brain barrier (BBB) is a crucial step in the disease pathogenesis of CNS autoimmunity, the consequences on brain endothelial barrier integrity upon interaction with such T cells and subsequent lesion formation and distribution are largely unknown. We made use of a transgenic spontaneous mouse model of CNS autoimmunity characterized by inflammatory demyelinating lesions confined to optic nerves and spinal cord (OSE mice). Genetic ablation of a single immune-regulatory molecule in this model [i.e., B7-homolog 1 (B7-H1, PD-L1)] not only significantly increased incidence of spontaneous CNS autoimmunity and aggravated disease course, especially in the later stages of disease, but also importantly resulted in encephalitogenic T-cell infiltration and lesion formation in normally unaffected brain regions, such as the cerebrum and cerebellum. Interestingly, B7-H1 ablation on myelin oligodendrocyte glycoprotein-specific CD4+ T cells, but not on antigen-presenting cells, amplified T-cell effector functions, such as IFN-γ and granzyme B production. Therefore, these T cells were rendered more capable of eliciting cell contact-dependent brain endothelial cell dysfunction and increased barrier permeability in an in vitro model of the BBB. Our findings suggest that a single immune-regulatory molecule on T cells can be ultimately responsible for localized BBB breakdown, and thus substantial changes in lesion topography in the context of CNS autoimmunity. PMID:27671636

  7. Effects of Lead and Cadmium on Brain Endothelial Cell Survival, Monolayer Permeability, and Crucial Oxidative Stress Markers in an in Vitro Model of the Blood-Brain Barrier

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    Shakila Tobwala

    2014-06-01

    Full Text Available Oxidative stress, which is the loss of balance between antioxidant defense and oxidant production in the cells, is implicated in the molecular mechanism of heavy metal-induced neurotoxicity. Given the key role of lead (Pb and cadmium (Cd in inducing oxidative stress, we investigated their role in disrupting the integrity and function of immortalized human brain microvascular endothelial cells (hCMEC/D3. To study this, hCMEC/D3 cells were exposed to control media or to media containing different concentrations of Pb or Cd. Those exposed to Pb or Cd showed significantly higher oxidative stress than the untreated group, as indicated by cell viability, reactive oxygen species (ROS, glutathione (GSH levels, and catalase enzyme activity. Pb also induced oxidative stress-related disruption of the hCMEC/D3 cell monolayer, as measured by trans-endothelial electrical resistance (TEER, the dextran permeability assay, and the level of tight junction protein, zona occluden protein (ZO-2. However, no significant disruption in the integrity of the endothelial monolayer was seen with cadmium at the concentrations used. Taken together, these results show that Pb and Cd induce cell death and dysfunction in hCMEC/D3 cells and, in the case of Pb, barrier disruption. This suggests blood brain barrier (BBB dysfunction as a contributing mechanism in Pb and Cd neurotoxicities.

  8. Endothelial cell laminin isoforms, laminins 8 and 10, play decisive roles in T cell recruitment across the blood-brain barrier in experimental autoimmune encephalomyelitis.

    Science.gov (United States)

    Sixt, M; Engelhardt, B; Pausch, F; Hallmann, R; Wendler, O; Sorokin, L M

    2001-05-28

    An active involvement of blood-brain barrier endothelial cell basement membranes in development of inflammatory lesions in the central nervous system (CNS) has not been considered to date. Here we investigated the molecular composition and possible function of the extracellular matrix encountered by extravasating T lymphocytes during experimental autoimmune encephalomyelitis (EAE). Endothelial basement membranes contained laminin 8 (alpha4beta1gamma1) and/or 10 (alpha5beta1gamma1) and their expression was influenced by proinflammatory cytokines or angiostatic agents. T cells emigrating into the CNS during EAE encountered two biochemically distinct basement membranes, the endothelial (containing laminins 8 and 10) and the parenchymal (containing laminins 1 and 2) basement membranes. However, inflammatory cuffs occurred exclusively around endothelial basement membranes containing laminin 8, whereas in the presence of laminin 10 no infiltration was detectable. In vitro assays using encephalitogenic T cell lines revealed adhesion to laminins 8 and 10, whereas binding to laminins 1 and 2 could not be induced. Downregulation of integrin alpha6 on cerebral endothelium at sites of T cell infiltration, plus a high turnover of laminin 8 at these sites, suggested two possible roles for laminin 8 in the endothelial basement membrane: one at the level of the endothelial cells resulting in reduced adhesion and, thereby, increased penetrability of the monolayer; and secondly at the level of the T cells providing direct signals to the transmigrating cells.

  9. [Endothelial cell adhesion molecules].

    Science.gov (United States)

    Ivanov, A N; Norkin, I A; Puchin'ian, D M; Shirokov, V Iu; Zhdanova, O Iu

    2014-01-01

    The review presents current data concerning the functional role of endothelial cell adhesion molecules belonging to different structural families: integrins, selectins, cadherins, and the immunoglobulin super-family. In this manuscript the regulatory mechanisms and factors of adhesion molecules expression and distribution on the surface of endothelial cells are discussed. The data presented reveal the importance of adhesion molecules in the regulation of structural and functional state of endothelial cells in normal conditions and in pathology. Particular attention is paid to the importance of these molecules in the processes of physiological and pathological angiogenesis, regulation of permeability of the endothelial barrier and cell transmigration.

  10. Generation of Brain Microvascular Endothelial-Like Cells from Human Induced Pluripotent Stem Cells by Co-Culture with C6 Glioma Cells.

    Science.gov (United States)

    Minami, Haruka; Tashiro, Katsuhisa; Okada, Atsumasa; Hirata, Nobue; Yamaguchi, Tomoko; Takayama, Kazuo; Mizuguchi, Hiroyuki; Kawabata, Kenji

    2015-01-01

    The blood brain barrier (BBB) is formed by brain microvascular endothelial cells (BMECs) and tightly regulates the transport of molecules from blood to neural tissues. In vitro BBB models from human pluripotent stem cell (PSCs)-derived BMECs would be useful not only for the research on the BBB development and function but also for drug-screening for neurological diseases. However, little is known about the differentiation of human PSCs to BMECs. In the present study, human induced PSCs (iPSCs) were differentiated into endothelial cells (ECs), and further maturated to BMECs. Interestingly, C6 rat glioma cell-conditioned medium (C6CM), in addition to C6 co-culture, induced the differentiation of human iPSC-derived ECs (iPS-ECs) to BMEC-like cells, increase in the trans-endothelial electrical resistance, decreased in the dextran transport and up-regulation of gene expression of tight junction molecules in human iPS-ECs. Moreover, Wnt inhibitors attenuated the effects of C6CM. In summary, we have established a simple protocol of the generation of BMEC-like cells from human iPSCs, and have demonstrated that differentiation of iPS-ECs to BMEC-like cells is induced by C6CM-derived signals, including canonical Wnt signals.

  11. Generation of Brain Microvascular Endothelial-Like Cells from Human Induced Pluripotent Stem Cells by Co-Culture with C6 Glioma Cells.

    Directory of Open Access Journals (Sweden)

    Haruka Minami

    Full Text Available The blood brain barrier (BBB is formed by brain microvascular endothelial cells (BMECs and tightly regulates the transport of molecules from blood to neural tissues. In vitro BBB models from human pluripotent stem cell (PSCs-derived BMECs would be useful not only for the research on the BBB development and function but also for drug-screening for neurological diseases. However, little is known about the differentiation of human PSCs to BMECs. In the present study, human induced PSCs (iPSCs were differentiated into endothelial cells (ECs, and further maturated to BMECs. Interestingly, C6 rat glioma cell-conditioned medium (C6CM, in addition to C6 co-culture, induced the differentiation of human iPSC-derived ECs (iPS-ECs to BMEC-like cells, increase in the trans-endothelial electrical resistance, decreased in the dextran transport and up-regulation of gene expression of tight junction molecules in human iPS-ECs. Moreover, Wnt inhibitors attenuated the effects of C6CM. In summary, we have established a simple protocol of the generation of BMEC-like cells from human iPSCs, and have demonstrated that differentiation of iPS-ECs to BMEC-like cells is induced by C6CM-derived signals, including canonical Wnt signals.

  12. T11TS inhibits Angiopoietin-1/Tie-2 signaling, EGFR activation and Raf/MEK/ERK pathway in brain endothelial cells restraining angiogenesis in glioma model.

    Science.gov (United States)

    Bhattacharya, Debanjan; Chaudhuri, Suhnrita; Singh, Manoj Kumar; Chaudhuri, Swapna

    2015-06-01

    Malignant gliomas represent one of the most aggressive and hypervascular primary brain tumors. Angiopoietin-1, the peptide growth factor activates endothelial Tie-2 receptor promoting vessel maturation and vascular stabilization steps of angiogenesis in glioma. Epidermal growth factor receptor (EGFR) and Tie-2 receptor on endothelial cells once activated transmits signals through downstream Raf/MEK/ERK pathway promoting endothelial cell proliferation and migration which are essential for angiogenesis induction. The in vivo effect of sheep erythrocyte membrane glycopeptide T11-target structure (T11TS) on angiopoietin-1/Tie-2 axis, EGFR signaling and Raf/MEK/ERK pathway in glioma associated endothelial cells has not been investigated previously. The present study performed with rodent glioma model aims to investigate the effect of T11TS treatment on angiopoietin-1/Tie-2 signaling, EGFR activity and Raf/MEK/ERK pathway in glioma associated endothelial cells within glioma milieu. T11TS administration in rodent glioma model inhibited angiopoietin-1 expression and attenuated Tie-2 expression and activation in glioma associated brain endothelial cells. T11TS treatment also downregulated total and phosphorylated EGFR expression in glioma associated endothelial cells. Additionally T11TS treatment inhibited Raf-1 expression, MEK-1 and ERK-1/2 expression and phosphorylation in glioma associated brain endothelial cells. Thus T11TS therapy remarkably inhibits endothelial angiopoietin-1/Tie-2 signaling associated with vessel maturation and simultaneously antagonizes endothelial cell proliferation signaling by blocking EGFR activation and components of Raf/MEK/ERK pathway. Collectively, the findings demonstrate a multi-targeted anti-angiogenic activity of T11TS which augments the potential for clinical translation of T11TS as an effective angiogenesis inhibitor for glioma treatment.

  13. Opiates Upregulate Adhesion Molecule Expression in Brain MicroVascular Endothelial Cells (BMVEC: Implications for Altered Blood Brain Barrier (BBB Permeability

    Directory of Open Access Journals (Sweden)

    Madhavan P.N. Nair

    2006-01-01

    Full Text Available The blood-brain barrier (BBB is an intricate cellular system composed of vascular endothelial cells and perivascular astrocytes that restrict the passage of immunocompetent cells into the central nervous system (CNS. Expression of the adhesion molecules, intercellular adhesion molecule 1 (ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1 on brain microvascular endothelial cells (BMVEC and their interaction with human immunodeficiency virus (HIV-1 viral proteins may help enhance viral adhesion and virus-cell fusion resulting in increased infectivity. Additionally, transmigration through the BBB is facilitated by both endothelial and monocyte/macrophage-derived nitric oxide (NO. Dysregulated production of NO by BMVEC due to opiates and HIV-1 viral protein interactions play a pivotal role in brain endothelial injury, resulting in the irreversible loss of BBB integrity, which may lead to enhanced infiltration of virus-carrying cells across the BBB. Opioids act as co-factors in the neuropathogenesis of HIV-1 by facilitating BBB dysfunction however, no studies have been done to investigate the role of opiates alone or in combination with HIV-1 viral proteins on adhesion molecule expression in BMVEC. We hypothesize that opiates such as heroin and morphine in conjunction with the HIV-1 viral protein gp120 increase the expression of adhesion molecules ICAM-1 and VCAM-1 and these effects are mediated via the modulation of NO. Results show that opiates alone and in synergy with gp120 increase both the genotypic and phenotypic expression of ICAM-1 and VCAM-1 by BMVEC, additionally, these opiate induced effects may be the result of increased NO production. These studies will provide a better understanding of how opiate abuse in conjunction with HIV-1 infection facilitates the breakdown of the BBB and exacerbates the neuropathogenesis of HIV-1. Elucidation of the mechanisms of BBB modulation will provide new therapeutic approaches to maintain BBB integrity

  14. Fucoidan Extracted from Hijiki Protects Brain Microvessel Endothelial Cells Against Diesel Exhaust Particle Exposure-Induced Disruption.

    Science.gov (United States)

    Choi, Young-Sook; Eom, Sang-Yong; Kim, In-Soo; Ali, Syed F; Kleinman, Michael T; Kim, Yong-Dae; Kim, Heon

    2016-05-01

    This study was performed to evaluate the protective effects of fucoidan against the decreased function of primary cultured bovine brain microvessel endothelial cells (BBMECs) after exposure to diesel exhaust particles (DEPs). BBMECs were extracted from bovine brains and cultured until confluent. To evaluate the function of BBMECs, we performed a permeability test using cell-by-cell equipment and by Western blot analysis for zonular occludens-1 (ZO-1), which is a tight junction protein of BMECs, and evaluated oxidative stress in BBMECs using the DCFH-DA assay and the CUPRAC-BCS assay. The increased oxidative stress in BBMECs following DEP exposure was suppressed by fucoidan. In addition, permeability of BBMECs induced by DEP exposure was decreased by fucoidan treatment. Our results showed that fucoidan protects against BBMEC disruption induced by DEP exposure. This study provides evidence that fucoidan might protect the central nervous system (CNS) against DEP exposure.

  15. Oxidative Stress Induced by Cigarette Smoke Extracts in Human Brain Cells (T98G) and Human Brain Microvascular Endothelial Cells (HBMEC) in Mono- and Co-Culture.

    Science.gov (United States)

    Kim, Ju-Hyeong; Cho, Myung-Haing; Choi, Kyung-Chul; Lee, Kyuhong; Kim, Kwang-Sik; Shim, Soon-Mi

    2015-01-01

    The objective of the current study was to examine oxidative stress induced by cigarette smoke extract (CSE) or cigarette smoke condensate (CSC) in human brain cells (T98G) and human brain microvascular endothelial cells (HBMEC) in mono- and co-culture systems. Cell viability of T98G cells exposed to CSC (0.05-4 mg/ml) was significantly decreased compared to CSE (0.025-20%). There were no marked differences between quantities of reactive oxygen species (ROS) generation by either CSE (2, 4, and 10%) or CSC (0.2, 0.4, and 0.8 mg/ml) treatment compared to control. However, a significant effect was noted in ROS generation following CSC incubation at 4mg/ml. Cellular integrity of HBMEC decreased to 74 and 64% within 120 h of exposure at the IC50 value of CSE and CSC, respectively. This study suggests that chronic exposure to cigarette smoking might initiate damage to the blood-brain barrier.

  16. Cancer-associated fibroblast promote transmigration through endothelial brain cells in three-dimensional in vitro models.

    Science.gov (United States)

    Choi, Yoon Pyo; Lee, Joo Hyun; Gao, Ming-Qing; Kim, Baek Gil; Kang, Suki; Kim, Se Hoon; Cho, Nam Hoon

    2014-11-01

    Brain metastases are associated with high morbidity as well as with poor prognosis and survival in breast cancer patients. Despite its clinical importance, metastasis of breast cancer cells through the blood-brain barrier (BBB) is poorly understood. The objective of our study was to investigate whether cancer-associated fibroblasts (CAFs) play crucial roles in breast cancer brain metastasis. Using a cell adhesion assays, in vitro BBB permeability and transmigration assays and soft agar colony formation assays, we investigated the physical roles of CAFs in breast cancer brain metastasis. We also performed immunofluorescence, flow cytometric analysis, Droplet Digital PCR and Simon™ Simple Western System to confirm changes in expression levels. We established two novel three-dimensional (3D) culture systems using a perpendicular slide chamber and applying 3D embedded culture method to reflect brain metastasis conditions. With a newly developed device, CAFs was proven to promote cell adhesion to human brain microvascular endothelial cells, in vitro BBB permeability and transmigration and colony formation of breast cancer cells. Furthermore, CAFs enhanced the invasive migration of breast cancer cells in two kinds of 3D cultures. These 3D models also reliably recapitulate the initial steps of BBB transmigration, micro-metastasis and colonization. Expression of integrin α5β1 and αvβ3, c-MET and α2,6-siayltransferase was increased in breast cancer cells that migrated through the BBB. In conclusion, based on our in vitro BBB and co-culture models, our data suggest that CAFs may play a role in breast cancer brain metastasis.

  17. Shear Stress Induces Differentiation of Endothelial Lineage Cells to Protect Neonatal Brain from Hypoxic-Ischemic Injury through NRP1 and VEGFR2 Signaling

    Directory of Open Access Journals (Sweden)

    Chia-Wei Huang

    2015-01-01

    Full Text Available Neonatal hypoxic-ischemic (HI brain injuries disrupt the integrity of neurovascular structure and lead to lifelong neurological deficit. The devastating damage can be ameliorated by preserving the endothelial network, but the source for therapeutic cells is limited. We aim to evaluate the beneficial effect of mechanical shear stress in the differentiation of endothelial lineage cells (ELCs from adipose-derived stem cells (ASCs and the possible intracellular signals to protect HI injury using cell-based therapy in the neonatal rats. The ASCs expressed early endothelial markers after biochemical stimulation of endothelial growth medium. The ELCs with full endothelial characteristics were accomplished after a subsequential shear stress application for 24 hours. When comparing the therapeutic potential of ASCs and ELCs, the ELCs treatment significantly reduced the infarction area and preserved neurovascular architecture in HI injured brain. The transplanted ELCs can migrate and engraft into the brain tissue, especially in vessels, where they promoted the angiogenesis. The activation of Akt by neuropilin 1 (NRP1 and vascular endothelial growth factor receptor 2 (VEGFR2 was important for ELC migration and following in vivo therapeutic outcomes. Therefore, the current study demonstrated importance of mechanical factor in stem cell differentiation and showed promising protection of brain from HI injury using ELCs treatment.

  18. Fatty acid transport protein expression in human brain and potential role in fatty acid transport across human brain microvessel endothelial cells.

    Science.gov (United States)

    Mitchell, Ryan W; On, Ngoc H; Del Bigio, Marc R; Miller, Donald W; Hatch, Grant M

    2011-05-01

    The blood-brain barrier (BBB), formed by the brain capillary endothelial cells, provides a protective barrier between the systemic blood and the extracellular environment of the CNS. Passage of fatty acids from the blood to the brain may occur either by diffusion or by proteins that facilitate their transport. Currently several protein families have been implicated in fatty acid transport. The focus of the present study was to identify the fatty acid transport proteins (FATPs) expressed in the brain microvessel endothelial cells and characterize their involvement in fatty acid transport across an in vitro BBB model. The major fatty acid transport proteins expressed in human brain microvessel endothelial cells (HBMEC), mouse capillaries and human grey matter were FATP-1, -4 and fatty acid binding protein 5 and fatty acid translocase/CD36. The passage of various radiolabeled fatty acids across confluent HBMEC monolayers was examined over a 30-min period in the presence of fatty acid free albumin in a 1 : 1 molar ratio. The apical to basolateral permeability of radiolabeled fatty acids was dependent upon both saturation and chain length of the fatty acid. Knockdown of various fatty acid transport proteins using siRNA significantly decreased radiolabeled fatty acid transport across the HBMEC monolayer. Our findings indicate that FATP-1 and FATP-4 are the predominant fatty acid transport proteins expressed in the BBB based on human and mouse expression studies. While transport studies in HBMEC monolayers support their involvement in fatty acid permeability, fatty acid translocase/CD36 also appears to play a prominent role in transport of fatty acids across HBMEC.

  19. 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.

  20. Regulation of store-operated Ca{sup 2+} entry activity by cell cycle dependent up-regulation of Orai2 in brain capillary endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Kito, Hiroaki [Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan); Department of Pharmacology, Division of Pathological Sciences, Kyoto Pharmaceutical University, Kyoto (Japan); Yamamura, Hisao; Suzuki, Yoshiaki; Yamamura, Hideto [Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan); Ohya, Susumu [Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan); Department of Pharmacology, Division of Pathological Sciences, Kyoto Pharmaceutical University, Kyoto (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)

    2015-04-10

    Store-operated Ca{sup 2+} entry (SOCE) via Orai1 and STIM1 complex is supposed to have obligatory roles in the regulation of cellular functions of vascular endothelial cells, while little is known about the contribution of Orai2. Quantitative PCR and Western blot analyses indicated the expression of Orai2 and STIM2, in addition to Orai1 and STIM1 in bovine brain capillary endothelial cell line, t-BBEC117. During the exponential growth of t-BBEC117, the knockdown of Orai1 and STIM1 significantly reduced the SOCE activity, whereas Orai2 and STIM2 siRNAs had no effect. To examine whether endogenous SOCE activity contributes to the regulation of cell cycle progression, t-BBEC117 were synchronized using double thymidine blockage. At the G2/M phase, Ca{sup 2+} influx via SOCE was decreased and Orai2 expression was increased compared to the G0/G1 phase. When Orai2 was knocked down at the G2/M phase, the decrease in SOCE was removed, and cell proliferation was partly attenuated. Taken together, Orai1 significantly contributes to cell proliferation via the functional expression, which is presumably independent of the cell cycle phases. In construct, Orai2 is specifically up-regulated during the G2/M phase, negatively modulates the SOCE activity, and may contribute to the regulation of cell cycle progression in brain capillary endothelial cells. - Highlights: • Orai1 is essential for SOCE activity in brain capillary endothelial cells (BCECs). • Cell cycle independent expression of Orai1 regulated SOCE and cell proliferation. • Orai2 was up-regulated only at G2/M phase and this consequently reduced SOCE. • Orai2 as well as Orai1 is a key player controlling SOCE and proliferation in BCECs.

  1. Hypoxia inducible factor-1alpha mediates protection of DL-3-n-butylphthalide in brain microvascular endothelial cells against oxygen glucose deprivation-induced injury

    Institute of Scientific and Technical Information of China (English)

    Weihong Yang; Ling Li; Ruxun Huang; Zhong Pei; Songjie Liao; Jinsheng Zeng

    2012-01-01

    Studies have demonstrated that DL-3-n-butylphthalide can significantly alleviate oxygen glucose deprivation-induced injury of human umbilical vein endothelial cells at least partly associated with its enhancement on oxygen glucose deprivation -induced hypoxia inducible factor-1α expression. In this study, we hypothesized that DL-3-n-butylphthalide can protect against oxygen glucose deprivation-induced injury of newborn rat brain microvascular endothelial cells by means of upregulating hypoxia inducible factor-1α expression. MTT assay and Hoechst staining results showed that DL-3-n-butylphthalide protected brain microvascular endothelial cells against oxygen glucose deprivation-induced injury in a dose-dependent manner. Western blot and immunofluorescent staining results further confirmed that the protective effect was related to upregulation of hypoxia inducible factor-1α. Real-time RT-PCR reaction results showed that DL-3-n-butylphthalide reduced apoptosis by inhibiting downregulation of pro-apoptotic gene caspase-3 mRNA expression and upregulation of apoptosis-executive protease bcl-2 mRNA expression; however, DL-3-n-butylphthalide had no protective effects on brain microvascular endothelial cells after knockdown of hypoxia inducible factor-1α by small interfering RNA. These findings suggest that DL-3-n-butylphthalide can protect brain microvascular endothelial cells against oxygen glucose deprivation-induced injury by upregulating bcl-2 expression and downregulating caspase-3 expression though hypoxia inducible factor-1α pathway.

  2. Brain endothelial TAK1 and NEMO safeguard the neurovascular unit

    Science.gov (United States)

    Ridder, Dirk A.; Wenzel, Jan; Müller, Kristin; Töllner, Kathrin; Tong, Xin-Kang; Assmann, Julian C.; Stroobants, Stijn; Weber, Tobias; Niturad, Cristina; Fischer, Lisanne; Lembrich, Beate; Wolburg, Hartwig; Grand’Maison, Marilyn; Papadopoulos, Panayiota; Korpos, Eva; Truchetet, Francois; Rades, Dirk; Sorokin, Lydia M.; Schmidt-Supprian, Marc; Bedell, Barry J.; Pasparakis, Manolis; Balschun, Detlef; D’Hooge, Rudi; Löscher, Wolfgang; Hamel, Edith

    2015-01-01

    Inactivating mutations of the NF-κB essential modulator (NEMO), a key component of NF-κB signaling, cause the genetic disease incontinentia pigmenti (IP). This leads to severe neurological symptoms, but the mechanisms underlying brain involvement were unclear. Here, we show that selectively deleting Nemo or the upstream kinase Tak1 in brain endothelial cells resulted in death of endothelial cells, a rarefaction of brain microvessels, cerebral hypoperfusion, a disrupted blood–brain barrier (BBB), and epileptic seizures. TAK1 and NEMO protected the BBB by activating the transcription factor NF-κB and stabilizing the tight junction protein occludin. They also prevented brain endothelial cell death in a NF-κB–independent manner by reducing oxidative damage. Our data identify crucial functions of inflammatory TAK1–NEMO signaling in protecting the brain endothelium and maintaining normal brain function, thus explaining the neurological symptoms associated with IP. PMID:26347470

  3. Interactions of primary neuroepithelial progenitor and brain endothelial cells: distinct effect on neural progenitor maintenance and differentiation by soluble factors and direct contact

    Institute of Scientific and Technical Information of China (English)

    Miguel A Gama Sosa; Rita De Gasperi; Anne B Rocher; Gissel M Perez; Keila Simons; Daniel E Cruz; Patrick R Hof; Gregory A Elder

    2007-01-01

    Neurovascular interactions are crucial for the normal development of the central nervous system. To study such interactions in primary cultures, we developed a procedure to simultaneously isolate neural progenitor and endothelial cell fractions from embryonic mouse brains. Depending on the culture conditions endothelial cells were found to favor maintenance of the neuroprogenitor phenotype through the production of soluble factors, or to promote neuronal differentiation of neural progenitors through direct contact. These apparently opposing effects could reflect differential cellular interactions needed for the proper development of the brain.

  4. Functional expression of choline transporter like-protein 1 (CTL1) and CTL2 in human brain microvascular endothelial cells.

    Science.gov (United States)

    Iwao, Beniko; Yara, Miki; Hara, Naomi; Kawai, Yuiko; Yamanaka, Tsuyoshi; Nishihara, Hiroshi; Inoue, Takeshi; Inazu, Masato

    2016-02-01

    In this study, we examined the molecular and functional characterization of choline transporter in human brain microvascular endothelial cells (hBMECs). Choline uptake into hBMECs was a saturable process that was mediated by a Na(+)-independent, membrane potential and pH-dependent transport system. The cells have two different [(3)H]choline transport systems with Km values of 35.0 ± 4.9 μM and 54.1 ± 8.1 μM, respectively. Choline uptake was inhibited by choline, acetylcholine (ACh) and the choline analog hemicholinium-3 (HC-3). Various organic cations also interacted with the choline transport system. Choline transporter-like protein 1 (CTL1) and CTL2 mRNA were highly expressed, while mRNA for high-affinity choline transporter 1 (CHT1) and organic cation transporters (OCTs) were not expressed in hBMECs. CTL1 and CTL2 proteins were localized to brain microvascular endothelial cells in human brain cortical sections. Both CTL1 and CTL2 proteins were expressed on the plasma membrane and mitochondria. CTL1 and CTL2 proteins are mainly expressed in plasma membrane and mitochondria, respectively. We conclude that choline is mainly transported via an intermediate-affinity choline transport system, CTL1 and CTL2, in hBMECs. These transporters are responsible for the uptake of extracellular choline and organic cations. CTL2 participate in choline transport mainly in mitochondria, and may be the major site for the control of choline oxidation.

  5. Exosomes contribute to the transmission of anti-HIV activity from TLR3-activated brain microvascular endothelial cells to macrophages

    Science.gov (United States)

    Sun, Li; Wang, Xu; Zhou, Yu; Zhou, Run-Hong; Ho, Wen-Zhe; Li, Jie-Liang

    2017-01-01

    Human brain microvascular endothelial cells (HBMECs), the major cell type in the blood-brain barrier (BBB), play a key role in maintaining brain homeostasis. However, their role in the BBB innate immunity against HIV invasion of the central nervous system (CNS) remains to be determined. Our early work showed that TLR3 signaling of HBMECs could produce the antiviral factors that inhibit HIV replication in macrophages. The present study examined whether exosomes from TLR3-activated HBMECs mediate the intercellular transfer of antiviral factors to macrophages. Primary human macrophages could take up exosomes from TLR3-activated HBMECs. HBMECs-derived exosomes contained multiple antiviral factors, including several key IFN-stimulated genes (ISGs; ISG15, ISG56, and Mx2) at mRNA and protein levels. The depletion of exosomes from TLR3-activated HBMECs culture supernatant diminished HBMECs-mediated anti-HIV activity in macrophages. In conclusion, we demonstrate that exosomes shed by HBMECs are able to transport the antiviral molecules to macrophages. This finding suggests the possibility that HIV nonpermissive BBB cells (HBMECs) can help to restore the antiviral state in HIV-infected macrophages, which may be a defense mechanism against HIV neuroinvasion. PMID:27496004

  6. CJZ3,a lomerizine derivative,modulates P-glycoprotein function in rat brain microvessel endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Bian-sheng JI; Ling HE; Xiao-qu LI; Guo-qing LIU

    2006-01-01

    Aim:To investigate the modulatory effect of CJZ3,a lomerizine derivative,on P-glycoprotein (P-gp) function in rat brain microvessel endothelial cells (RBMEC).Methods:RBMEC were isolated and cultured in Dulbecco's modified Eagle's medium/F12 (1∶1) medium,and the amount of intracellular rhodamine 123 (Rh123) was determined using a fluorescence spectrophotometer to evaluate the modulatory effect of CJZ3 on P-gp function.Results:The accumulation of Rh123 was 190ten. tiated in a concentration-dependent manner after incubation witll CJZ3 for RBMEC.but not for human umbilical vein endothelial cells (HUVEC).CJZ3 caused the accumulation of intracellular Rh 123 in a time.dependent manner and significantly decreased the effiUX of Rhl 23 from the cells.The inhibitory effect of CJZ3 on P-gp function was reversible and remained for 120 min after CJZ3 (2.5 μmol/L) was removed from the medium.Conclusion:CJZ3 has a potent in vitro effect on the inhibition of P-gp function.

  7. Reversible Opening of Intercellular Junctions of Intestinal Epithelial and Brain Endothelial Cells With Tight Junction Modulator Peptides.

    Science.gov (United States)

    Bocsik, Alexandra; Walter, Fruzsina R; Gyebrovszki, Andrea; Fülöp, Lívia; Blasig, Ingolf; Dabrowski, Sebastian; Ötvös, Ferenc; Tóth, András; Rákhely, Gábor; Veszelka, Szilvia; Vastag, Monika; Szabó-Révész, Piroska; Deli, Mária A

    2016-02-01

    The intercellular junctions restrict the free passage of hydrophilic compounds through the paracellular clefts. Reversible opening of the tight junctions of biological barriers is investigated as one of the ways to increase drug delivery to the systemic circulation or the central nervous system. Six peptides, ADT-6, HAV-6, C-CPE, 7-mer (FDFWITP, PN-78), AT-1002, and PN-159, acting on different integral membrane and linker junctional proteins were tested on Caco-2 intestinal epithelial cell line and a coculture model of the blood-brain barrier. All peptides tested in nontoxic concentrations showed a reversible tight junctions modulating effect and were effective to open the paracellular pathway for the marker molecules fluorescein and albumin. The change in the structure of cell-cell junctions was verified by immunostaining for occludin, claudin-4,-5, ZO-1, β-catenin, and E-cadherin. Expression levels of occludin and claudins were measured in both models. We could demonstrate a selectivity of C-CPE, ADT-6, and HAV-6 peptides for epithelial cells and 7-mer and AT-1002 peptides for brain endothelial cells. PN-159 was the most effective modulator of junctional permeability in both models possibly acting via claudin-1 and -5. Our results indicate that these peptides can be effectively and selectively used as potential pharmaceutical excipients to improve drug delivery across biological barriers.

  8. Initial contact of glioblastoma cells with existing normal brain endothelial cells strengthen the barrier function via fibroblast growth factor 2 secretion: a new in vitro blood-brain barrier model.

    Science.gov (United States)

    Toyoda, Keisuke; Tanaka, Kunihiko; Nakagawa, Shinsuke; Thuy, Dinh Ha Duy; Ujifuku, Kenta; Kamada, Kensaku; Hayashi, Kentaro; Matsuo, Takayuki; Nagata, Izumi; Niwa, Masami

    2013-05-01

    Glioblastoma multiforme (GBM) cells invade along the existing normal capillaries in brain. Normal capillary endothelial cells function as the blood-brain barrier (BBB) that limits permeability of chemicals into the brain. To investigate whether GBM cells modulate the BBB function of normal endothelial cells, we developed a new in vitro BBB model with primary cultures of rat brain endothelial cells (RBECs), pericytes, and astrocytes. Cells were plated on a membrane with 8 μm pores, either as a monolayer or as a BBB model with triple layer culture. The BBB model consisted of RBEC on the luminal side as a bottom, and pericytes and astrocytes on the abluminal side as a top of the chamber. Human GBM cell line, LN-18 cells, or lung cancer cell line, NCI-H1299 cells, placed on either the RBEC monolayer or the BBB model increased the transendothelial electrical resistance (TEER) values against the model, which peaked within 72 h after the tumor cell application. The TEER value gradually returned to baseline with LN-18 cells, whereas the value quickly dropped to the baseline in 24 h with NCI-H1299 cells. NCI-H1299 cells invaded into the RBEC layer through the membrane, but LN-18 cells did not. Fibroblast growth factor 2 (FGF-2) strengthens the endothelial cell BBB function by increased occludin and ZO-1 expression. In our model, LN-18 and NCI-H1299 cells secreted FGF-2, and a neutralization antibody to FGF-2 inhibited LN-18 cells enhanced BBB function. These results suggest that FGF-2 would be a novel therapeutic target for GBM in the perivascular invasive front.

  9. Neisseria meningitidis causes cell cycle arrest of human brain microvascular endothelial cells at S phase via p21 and cyclin G2.

    Science.gov (United States)

    Oosthuysen, Wilhelm F; Mueller, Tobias; Dittrich, Marcus T; Schubert-Unkmeir, Alexandra

    2016-01-01

    Microbial pathogens have developed several mechanisms to modulate and interfere with host cell cycle progression. In this study, we analysed the effect of the human pathogen Neisseria meningitidis on cell cycle in a brain endothelial cell line as well as in primary brain endothelial cells. We found that N.  Meningitidis causes an accumulation of cells in the S phase early at 3 and at 24 h post-infection that was paralleled by a decrease of cells in G2/M phase. Importantly, the outer membrane proteins of the colony opacity-associated (Opa) protein family as well as the Opc protein proved to trigger the accumulation of cells in the S phase. A focused cell cycle reverse transcription quantitative polymerase chain reaction-based array and integrated network analysis revealed changes in the abundance of several cell cycle regulatory mRNAs, including the cell cycle inhibitors p21(WAF1/CIP1) and cyclin G2. These alterations were reflected in changes in protein expression levels and/or relocalization in N. meningitidis-infected cells. Moreover, an increase in p21(WAF1/CIP1) expression was found to be p53 independent. Genetic ablation of p21(WAF1/CIP1) and cyclin G2 abrogated N. meningitidis-induced S phase accumulation. Finally, by measuring the levels of the biomarker 8-hydroxydeoxyguanosine and phosphorylation of the histone variant H2AX, we provide evidence that N. meningitidis induces oxidative DNA damage in infected cells.

  10. Exogenous arachidonic acid mediates permeability of human brain microvessel endothelial cells through prostaglandin E2 activation of EP3 and EP4 receptors.

    Science.gov (United States)

    Dalvi, Siddhartha; Nguyen, Hieu H; On, Ngoc; Mitchell, Ryan W; Aukema, Harold M; Miller, Donald W; Hatch, Grant M

    2015-12-01

    The blood-brain barrier, formed by microvessel endothelial cells, is the restrictive barrier between the brain parenchyma and the circulating blood. Arachidonic acid (ARA; 5,8,11,14-cis-eicosatetraenoic acid) is a conditionally essential polyunsaturated fatty acid [20:4(n-6)] and is a major constituent of brain lipids. The current study examined the transport processes for ARA in confluent monolayers of human brain microvascular endothelial cells (HBMEC). Addition of radioactive ARA to the apical compartment of HBMEC cultured on Transwell(®) inserts resulted in rapid incorporation of radioactivity into the basolateral medium. Knock down of fatty acid transport proteins did not alter ARA passage into the basolateral medium as a result of the rapid generation of prostaglandin E2 (PGE2 ), an eicosanoid known to facilitate opening of the blood-brain barrier. Permeability following ARA or PGE2 exposure was confirmed by an increased movement of fluorescein-labeled dextran from apical to basolateral medium. ARA-mediated permeability was attenuated by specific cyclooxygenase-2 inhibitors. EP3 and EP4 receptor antagonists attenuated the ARA-mediated permeability of HBMEC. The results indicate that ARA increases permeability of HBMEC monolayers likely via increased production of PGE2 which acts upon EP3 and EP4 receptors to mediate permeability. These observations may explain the rapid influx of ARA into the brain previously observed upon plasma infusion with ARA. The blood-brain barrier, formed by microvessel endothelial cells, is a restrictive barrier between the brain parenchyma and the circulating blood. Radiolabeled arachidonic acid (ARA) movement across, and monolayer permeability in the presence of ARA, was examined in confluent monolayers of primary human brain microvessel endothelial cells (HBMECs) cultured on Transwell(®) plates. Incubation of HBMECs with ARA resulted in a rapid increase in HBMEC monolayer permeability. The mechanism was mediated, in part

  11. Effect of low-dose methylprednisolone on peripheral blood endothelial progenitor cells and its significance in rats after brain injury

    Directory of Open Access Journals (Sweden)

    Bin ZHANG

    2011-05-01

    Full Text Available Objective To explore the effects of low-dose methylprednisolone(MP treatment after traumatic brain injury(TBI in rats on the number of peripheral blood endothelial progenitor cells(EPCs and injury area of the brain.Methods One hundred and fifty-four adult male Wistar rats were involved in the present study,and they were randomly divided into normal control group(n=18,TBI control group(n=38,MP control group(n=30,MP+TBI group(n=30 and TBI+MP group(n=38.The TBI model was reproduced by fluid percussion injury(FPI.MP(5mg/kg was intraperitoneally administered once a day for 4 days.Peripheral venous blood samples were taken on day 1,3,7 and 14,and the counts of EPCs were determined by flow cytometry.The rats were sacrificed on day 1 and 3,brain edema was estimated by dry-wet weight method,and the blood-brain barrier(BBB permeability was determined by Evans-blue extravasation.Results The counts of peripheral blood EPCs were significantly higher in MP control group,MP+TBI group and TBI+MP group on day 1,3 and 7 than that in normal control and TBI control group,and it returned to the level of normal control group on day 14.The BBB permeability was improved and brain edema alleviated in MP+TBI and TBI+MP group on day 3.Conclusion The administration of low-dose MP may increase the count of peripheral blood EPCs in rats,decrease BBB damage,and alleviate brain edema.

  12. Inhibition of glutathione synthesis in brain endothelial cells lengthens S-phase transit time in the cell cycle: Implications for proliferation in recovery from oxidative stress and endothelial cell damage

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    Carmina Buşu

    2013-01-01

    Full Text Available Oxidative stress-induced decrease in tissue or systemic glutathione (GSH and damage to the vascular endothelium of the blood-brain barrier such as occurs in diabetes or stroke will have important implications for brain homeostasis. Endothelial proliferation or repair is crucial to preserving barrier function. Cell proliferation has been associated with increased intracellular GSH, but the kinetic and distribution of GSH during cell cycle is poorly understood. Here, we determined the influence of cellular GSH status on the early dynamics of nuclear-to-cytosol (N-to-C GSH distribution (6-h interval during proliferation in a human brain microvascular endothelial cell line (IHEC. Control IHECs exhibited two peak S-phases of the cell cycle at 48 and 60 h post seeding that temporally corresponded to peak nuclear GSH levels and expression of cdk1, the S-to-G2-to-M checkpoint controller, suggesting a link between cell cycle progression and nuclear GSH. Sustained inhibition of GSH synthesis delayed S-to-G2/M cell transition; cell arrest in the S-phase was correlated with decreased total nuclear GSH and increased nuclear expressions of chk2/phospho-chk2 and GADPH. The temporal correspondence of nuclear chk2 activation and GAPDH expression with S-phase prolongation is consistent with enhanced DNA damage response and extended time for DNA repair. Strikingly, when GSH synthesis was restored, cell transit time through S-phase remained delayed. Significantly, total nuclear GSH remained depressed, indicating a time lag between restored cellular GSH synthetic capacity and recovery of the nuclear GSH status. Interestingly, despite a delay in cell cycle recovery, nuclear expressions of chk2/phospho-chk2 and GAPDH resembled those of control cells. This means that restoration of nuclear DNA integrity preceded normalization of the cell cycle. The current results provide important insights into GSH control of endothelial proliferation with implications for cell

  13. Up-regulation of K{sub ir}2.1 by ER stress facilitates cell death of brain capillary endothelial cells

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    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. Lipid raft/caveolae signaling is required for Cryptococcus neoformans invasion into human brain microvascular endothelial cells

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    Long Min

    2012-02-01

    Full Text Available Abstract Background Cryptococcus neoformans has a predilection for central nervous system infection. C. neoformans traversal of the blood brain barrier, composed of human brain microvascular endothelial cells (HBMEC, is the crucial step in brain infection. However, the molecular mechanism of the interaction between Cryptococcus neoformans and HBMEC, relevant to its brain invasion, is still largely unknown. Methods In this report, we explored several cellular and molecular events involving the membrane lipid rafts and caveolin-1 (Cav1 of HBMEC during C. neoformans infection. Immunofluorescence microscopy was used to examine the roles of Cav1. The knockdown of Cav1 by the siRNA treatment was performed. Phosphorylation of Cav1 relevant to its invasion functions was investigated. Results We found that the host receptor CD44 colocalized with Cav1 on the plasma membrane, and knockdown of Cav1 significantly reduced the fungal ability to invade HBMEC. Although the CD44 molecules were still present, HBMEC membrane organization was distorted by Cav1 knockdown. Concomitantly, knockdown of Cav1 significantly reduced the fungal crossing of the HBMEC monolayer in vitro. Upon C. neoformans engagement, host Cav1 was phosphorylated in a CD44-dependent manner. This phosphorylation was diminished by filipin, a disrupter of lipid raft structure. Furthermore, the phosphorylated Cav1 at the lipid raft migrated inward to the perinuclear localization. Interestingly, the phospho-Cav1 formed a thread-like structure and colocalized with actin filaments but not with the microtubule network. Conclusion These data support that C. neoformans internalization into HBMEC is a lipid raft/caveolae-dependent endocytic process where the actin cytoskeleton is involved, and the Cav1 plays an essential role in C. neoformans traversal of the blood-brain barrier.

  15. Up-regulation of COX-2/PGE2 by endothelin-1 via MAPK-dependent NF-κB pathway in mouse brain microvascular endothelial cells

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    Lin Chih-Chung

    2013-01-01

    Full Text Available Abstract Background Endothelin-1 (ET-1 is a proinflammatory mediator and elevated in the regions of several brain injury and inflammatory diseases. The deleterious effects of ET-1 on endothelial cells may aggravate brain inflammation mediated through the regulation of cyclooxygenase-2 (COX-2/prostaglandin E2 (PGE2 system in various cell types. However, the signaling mechanisms underlying ET-1-induced COX-2 expression in brain microvascular endothelial cells remain unclear. Herein we investigated the effects of ET-1 in COX-2 regulation in mouse brain microvascular endothelial (bEnd.3 cells. Results The data obtained with Western blotting, RT-PCR, and immunofluorescent staining analyses showed that ET-1-induced COX-2 expression was mediated through an ETB-dependent transcriptional activation. Engagement of Gi- and Gq-protein-coupled ETB receptors by ET-1 led to phosphorylation of ERK1/2, p38 MAPK, and JNK1/2 and then activated transcription factor NF-κB. Moreover, the data of chromatin immunoprecipitation (ChIP and promoter reporter assay demonstrated that the activated NF-κB was translocated into nucleus and bound to its corresponding binding sites in COX-2 promoter, thereby turning on COX-2 gene transcription. Finally, up-regulation of COX-2 by ET-1 promoted PGE2 release in these cells. Conclusions These results suggested that in mouse bEnd.3 cells, activation of NF-κB by ETB-dependent MAPK cascades is essential for ET-1-induced up-regulation of COX-2/PGE2 system. Understanding the mechanisms of COX-2 expression and PGE2 release regulated by ET-1/ETB system on brain microvascular endothelial cells may provide rationally therapeutic interventions for brain injury or inflammatory diseases.

  16. Focal adhesion kinase is involved in type III group B streptococcal invasion of human brain microvascular endothelial cells.

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    Shin, Sooan; Paul-Satyaseela, Maneesh; Maneesh, Paul-Satyaseela; Lee, Jong-Seok; Romer, Lewis H; Kim, Kwang Sik

    2006-01-01

    Group B streptococcus (GBS), the leading cause of neonatal meningitis, has been shown to invade human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. GBS invasion of HBMEC has been shown to require the host cell actin cytoskeleton rearrangements. The present study examined the mechanisms underlying actin cytoskeleton rearrangements that are involved in type III GBS invasion of HBMEC. We showed that type III GBS invasion was inhibited by genistein, a general tyrosine kinase inhibitor (mean 54% invasion decrease at 100 microM), and LY294002, a phosphatidylinositol 3 (PI3) kinase inhibitor (mean 70% invasion decrease at 50 microM), but not by PP2, an inhibitor of the Src family tyrosine kinases. We subsequently showed that the focal adhesion kinase (FAK) was the one of the host proteins tyrosine phosphorylated by type III GBS. Over-expression of a dominant negative form of the FAK C-terminal domain significantly decreased type III GBS invasion of HBMEC (mean 51% invasion decrease). In addition, we showed that FAK phosphorylation correlated with its association of paxillin, an adapter protein of actin filament, and PI3-kinase subunit p85. This is the first demonstration that FAK phosphorylation and its association with paxillin and PI3 kinase play a key role in type III GBS invasion of HBMEC.

  17. Effect of baicalin and berberine on transport of nimodipine on primary-cultured, rat brain microvascular endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Dong-mei ZHANG; Hai-yan LIU; Lin XIE; Xiao-dong LIU

    2007-01-01

    Aim: To investigate whether baicalin and berberine affects the transport of nimodipine (NMD) across the blood-brain barrier (BBB). Methods: Primary-cultured, rat brain microvascular endothelial cells (rBMEC) were used as an in vitro model of the BBB. When cells became confluent, the steady-state uptake of NMD by rBMEC with or without baicalin and berberine was measured. The ef-fects of baicalin and berberine on the efflux of NMD from rBMEC were also studied.Results: Baicalin (2-5 μg/mL) increased the uptake of NMD, and baicalin (10-20 μg/mL) decreased the uptake. The steady-state uptake of NMD was higher than that of control group in the presence of 0.01-1 μg/mL berberine, but was lower in the presence of 2-10 μg/mL berberine. Conclusion: The bidirectional effect of baicalin and berberine on the uptake of NMD by rBMEC was found. Higher concentration showed an inhibitory effect, and lower concentration demonstrated an increasing effect.

  18. eEF-2 Phosphorylation Down-Regulates P-Glycoprotein Over-Expression in Rat Brain Microvessel Endothelial Cells.

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    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.

  19. Regulation of Monocarboxylic Acid Transporter 1 Trafficking by the Canonical Wnt/β-Catenin Pathway in Rat Brain Endothelial Cells Requires Cross-talk with Notch Signaling.

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    Liu, Zejian; Sneve, Mary; Haroldson, Thomas A; Smith, Jeffrey P; Drewes, Lester R

    2016-04-01

    The transport of monocarboxylate fuels such as lactate, pyruvate, and ketone bodies across brain endothelial cells is mediated by monocarboxylic acid transporter 1 (MCT1). Although the canonical Wnt/β-catenin pathway is required for rodent blood-brain barrier development and for the expression of associated nutrient transporters, the role of this pathway in the regulation of brain endothelial MCT1 is unknown. Here we report expression of nine members of the frizzled receptor family by the RBE4 rat brain endothelial cell line. Furthermore, activation of the canonical Wnt/β-catenin pathway in RBE4 cells via nuclear β-catenin signaling with LiCl does not alter brain endothelialMct1mRNA but increases the amount of MCT1 transporter protein. Plasma membrane biotinylation studies and confocal microscopic examination of mCherry-tagged MCT1 indicate that increased transporter results from reduced MCT1 trafficking from the plasma membrane via the endosomal/lysosomal pathway and is facilitated by decreased MCT1 ubiquitination following LiCl treatment. Inhibition of the Notch pathway by the γ-secretase inhibitorN-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycinet-butyl ester negated the up-regulation of MCT1 by LiCl, demonstrating a cross-talk between the canonical Wnt/β-catenin and Notch pathways. Our results are important because they show, for the first time, the regulation of MCT1 in cerebrovascular endothelial cells by the multifunctional canonical Wnt/β-catenin and Notch signaling pathways.

  20. Proteomic Analysis of Human Brain Microvascular Endothelial Cells Reveals Differential Protein Expression in Response to Enterovirus 71 Infection

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    Wenying Luo

    2015-01-01

    Full Text Available 2D DIGE technology was employed on proteins prepared from human brain microvascular endothelial cells (HBMEC, to study the differentially expressed proteins in cells at 0 h, 1 h, 16 h, and 24 h after infection. Proteins found to be differentially expressed were identified with matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDITOF/TOF MS analysis. We identified 43 spots showing changes of at least 2.5 fold up- or downregulated expressions in EV71-infected cells at different time when comparing to control, and 28 proteins could be successfully identified by MALDI TOF/TOF mass spectrometry analysis. 4 proteins were significantly upregulated, and 6 proteins were downregulated, another 18 proteins were different expression at different incubation time. We identified changes in the expression of 12 cellular metabolism-related proteins, 5 molecules involved in cytoskeleton, 3 molecules involved in energy metabolism, 2 molecules involved in signal transduction, 1 molecule involved in the ubiquitin-proteasome pathway, 1 molecule involved in cell cycle, 1 molecule involved in apoptosis-related protein, 1 molecular chaperone, and 2 unknown proteins. These findings build up a comprehensive profile of the HBMEC proteome and provide a useful basis for further analysis of the pathogenic mechanism that underlies EV71 infections to induce severe neural complications.

  1. Protective Effects of Inducible HO-1 on Oxygen Toxicity in Rat Brain Endothelial Microvessel Cells

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    Seung-Jun Yoo

    2014-09-01

    Full Text Available BackgroundReperfusion in ischemia is believed to generate cytotoxic oxidative stress, which mediates reperfusion injury. These stress conditions can initiate lipid peroxidation and damage to proteins, as well as promote DNA strand breaks. As biliverdin and bilirubin produced by heme oxygenase isoform 1 (HO-1 have antioxidant properties, the production of both antioxidants by HO-1 may help increase the resistance of the ischemic brain to oxidative stress. In the present study, the survival effect of HO-1 was confirmed using hemin.MethodsTo confirm the roles of HO-1, carbon monoxide, and cyclic guanosine monophosphate further in the antioxidant effect of HO-1 and bilirubin, cells were treated with cycloheximide, desferoxamine, and zinc deuteroporphyrin IX 2,4 bis glycol, respectively.ResultsHO-1 itself acted as an antioxidant. Furthermore, iron, rather than carbon monoxide, was involved in the HO-1-mediated survival effect. HO-1 activity was also important in providing bilirubin as an antioxidant.ConclusionOur results suggested that HO-1 helped to increase the resistance of the ischemic brain to oxidative stress.

  2. Insulin-like growth factor-1 secreted by brain microvascular endothelial cells attenuates neuron injury upon ischemia.

    Science.gov (United States)

    Wang, Jun; Tang, Yibo; Zhang, Wei; Zhao, Haiping; Wang, Runjun; Yan, Yangyang; Xu, Liwei; Li, Pengtao

    2013-08-01

    Insulin-like growth factor (IGF)-1 is essential for the development of the nervous system, and is present in many cell types. Relatively little is known about IGF-1 expression in brain microvascular endothelial cells (BMECs). For in vivo studies, we examined the expression of IGF-1 and insulin-like growth factor-binding protein (IGFBP)-2 after focal cerebral ischemia for 12 h, 24 h, 3 days and 7 days, utilizing a permanent middle cerebral artery occlusion (MCAO) model in rats. For in vitro studies, we examined the levels of IGF-1 and IGFBP-2 in the culture medium or primary culture of BMECs injured by oxygen-glucose deprivation (OGD). Then, we elucidated the protective effects of IGF-1 on cortical neurons injured by OGD and the possible mechanism. In addition, we investigated the effect of BMEC-conditioned medium on IGF-1 receptor expression in neurons. The results showed that IGF-1 expression increased in serum and brain tissue, whereas IGFBP-2 expression decreased in brain tissue of MCAO-injured rats. In primary culture of BMECs, the expression levels of IGF-1 and IGFBP-2 were significantly higher under OGD conditions in culture. IGF-1 administration improved neuron viability upon normoxia or OGD, and upregulated p-Akt expression. This effect was reversed by LY294002, a specific inhibitor of the phosphoinositide 3-kinase-Akt signaling pathway. Furthermore, conditioned medium from OGD-treated BMECs substantially suppressed neuron viability and the expression of IGF-1 receptor simultaneously. These data demonstrate that therapeutic strategies that prioritize the early recovery of the IGF-1 system in BMECs might be promising in ischemic injury.

  3. Synergy of endothelial and neural progenitor cells from adipose-derived stem cells to preserve neurovascular structures in rat hypoxic-ischemic brain injury.

    Science.gov (United States)

    Hsueh, Yuan-Yu; Chang, Ya-Ju; Huang, Chia-Wei; Handayani, Fitri; Chiang, Yi-Lun; Fan, Shih-Chen; Ho, Chien-Jung; Kuo, Yu-Min; Yang, Shang-Hsun; Chen, Yuh-Ling; Lin, Sheng-Che; Huang, Chao-Ching; Wu, Chia-Ching

    2015-10-08

    Perinatal cerebral hypoxic-ischemic (HI) injury damages the architecture of neurovascular units (NVUs) and results in neurological disorders. Here, we differentiated adipose-derived stem cells (ASCs) toward the progenitor of endothelial progenitor cells (EPCs) and neural precursor cells (NPCs) via microenvironmental induction and investigated the protective effect by transplanting ASCs, EPCs, NPCs, or a combination of EPCs and NPCs (E+N) into neonatal HI injured rat pups. The E+N combination produced significant reduction in brain damage and cell apoptosis and the most comprehensive restoration in NVUs regarding neuron number, normal astrocytes, and vessel density. Improvements in cognitive and motor functions were also achieved in injured rats with E+N therapy. Synergistic interactions to facilitate transmigration under in vitro hypoxic microenvironment were discovered with involvement of the neuropilin-1 (NRP1) signal in EPCs and the C-X-C chemokine receptor 4 (CXCR4) and fibroblast growth factor receptor 1 (FGFR1) signals in NPCs. Therefore, ASCs exhibit great potential for cell sources in endothelial and neural lineages to prevent brain from HI damage.

  4. 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

    The blood–brain-barrier (BBB) is a formidable obstacle for successful translocation of many drug molecules from the systemic circulation into the brain, and therefore a major challenge for neurotherapeutics. Nanoparticles may offer some opportunities for delivery of bioactive molecules into brain...

  5. 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?

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    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.

  6. Trafficking of adeno-associated virus vectors across a model of the blood-brain barrier; a comparative study of transcytosis and transduction using primary human brain endothelial cells.

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    Merkel, Steven F; Andrews, Allison M; Lutton, Evan M; Mu, Dakai; Hudry, Eloise; Hyman, Bradley T; Maguire, Casey A; Ramirez, Servio H

    2017-01-01

    Developing therapies for central nervous system (CNS) diseases is exceedingly difficult because of the blood-brain barrier (BBB). Notably, emerging technologies may provide promising new options for the treatment of CNS disorders. Adeno-associated virus serotype 9 (AAV9) has been shown to transduce cells in the CNS following intravascular administration in rodents, cats, pigs, and non-human primates. These results suggest that AAV9 is capable of crossing the BBB. However, mechanisms that govern AAV9 transendothelial trafficking at the BBB remain unknown. Furthermore, possibilities that AAV9 may transduce brain endothelial cells or affect BBB integrity still require investigation. Using primary human brain microvascular endothelial cells as a model of the human BBB, we performed transduction and transendothelial trafficking assays comparing AAV9 to AAV2, a serotype that does not cross the BBB or transduce endothelial cells effectively in vivo. Results of our in vitro studies indicate that AAV9 penetrates brain microvascular endothelial cells barriers more effectively than AAV2, but has reduced transduction efficiency. In addition, our data suggest that (i) AAV9 penetrates endothelial barriers through an active, cell-mediated process, and (ii) AAV9 fails to disrupt indicators of BBB integrity such as transendothelial electrical resistance, tight junction protein expression/localization, and inflammatory activation status. Overall, this report shows how human brain endothelial cells configured in BBB models can be utilized for evaluating transendothelial movement and transduction kinetics of various AAV capsids. Importantly, the use of a human in vitro BBB model can provide import insight into the possible effects that candidate AVV gene therapy vectors may have on the status of BBB integrity. Read the Editorial Highlight for this article on page 192.

  7. Targeting Cells With MR Imaging Probes: Cellular Interaction And Intracellular Magnetic Iron Oxide Nanoparticles Uptake In Brain Capillary Endothelial and Choroidal Plexus Epithelial Cells

    Science.gov (United States)

    Cambianica, I.; Bossi, M.; Gasco, P.; Gonzalez, W.; Idee, J. M.; Miserocchi, G.; Rigolio, R.; Chanana, M.; Morjan, I.; Wang, D.; Sancini, G.

    2010-10-01

    Magnetic iron oxide nanoparticles (NPs) are considered for various diagnostic and therapeutic applications in brain including their use as contrast agent for magnetic resonance imaging. In delivery application, the critical step is the transport across cell layers and the internalization of NPs into specific cells, a process often limited by poor targeting specificity and low internalization efficiency. The development of the models of brain endothelial cells and choroidal plexus epithelial cells in culture has allowed us to investigate into these mechanisms. Our strategy is aimed at exploring different routes to the entrapment of iron oxide NPs in these brain related cells. Here we demonstrated that not only cells endowed with a good phagocytic activity like activated macrophages but also endothelial brain capillary and choroidal plexus epithelial cells do internalize iron oxide NPs. Our study of the intracellular trafficking of NPs by TEM, and confocal microscopy revealed that NPs are mainly internalized by the endocytic pathway. Iron oxide NPs were dispersed in water and coated with 3,4-dihydroxyl-L-phenylalanine (L-DOPA) using standard procedures. Magnetic lipid NPs were prepared by NANOVECTOR: water in oil in water (W/O/W) microemulsion process has been applied to directly coat different iron based NPs by lipid layer or to encapsulate them into Solid Lipid Nanoparticles (SLNs). By these coating/loading the colloidal stability was improved without strong alteration of the particle size distribution. Magnetic lipid NPs could be reconstituted after freeze drying without appreciable changes in stability. L-DOPA coated NPs are stable in PBS and in MEM (Modified Eagle Medium) medium. The magnetic properties of these NPs were not altered by the coating processes. We investigated the cellular uptake, cytotoxicity, and interaction of these NPs with rat brain capillary endothelial (REB4) and choroidal plexus epithelial (Z310) cells. By means of widefield, confocal

  8. Brain pericytes increase the lipopolysaccharide-enhanced transcytosis of HIV-1 free virus across the in vitro blood–brain barrier: evidence for cytokine-mediated pericyte-endothelial cell crosstalk

    Science.gov (United States)

    2013-01-01

    Background Human immunodeficiency virus-1 (HIV-1) enters the brain by crossing the blood–brain barrier (BBB) as both free virus and within infected immune cells. Previous work showed that activation of the innate immune system with lipopolysaccharide (LPS) enhances free virus transport both in vivo and across monolayer monocultures of brain microvascular endothelial cells (BMECs) in vitro. Methods Here, we used monocultures and co-cultures of brain pericytes and brain endothelial cells to examine the crosstalk between these cell types in mediating the LPS-enhanced permeation of radioactively-labeled HIV-1 (I-HIV) across BMEC monolayers. Results We found that brain pericytes when co-cultured with BMEC monolayers magnified the LPS-enhanced transport of I-HIV without altering transendothelial electrical resistance, indicating that pericytes affected the transcytotic component of HIV-1 permeation. As LPS crosses the BBB poorly if at all, and since pericytes are on the abluminal side of the BBB, we postulated that luminal LPS acts indirectly on pericytes through abluminal secretions from BMECs. Consistent with this, we found that the pattern of secretion of cytokines by pericytes directly exposed to LPS was different than when the pericytes were exposed to the abluminal fluid from LPS-treated BMEC monolayers. Conclusion These results are evidence for a cellular crosstalk in which LPS acts at the luminal surface of the brain endothelial cell, inducing abluminal secretions that stimulate pericytes to release substances that enhance the permeability of the BMEC monolayer to HIV. PMID:23816186

  9. Brain-derived neurotrophic factor increases vascular endothelial growth factor expression and enhances angiogenesis in human chondrosarcoma cells.

    Science.gov (United States)

    Lin, Chih-Yang; Hung, Shih-Ya; Chen, Hsien-Te; Tsou, Hsi-Kai; Fong, Yi-Chin; Wang, Shih-Wei; Tang, Chih-Hsin

    2014-10-15

    Chondrosarcomas are a type of primary malignant bone cancer, with a potent capacity for local invasion and distant metastasis. Brain-derived neurotrophic factor (BDNF) is commonly upregulated during neurogenesis. The aim of the present study was to examine the mechanism involved in BDNF-mediated vascular endothelial growth factor (VEGF) expression and angiogenesis in human chondrosarcoma cells. Here, we knocked down BDNF expression in chondrosarcoma cells and assessed their capacity to control VEGF expression and angiogenesis in vitro and in vivo. We found knockdown of BDNF decreased VEGF expression and abolished chondrosarcoma conditional medium-mediated angiogenesis in vitro as well as angiogenesis effects in vivo in the chick chorioallantoic membrane and Matrigel plug nude mouse models. In addition, in the xenograft tumor angiogenesis model, the knockdown of BDNF significantly reduced tumor growth and tumor-associated angiogenesis. BDNF increased VEGF expression and angiogenesis through the TrkB receptor, PLCγ, PKCα, and the HIF-1α signaling pathway. Finally, we analyzed samples from chondrosarcoma patients by immunohistochemical staining. The expression of BDNF and VEGF protein in 56 chondrosarcoma patients was significantly higher than in normal cartilage. In addition, the high level of BDNF expression correlated strongly with VEGF expression and tumor stage. Taken together, our results indicate that BDNF increases VEGF expression and enhances angiogenesis through a signal transduction pathway that involves the TrkB receptor, PLCγ, PKCα, and the HIF-1α. Therefore, BDNF may represent a novel target for anti-angiogenic therapy for human chondrosarcoma.

  10. Mecanotransduction and Endothelial Cells

    Institute of Scientific and Technical Information of China (English)

    S.MULLER; JF.; STOLTZ2

    2005-01-01

    1 IntroductionAtherosclerosis preferentially occurs in areas of complex blood flow where there are disturbed flow and low fluid shear stress, whereas laminar blood flow and high shear stress are atheroprotective~([1]). Reports of others and our studies suggest a steady laminar flow decreases some molecules and genes expression of vascular endothelial cells (EC) that may promote atherosclerosis, as well as it can differentially regulate production of many vasoactive factors at the level of gene expression an...

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

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    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.

  12. Brain-derived neurotrophic factor induces migration of endothelial cells through a TrkB-ERK-integrin αVβ3-FAK cascade.

    Science.gov (United States)

    Matsuda, Shinji; Fujita, Tsuyoshi; Kajiya, Mikihito; Takeda, Katsuhiro; Shiba, Hideki; Kawaguchi, Hiroyuki; Kurihara, Hidemi

    2012-05-01

    Brain-derived neurotrophic factor (BDNF) promotes the regeneration of periodontal tissue. Since angiogenesis is important for tissue regeneration, investigating effect of BDNF on endothelial cell function may help to reveal its mechanism, whereby, BDNF promotes periodontal tissue regeneration. In this study, we examined the influence of BDNF on migration in human microvascular endothelial cells (HMVECs), focusing on the effects on extracellular signal-regulated kinase (ERK), integrin α(V)β(3), and focal adhesion kinase (FAK). The migration of endothelial cells was assessed with a modified Boyden chamber and a wound healing assay. The expression of integrin α(V)β(3) and the phosphorylation of ERK and FAK were analyzed by immunoblotting and immunofluorescence microscopy. BDNF (25 ng/ml) induced cell migration. PD98059, an ERK inhibitor, K252a, a specific inhibitor for TrkB, a high affinity receptor of BDNF, and an anti-integrin α(V)β(3) antibody suppressed the BDNF-induced migration. BDNF increased the levels of integrin α(V)β(3) and phosphorylated ERK1/2 and FAK. The ERK inhibitor and TrkB inhibitor also reduced levels of integrin α(V)β(3) and phosphorylated FAK. We propose that BDNF stimulates endothelial cell migration by a process involving TrkB/ERK/integrin α(V)β(3)/FAK, and this may help to enhance the regeneration of periodontal tissue.

  13. Interactions of Pseudomonas aeruginosa and Corynebacterium spp. with non-phagocytic brain microvascular endothelial cells and phagocytic Acanthamoeba castellanii.

    Science.gov (United States)

    Siddiqui, Ruqaiyyah; Lakhundi, Sahreena; Khan, Naveed Ahmed

    2015-06-01

    Several lines of evidence suggest that Acanthamoeba interact with bacteria, which may aid in pathogenic bacterial transmission to susceptible hosts, and these interactions may have influenced evolution of bacterial pathogenicity. In this study, we tested if Gram-negative Pseudomonas aeruginosa and Gram-positive Corynebacterium spp. can associate/invade and survive inside Acanthamoeba castellanii trophozoites and cysts, as well as non-phagocytic human brain microvascular endothelial cells. The results revealed that both Corynebacterium spp. and P. aeruginosa were able to associate as well as invade and/or taken up by the phagocytic A. castellanii trophozoite. In contrast, P. aeruginosa exhibited higher association as well as invasion of non-phagocytic HBMEC compared with Corynebacterium spp. Notably, P. aeruginosa remained viable during the encystment process and exhibited higher levels of recovery from mature cysts (74.54 bacteria per amoebae) compared with Corynebacterium spp. (2.69 bacteria per amoeba) (P < 0.05). As Acanthamoeba cysts can be airborne, these findings suggest that Acanthamoeba is a potential vector in the transmission of P. aeruginosa to susceptible hosts. When bacterial-ridden amoebae were exposed to favourable (nutrient-rich) conditions, A. castellanii emerged as vegetative trophozoites and remained viable, and likewise viable P. aeruginosa were also observed but rarely any Corynebacterium spp. were observed. Correspondingly, P. aeruginosa but not Corynebacterium spp. exhibited higher cytotoxicity to non-phagocytic HBMEC, producing more than 75% cell death in 24 h, compared to 20% cell death observed with Corynebacterium spp. Additionally, it was observed that the bacterial conditioned medium had no negative effect on A. castellanii growth. Further characterization of amoebal and bacterial interactions will assist in identifying the role of Acanthamoeba in the transmission and evolution of pathogenic bacteria.

  14. Interactions between antiparkinsonian drugs and ABCB1/P-glycoprotein at the blood-brain barrier in a rat brain endothelial cell model.

    Science.gov (United States)

    Vautier, Sarah; Milane, Aline; Fernandez, Christine; Buyse, Marion; Chacun, Helene; Farinotti, Robert

    2008-09-05

    Parkinson's disease is a neurodegenerative disorder that requires treatment by dopaminergic agonists, which may be responsible for central side effects. We hypothesized that the efflux transporter ABCB1/P-glycoprotein played a role in brain disposition of antiparkinsonian drugs and could control central toxicity. We aimed to evaluate antiparkinsonian drugs as ABCB1 substrates and/or inhibitors in rat brain endothelial cells GPNT, in order to predict potential clinical drug-drug interactions. Among the antiparkinsonian drugs tested, levodopa, bromocriptine, pergolide and pramipexole were ABCB1 substrates. However, only bromocriptine could inhibit ABCB1 functionality with an IC(50) of 6.71 microM on Rhodamine 123 uptake and an IC(50) of 1.71 microM on digoxine uptake. Thus, bromocriptine at 100 microM is responsible for an increase of levodopa intracellular transport of about 2.05-fold versus control. Therefore, we can conclude that bromocriptine is a potent drug for medicinal interactions in vitro. Hence, in patients with Parkinson's disease, these results may be considered to optimise treatments individually.

  15. Induction of heme oxygenase-1 attenuates lipopolysaccharide-induced cyclooxygenase-2 expression in mouse brain endothelial cells

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    Yang Chuen-Mao

    2010-11-01

    Full Text Available Abstract Background Prostaglandin E2 (PGE2, an arachidonic acid metabolite converted by cyclooxygenase-2 (COX-2, plays important roles in the regulation of endothelial functions in response to bacterial infection. The enzymatic activity of COX-2 can be down-regulated by heme oxygenase-1 (HO-1 induction. However, the mechanisms underlying HO-1 modulating COX-2 protein expression are not known. Objective The aim of the present study was to investigate whether the up-regulation of HO-1 regulates COX-2 expression induced by lipopolysaccharide (LPS, an endotoxin produced by Gram negative bacteria, in mouse brain endothelial cells (bEnd.3 Methods Cultured bEnd.3 cells were used to investigate LPS-induced COX-2 expression and PGE2 production. Cobalt protoporphyrin IX (CoPP, an HO-1 inducer, infection with a recombinant adenovirus carried with HO-1 gene (Adv-HO-1, or zinc protoporphyrin (ZnPP, an HO-1 inhibitor was used to stimulate HO-1 induction or inhibit HO-1 activity. The expressions of COX-2 and HO-1 were evaluated by western blotting. PGE2 levels were detected by an enzyme-linked immunoassay. Hemoglobin (a chelator of carbon monoxide, CO, one of metabolites of HO-1 and CO-RM2 (a CO releasing molecule were used to investigate the mechanisms of HO-1 regulating COX-2 expression. Results We found that LPS-induced COX-2 expression and PGE2 production were mediated through NF-κB (p65 via activation of Toll-like receptor 4 (TLR4. LPS-induced COX-2 expression was inhibited by HO-1 induction by pretreatment with CoPP or infection with Adv-HO-1. This inhibitory effect of HO-1 was reversed by pretreatment with either ZnPP or hemoglobin. Pretreatment with CO-RM2 also inhibited TLR4/MyD88 complex formation, NF-κB (p65 activation, COX-2 expression, and PGE2 production induced by LPS. Conclusions We show here a novel inhibition of HO-1 on LPS-induced COX-2/PGE2 production in bEnd.3. Our results reinforce the emerging role of cerebral endothelium-derived HO-1

  16. The Physiochemistry of Capped Nanosilver Predicts Its Biological Activity in Rat Brain Endothelial Cells (REBEC4)

    Science.gov (United States)

    The “capping” or coating of nanosilver (nanoAg) extends its potency by limiting its oxidation and aggregation and stabilizing its size and shape. The ability of such coated nanoAg to alter the permeability and activate oxidative stress pathways in rat brain endothelia...

  17. HCdc14A is involved in cell cycle regulation of human brain vascular endothelial cells following injury induced by high glucose, free fatty acids and hypoxia.

    Science.gov (United States)

    Su, Jingjing; Zhou, Houguang; Tao, Yinghong; Guo, Zhuangli; Zhang, Shuo; Zhang, Yu; Huang, Yanyan; Tang, Yuping; Hu, Renming; Dong, Qiang

    2015-01-01

    Cell cycle processes play a vital role in vascular endothelial proliferation and dysfunction. Cell division cycle protein 14 (Cdc14) is an important cell cycle regulatory phosphatase. Previous studies in budding yeast demonstrated that Cdc14 could trigger the inactivation of mitotic cyclin-dependent kinases (Cdks), which are required for mitotic exit and cytokinesis. However, the exact function of human Cdc14 (hCdc14) in cell cycle regulation during vascular diseases is yet to be elucidated. There are two HCdc14 homologs: hCdc14A and hCdc14B. In the current study, we investigated the potential role of hCdc14A in high glucose-, free fatty acids (FFAs)-, and hypoxia-induced injury in cultured human brain vascular endothelial cells (HBVECs). Data revealed that high glucose, FFA, and hypoxia down-regulated hCdc14A expression remarkably, and also affected the expression of other cell cycle-related proteins such as cyclin B, cyclin D, cyclin E, and p53. Furthermore, the combined addition of the three stimuli largely blocked cell cycle progression, decreased cell proliferation, and increased apoptosis. We also determined that hCdc14A was localized mainly to centrosomes during interphase and spindles during mitosis using confocal microscopy, and that it could affect the expression of other cycle-related proteins. More importantly, the overexpression of hCdc14A accelerated cell cycle progression, enhanced cell proliferation, and promoted neoplastic transformation, whereas the knockdown of hCdc14A using small interfering RNA produced the opposite effects. Therefore, these findings provide novel evidence that hCdc14A might be involved in cell cycle regulation in cultured HBVECs during high glucose-, FFA-, and hypoxia-induced injury.

  18. Endothelial cells derived from the blood-brain barrier and islets of Langerhans differ in their response to the effects of bilirubin on oxidative stress under hyperglycemic conditions

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    Jaime eKapitulnik

    2012-07-01

    Full Text Available Unconjugated bilirubin (UCB is a neurotoxic degradation product of heme. Its toxic effects include induction of apoptosis, and ultimately neuronal cell death. However, at low concentrations, UCB is a potent antioxidant that may protect cells and tissues against oxidative stress by neutralizing toxic metabolites such as reactive oxygen species (ROS. High glucose levels (hyperglycemia generate reactive metabolites. Endothelial cell dysfunction, an early vascular complication in diabetes, has been associated with hyperglycemia-induced oxidative stress. Both glucose and UCB are substrates for transport proteins in microvascular endothelial cells of the blood-brain barrier (BBB. In the current study we show that UCB (1-40 M induces apoptosis and reduces survival of bEnd3 cells, a mouse brain endothelial cell line which serves as an in vitro model of the BBB. These deleterious effects of UCB were enhanced in the presence of high glucose (25 mM levels. Interestingly, the bEnd3 cells exhibited an increased sensitivity to the apoptotic effects of UCB when compared to the MS1 microcapillary endothelial cell line. MS1 cells originate from murine pancreatic islets of Langherans, and are devoid of the barrier characteristics of BBB-derived endothelial cells. ROS production was increased in both bEnd3 and MS1 cells exposed to high glucose, as compared with cells exposed to normal (5.5 mM glucose levels. While UCB (0.1-40 M did not alter ROS production in cells exposed to normal glucose, relatively low ('physiological' UCB concentrations (0.1-5 M attenuated ROS generation in both cell lines exposed to high glucose levels. Most strikingly, higher UCB concentrations (20-40 M increased ROS generation in bEnd3 cells exposed to high glucose, but not in similarly treated MS1 cells. These results may be of critical importance for understanding the vulnerability of the BBB endothelium upon exposure to increasing UCB levels under hyperglycemic conditions.

  19. Murine cerebrovascular cells as a cell culture model for cerebral amyloid angiopathy: isolation of smooth muscle and endothelial cells from mouse brain.

    Science.gov (United States)

    Gauthier, Sebastien A; Sahoo, Susmita; Jung, Sonia S; Levy, Efrat

    2012-01-01

    The use of murine cerebrovascular endothelial and smooth muscle cells has not been widely employed as a cell culture model for the investigation of cellular mechanisms involved in cerebral amyloid angiopathy (CAA). Difficulties in isolation and propagation of murine cerebrovascular cells and insufficient yields for molecular and cell culture studies have deterred investigators from using mice as a source for cerebrovascular cells in culture. Instead, cerebrovascular cells from larger mammals are preferred and several methods describing the isolation of endothelial and smooth muscle cells from human, canine, rat, and guinea pig have been published. In recent years, several transgenic mouse lines showing CAA pathology have been established; consequently murine cerebrovascular cells derived from these animals can serve as a key cellular model to study CAA. Here, we describe a procedure for isolating murine microvessels that yields healthy smooth muscle and endothelial cell populations and produce sufficient material for experimental purposes. Murine smooth muscle cells isolated using this protocol exhibit the classic "hill and valley" morphology and are immunoreactive for the smooth muscle cell marker α-actin. Endothelial cells display a "cobblestone" pattern phenotype and show the characteristic immunostaining for the von Willebrand factor and the factor VIII-related antigen. In addition, we describe methods designed to preserve these cells by storage in liquid nitrogen and reestablishing viable cell cultures. Finally, we compare our methods with protocols designed to isolate and maintain human cerebrovascular cell cultures.

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

    OpenAIRE

    Haqqani Arsalan S; Delaney Christie E; Tremblay Tammy-Lynn; Sodja Caroline; Sandhu Jagdeep K; Stanimirovic Danica B

    2013-01-01

    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 ...

  1. 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.

  2. Sodium-dependent vitamin C transporter 2 (SVCT2 expression and activity in brain capillary endothelial cells after transient ischemia in mice.

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    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.

  3. Matrix metalloproteinase 2 (MMP-2) degrades soluble vasculotropic amyloid-beta E22Q and L34V mutants, delaying their toxicity for human brain microvascular endothelial cells.

    Science.gov (United States)

    Hernandez-Guillamon, Mar; Mawhirt, Stephanie; Fossati, Silvia; Blais, Steven; Pares, Mireia; Penalba, Anna; Boada, Merce; Couraud, Pierre-Olivier; Neubert, Thomas A; Montaner, Joan; Ghiso, Jorge; Rostagno, Agueda

    2010-08-27

    Patients carrying mutations within the amyloid-beta (Abeta) sequence develop severe early-onset cerebral amyloid angiopathy with some of the related variants manifesting primarily with hemorrhagic phenotypes. Matrix metalloproteases (MMPs) are typically associated with blood brain barrier disruption and hemorrhagic transformations after ischemic stroke. However, their contribution to cerebral amyloid angiopathy-related hemorrhage remains unclear. Human brain endothelial cells challenged with Abeta synthetic homologues containing mutations known to be associated in vivo with hemorrhagic manifestations (AbetaE22Q and AbetaL34V) showed enhanced production and activation of MMP-2, evaluated via Multiplex MMP antibody arrays, gel zymography, and Western blot, which in turn proteolytically cleaved in situ the Abeta peptides. Immunoprecipitation followed by mass spectrometry analysis highlighted the generation of specific C-terminal proteolytic fragments, in particular the accumulation of Abeta-(1-16), a result validated in vitro with recombinant MMP-2 and quantitatively evaluated using deuterium-labeled internal standards. Silencing MMP-2 gene expression resulted in reduced Abeta degradation and enhanced apoptosis. Secretion and activation of MMP-2 as well as susceptibility of the Abeta peptides to MMP-2 degradation were dependent on the peptide conformation, with fibrillar elements of AbetaE22Q exhibiting negligible effects. Our results indicate that MMP-2 release and activation differentially degrades Abeta species, delaying their toxicity for endothelial cells. However, taking into consideration MMP ability to degrade basement membrane components, these protective effects might also undesirably compromise blood brain barrier integrity and precipitate a hemorrhagic phenotype.

  4. Low intracellular ATP levels exacerbate carcinogen-induced inflammatory stress response and inhibit in vitro tubulogenesis in human brain endothelial cells

    Directory of Open Access Journals (Sweden)

    Elizabeth Tahanian

    2011-01-01

    Full Text Available Elizabeth Tahanian, Sabrina Peiro, Borhane AnnabiLaboratoire d'Oncologie Moléculaire, Centre de Recherche BioMED, Département de Chimie, Université du Québec à Montréal, Montréal, Québec, CanadaAbstract: Solid tumor development requires angiogenesis and is correlated to the expression of inflammatory markers through cellular metabolic and energetic adaptation. While high glycolysis rates enable the cancer cell compartment to generate adenosine triphosphate (ATP, very little is known about the impact of low intracellular ATP concentrations within the vascular endothelial cell compartment, which is responsible for tumor angiogenesis. Here, we investigated the effect of 2-deoxy-D-glucose (2-DG, a glucose analog that inhibits glycolysis through intracellular ATP depletion, on human brain microvascular endothelial cell (HBMEC angiogenic properties. While preformed capillaries remained unaffected, we found that in vitro tubulogenesis was dose-dependently decreased by 2-DG and that this correlated with reduced intracellular ATP levels. Procarcinogenic signaling was induced with phorbol 12-myristate 13-acetate (PMA and found to trigger the proinflammatory marker cyclooxygenase-2 (COX-2 and endoplasmic reticulum (ER stress marker GRP78 expression, whose inductions were potentiated when PMA was combined with 2-DG treatment. Inversely, PMA-induced matrix-metalloproteinase-9 (MMP-9 gene expression and protein secretion were abrogated in the presence of 2-DG, and this can be partially explained by reduced nuclear factor-κB signaling. Collectively, we provide evidence for an intracellular ATP requirement in order for tubulogenesis to occur, and we link increases in ER stress to inflammation. A better understanding of the metabolic adaptations of the vascular endothelial cells that mediate tumor vascularization will help the development of new drugs and therapies.Keywords: endoplasmic reticulum stress, MMP-9, COX-2, 2-deoxy-D-glucose, endothelial

  5. Transport of monocarboxylic acids at the blood-brain barrier: Studies with monolayers of primary cultured bovine brain capillary endothelial cells

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    Terasaki, T.; Takakuwa, S.; Moritani, S.; Tsuji, A. (Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Kanazawa University (Japan))

    1991-09-01

    The kinetics and mechanism of the transport of monocarboxylic acids (MCAs) were studied by using primary cultured bovine brain capillary endothelial cells. Concentration-dependent uptake of acetic acid was observed, and the kinetic parameters were estimated as follows: the Michaelis constant, Kt, was 3.41 {plus minus} 1.87 mM, the maximum uptake rate, Jmax, was 144.7 {plus minus} 55.7 nmol/mg of protein/min and the nonsaturable first-order rate constant, Kd, was 6.66 {plus minus} 1.98 microliters/mg of protein/min. At medium pH below 7.0, the uptake rate of (3H)acetic acid increased markedly with decreasing medium pH, whereas pH-independent uptake was observed in the presence of 10 mM acetic acid. An energy requirement for (3H)acetic acid uptake was also demonstrated, because metabolic inhibitors (2,4-dinitrophenol and rotenone) reduced significantly the uptake rate (P less than .05). Carbonylcyanide-p-trifluoro-methoxyphenylhydrazone, a protonophore, inhibited significantly the uptake of (3H)acetic acid at medium pH of 5.0 and 6.0, whereas 4,4{prime}-diisothiocyanostilben-2,2{prime}-disulfonic acid did not. Several MCAs inhibited significantly the uptake rate of (3H)acetic acid, whereas di- and tricarboxylic acids did not. The uptake of (3H)acetic acid was competitively inhibited by salicylic acid, with an inhibition constant, Ki, of 3.60 mM, suggesting a common transport system between acetic acid and salicylic acid. Moreover, at the medium pH of 7.4, salicylic acid and valproic acid inhibited significantly the uptake of (3H)acetic acid, demonstrating that the transport of MCA drugs could also be ascribed to the MCA transport system at the physiologic pH.

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

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    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

  7. 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.

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

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    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.

  9. 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

    2017-02-20

    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.

  10. 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

  11. Oxidative and pro-inflammatory impact of regular and denicotinized cigarettes on blood brain barrier endothelial cells: is smoking reduced or nicotine-free products really safe?

    Science.gov (United States)

    2014-01-01

    Background Both active and passive tobacco smoke (TS) potentially impair the vascular endothelial function in a causative and dose-dependent manner, largely related to the content of reactive oxygen species (ROS), nicotine, and pro-inflammatory activity. Together these factors can compromise the restrictive properties of the blood–brain barrier (BBB) and trigger the pathogenesis/progression of several neurological disorders including silent cerebral infarction, stroke, multiple sclerosis and Alzheimer’s disease. Based on these premises, we analyzed and assessed the toxic impact of smoke extract from a range of tobacco products (with varying levels of nicotine) on brain microvascular endothelial cell line (hCMEC/D3), a well characterized human BBB model. Results Initial profiling of TS showed a significant release of reactive oxygen (ROS) and reactive nitrogen species (RNS) in full flavor, nicotine-free (NF, “reduced-exposure” brand) and ultralow nicotine products. This release correlated with increased oxidative cell damage. In parallel, membrane expression of endothelial tight junction proteins ZO-1 and occludin were significantly down-regulated suggesting the impairment of barrier function. Expression of VE-cadherin and claudin-5 were also increased by the ultralow or nicotine free tobacco smoke extract. TS extract from these cigarettes also induced an inflammatory response in BBB ECs as demonstrated by increased IL-6 and MMP-2 levels and up-regulation of vascular adhesion molecules, such as VCAM-1 and PECAM-1. Conclusions In summary, our results indicate that NF and ultralow nicotine cigarettes are potentially more harmful to the BBB endothelium than regular tobacco products. In addition, this study demonstrates that the TS-induced toxicity at BBB ECs is strongly correlated to the TAR and NO levels in the cigarettes rather than the nicotine content. PMID:24755281

  12. Hypertonic saline protects brain endothelial cells against hypoxia correlated to the levels of estimated glomerular filtration rate and interleukin-1β

    Science.gov (United States)

    Chen, Sheng-Long; Deng, Yi-Yu; Wang, Qiao-Sheng; Han, Yong-Li; Jiang, Wen-Qiang; Fang, Ming; Hu, Bei; Wu, Zhi-Xin; Huang, Lin-Qiang; Zeng, Hong-Ke

    2017-01-01

    Abstract Objective: The aim of this study was to verify the protective effect of hypertonic saline (HS) on brain endothelial cells under hypoxic conditions and the relevant underlying mechanism. Methods: bEnd.3 cells were treated with oxygen-glucose deprivation (OGD)-induced injury. To measure HS performance, cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt assay, and cell apoptosis was assessed by flow cytometry and Terminal deoxynucleotidyl transferase UTP nick-end labeling staining. RNA-seq was performed to assess the expression profiles and screen the candidate genes that participated in OGD-induced injury and the HS protective effect. Quantitative real-time polymerase chain reaction (qPCR) and western blot analysis were used to confirm the expression of candidate genes, and enzyme-linked immunosorbent assay was used to measure the level of interleukin (IL)-1β. Overexpression analyses were performed to confirm the functions of the differentially expressed genes. Results: HS with a concentration of 40 mmol/L NaCl had an obvious protective effect on bEnd.3 cells after OGD-induced injury, resulting in increased cell viability and a smaller percentage of apoptotic cells. According to the RNA-seq results, epidermal growth factor receptor (EGFR) was chosen as the differentially expressed gene target in this study. The qPCR and western blot analyses further confirmed that the levels of EGFR/phosphorylated epidermal growth factor receptor and IL-1β were enhanced after OGD-induced injury, but attenuated after treatment with 40 mmol/L of NaCl HS. Overexpressed EGFR reversed the protective effect of HS that caused low viability and high rates of apoptosis in cells. Conclusion: HS can protect endothelial cells against OGD-induced injury, but is affected by the expression of EGFR/p-EGFR and IL-1β. PMID:28072729

  13. Nanomolar aluminum induces expression of the inflammatory systemic biomarker C-reactive protein (CRP) in human brain microvessel endothelial cells (hBMECs).

    Science.gov (United States)

    Alexandrov, Peter N; Kruck, Theodore P A; Lukiw, Walter J

    2015-11-01

    C-reactive protein (CRP; also known as pentraxin 1, PTX1), a 224 amino acid soluble serum protein organized into a novel pentameric ring-shaped structure, is a highly sensitive pathogenic biomarker for systemic inflammation. High CRP levels are found in practically every known inflammatory state, and elevated CRP levels indicate an increased risk for several common age-related human degenerative disorders, including cardiovascular disease, cancer, diabetes, and Alzheimer's disease (AD). While the majority of CRP is synthesized in the liver for secretion into the systemic circulation, it has recently been discovered that an appreciable amount of CRP is synthesized in highly specialized endothelial cells that line the vasculature of the brain and central nervous system (CNS). These highly specialized cells, the major cell type lining the human CNS vasculature, are known as human brain microvessel endothelial cells (hBMECs). In the current pilot study we examined (i) CRP levels in human serum obtained from AD and age-matched control patients; and (ii) analyzed the effects of nanomolar aluminum sulfate on CRP expression in primary hBMECs. The three major findings in this short communication are: (i) that CRP is up-regulated in AD serum; (ii) that CRP serum levels increased in parallel with AD progression; and (iii) for the first time show that nanomolar aluminum potently up-regulates CRP expression in hBMECs to many times its 'basal abundance'. The results suggest that aluminum-induced CRP may in part contribute to a pathophysiological state associated with a chronic systemic inflammation of the human vasculature.

  14. 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

  15. 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.

  16. Fumaric Acid Esters Do Not Reduce Inflammatory NF-κB/p65 Nuclear Translocation, ICAM-1 Expression and T-Cell Adhesiveness of Human Brain Microvascular Endothelial Cells.

    Science.gov (United States)

    Haarmann, Axel; Nehen, Mathias; Deiß, Annika; Buttmann, Mathias

    2015-08-13

    Dimethyl fumarate (DMF) is approved for disease-modifying treatment of patients with relapsing-remitting multiple sclerosis. Animal experiments suggested that part of its therapeutic effect is due to a reduction of T-cell infiltration of the central nervous system (CNS) by uncertain mechanisms. Here we evaluated whether DMF and its primary metabolite monomethyl fumarate (MMF) modulate pro-inflammatory intracellular signaling and T-cell adhesiveness of nonimmortalized single donor human brain microvascular endothelial cells at low passages. Neither DMF nor MMF at concentrations of 10 or 50 µM blocked the IL-1β-induced nuclear translocation of NF-κB/p65, whereas the higher concentration of DMF inhibited the nuclear entry of p65 in human umbilical vein endothelium cultured in parallel. DMF and MMF also did not alter the IL-1β-stimulated activation of p38 MAPK in brain endothelium. Furthermore, neither DMF nor MMF reduced the basal or IL-1β-inducible expression of ICAM-1. In accordance, both fumaric acid esters did not reduce the adhesion of activated Jurkat T cells to brain endothelium under basal or inflammatory conditions. Therefore, brain endothelial cells probably do not directly mediate a potential blocking effect of fumaric acid esters on the inflammatory infiltration of the CNS by T cells.

  17. Fumaric Acid Esters Do Not Reduce Inflammatory NF-κB/p65 Nuclear Translocation, ICAM-1 Expression and T-Cell Adhesiveness of Human Brain Microvascular Endothelial Cells

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    Axel Haarmann

    2015-08-01

    Full Text Available Dimethyl fumarate (DMF is approved for disease-modifying treatment of patients with relapsing-remitting multiple sclerosis. Animal experiments suggested that part of its therapeutic effect is due to a reduction of T-cell infiltration of the central nervous system (CNS by uncertain mechanisms. Here we evaluated whether DMF and its primary metabolite monomethyl fumarate (MMF modulate pro-inflammatory intracellular signaling and T-cell adhesiveness of nonimmortalized single donor human brain microvascular endothelial cells at low passages. Neither DMF nor MMF at concentrations of 10 or 50 µM blocked the IL-1β-induced nuclear translocation of NF-κB/p65, whereas the higher concentration of DMF inhibited the nuclear entry of p65 in human umbilical vein endothelium cultured in parallel. DMF and MMF also did not alter the IL-1β-stimulated activation of p38 MAPK in brain endothelium. Furthermore, neither DMF nor MMF reduced the basal or IL-1β-inducible expression of ICAM-1. In accordance, both fumaric acid esters did not reduce the adhesion of activated Jurkat T cells to brain endothelium under basal or inflammatory conditions. Therefore, brain endothelial cells probably do not directly mediate a potential blocking effect of fumaric acid esters on the inflammatory infiltration of the CNS by T cells.

  18. In Vitro Endothelialization Test of Biomaterials Using Immortalized Endothelial Cells.

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    Ken Kono

    Full Text Available Functionalizing biomaterials with peptides or polymers that enhance recruitment of endothelial cells (ECs can reduce blood coagulation and thrombosis. To assess endothelialization of materials in vitro, primary ECs are generally used, although the characteristics of these cells vary among the donors and change with time in culture. Recently, primary cell lines immortalized by transduction of simian vacuolating virus 40 large T antigen or human telomerase reverse transcriptase have been developed. To determine whether immortalized ECs can substitute for primary ECs in material testing, we investigated endothelialization on biocompatible polymers using three lots of primary human umbilical vein endothelial cells (HUVEC and immortalized microvascular ECs, TIME-GFP. Attachment to and growth on polymer surfaces were comparable between cell types, but results were more consistent with TIME-GFP. Our findings indicate that TIME-GFP is more suitable for in vitro endothelialization testing of biomaterials.

  19. Neuron-specific enolase expression in a rat model of radiation-induced brain injury following vascular endothelial growth factor-modified neural stem cell transplantation

    Institute of Scientific and Technical Information of China (English)

    Songhua Xiao; Chaohui Duan; Qingyu Shen; Yigang Xing; Ying Peng; Enxiang Tao; Jun Liu

    2009-01-01

    BACKGROUND:Previous studies have shown that transplantation of vascular endothelial growth factor (VEGF)-modified neural stem cells (NSC) provides better outcomes,compared with neural stem cells,in the treatment of brain damage.OBJECTIVE:To compare the effects of VEGF-modified NSC transplantation and NSC transplantation on radiation-induced brain injury,and to determine neuron-specific enolase (NSE) expression in the brain.DESIGN,TIME,AND SETTING:The randomized,controlled study was performed at the Linbaixin Experimental Center,Second Affiliated Hospital,Sun Yat-sen University,China from November 2007 to October 2008.MATERIALS:VEGF-medified C17.2 NSCs were supplied by Harvard Medical School,USA.Streptavidin-biotin-peroxidase-complex kit (Boster,China) and 5,6-carboxyfluorescein diacetate succinimidyl ester (Fluka,USA) were used in this study.METHODS:A total of 84 Sprague Dawley rats were randomly assigned to a blank control group (n=20),model group (n=20),NSC group (n=20),and a VEGF-modified NSC group (n=24).Rat models of radiation-induced brain injury were established in the model,NSC,and VEGF-modified NSC groups.At 1 week following model induction,10 μL (5×10~4 cells/μL) VEGF-modified NSCs or NSCs were respectively infused into the striatum and cerebral cortex of rats from the VEGF-modified NSC and NSC groups.A total of 10 μL saline was injected into rats from the blank control and model groups.MAIN OUTCOME MEASURES:NSE expression in the brain was detected by immunohistochemistry following VEGF-modified NSC transplantation.RESULTS:NSE expression was significantly decreased in the brains of radiation-induced brain injury rats (P<0.05).The number of NSE-positive neurons significantly increased in the NSC and VEGF-modified NSC groups,compared with the model group (P<0.05).NSE expression significantly increased in the VEGF-modified NSC group,compared with the NSC group,at 6 weeks following transplantation (P<0.05).CONCLUSION:VEGF-modified NSC

  20. Inflammation Modulates RLIP76/RALBP1 Electrophile-Glutathione Conjugate Transporter and Housekeeping Genes in Human Blood-Brain Barrier Endothelial Cells.

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    Barbara Bennani-Baiti

    Full Text Available Endothelial cells are often present at inflammation sites. This is the case of endothelial cells of the blood-brain barrier (BBB of patients afflicted with neurodegenerative disorders such as Alzheimer's, Parkinson's, or multiple sclerosis, as well as in cases of bacterial meningitis, trauma, or tumor-associated ischemia. Inflammation is a known modulator of gene expression through the activation of transcription factors, mostly NF-κB. RLIP76 (a.k.a. RALBP1, an ATP-dependent transporter of electrophile-glutathione conjugates, modulates BBB permeability through the regulation of tight junction function, cell adhesion, and exocytosis. Genes and pathways regulated by RLIP76 are transcriptional targets of tumor necrosis factor alpha (TNF-α pro-inflammatory molecule, suggesting that RLIP76 may also be an inflammation target. To assess the effects of TNF-α on RLIP76, we faced the problem of choosing reference genes impervious to TNF-α. Since such genes were not known in human BBB endothelial cells, we subjected these to TNF-α, and measured by quantitative RT-PCR the expression of housekeeping genes commonly used as reference genes. We find most to be modulated, and analysis of several inflammation datasets as well as a metaanalysis of more than 5000 human tissue samples encompassing more than 300 cell types and diseases show that no single housekeeping gene may be used as a reference gene. Using three different algorithms, however, we uncovered a reference geneset impervious to TNF-α, and show for the first time that RLIP76 expression is induced by TNF-α and follows the induction kinetics of inflammation markers, suggesting that inflammation can influence RLIP76 expression at the BBB. We also show that MRP1 (a.k.a. ABCC1, another electrophile-glutathione transporter, is not modulated in the same cells and conditions, indicating that RLIP76 regulation by TNF-α is not a general property of glutathione transporters. The reference geneset

  1. Inflammation Modulates RLIP76/RALBP1 Electrophile-Glutathione Conjugate Transporter and Housekeeping Genes in Human Blood-Brain Barrier Endothelial Cells.

    Science.gov (United States)

    Bennani-Baiti, Barbara; Toegel, Stefan; Viernstein, Helmut; Urban, Ernst; Noe, Christian R; Bennani-Baiti, Idriss M

    2015-01-01

    Endothelial cells are often present at inflammation sites. This is the case of endothelial cells of the blood-brain barrier (BBB) of patients afflicted with neurodegenerative disorders such as Alzheimer's, Parkinson's, or multiple sclerosis, as well as in cases of bacterial meningitis, trauma, or tumor-associated ischemia. Inflammation is a known modulator of gene expression through the activation of transcription factors, mostly NF-κB. RLIP76 (a.k.a. RALBP1), an ATP-dependent transporter of electrophile-glutathione conjugates, modulates BBB permeability through the regulation of tight junction function, cell adhesion, and exocytosis. Genes and pathways regulated by RLIP76 are transcriptional targets of tumor necrosis factor alpha (TNF-α) pro-inflammatory molecule, suggesting that RLIP76 may also be an inflammation target. To assess the effects of TNF-α on RLIP76, we faced the problem of choosing reference genes impervious to TNF-α. Since such genes were not known in human BBB endothelial cells, we subjected these to TNF-α, and measured by quantitative RT-PCR the expression of housekeeping genes commonly used as reference genes. We find most to be modulated, and analysis of several inflammation datasets as well as a metaanalysis of more than 5000 human tissue samples encompassing more than 300 cell types and diseases show that no single housekeeping gene may be used as a reference gene. Using three different algorithms, however, we uncovered a reference geneset impervious to TNF-α, and show for the first time that RLIP76 expression is induced by TNF-α and follows the induction kinetics of inflammation markers, suggesting that inflammation can influence RLIP76 expression at the BBB. We also show that MRP1 (a.k.a. ABCC1), another electrophile-glutathione transporter, is not modulated in the same cells and conditions, indicating that RLIP76 regulation by TNF-α is not a general property of glutathione transporters. The reference geneset uncovered herein should

  2. 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

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    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.

  3. G-CSF Protects Human Brain Vascular Endothelial Cells Injury Induced by High Glucose, Free Fatty Acids and Hypoxia through MAPK and Akt Signaling

    Science.gov (United States)

    Tao, Yinghong; Guo, Jingchun; Guo, Zhuangli; Zhang, Shuo; Zhang, Yu; Huang, Yanyan; Tang, Yuping; Dong, Qiang; Hu, Renming

    2015-01-01

    Granulocyte-colony stimulating factor (G-CSF) has been shown to play a neuroprotective role in ischemic stroke by mobilizing bone marrow (BM)-derived endothelial progenitor cells (EPCs), promoting angiogenesis, and inhibiting apoptosis. Impairments in mobilization and function of the BM-derived EPCs have previously been reported in animal and human studies of diabetes where there is both reduction in the levels of the BM-derived EPCs and its ability to promote angiogenesis. This is hypothesized to account for the pathogenesis of diabetic vascular complications such as stroke. Here, we sought to investigate the effects of G-CSF on diabetes-associated cerebral vascular defect. We observed that pretreatment of the cultured human brain vascular endothelial cells (HBVECs) with G-CSF largely prevented cell death induced by the combination stimulus with high glucose, free fatty acids (FFA) and hypoxia by increasing cell viability, decreasing apoptosis and caspase-3 activity. Cell ultrastructure measured by transmission electron microscope (TEM) revealed that G-CSF treatment nicely reduced combination stimulus-induced cell apoptosis. The results from fluorescent probe Fluo-3/AM showed that G-CSF greatly suppressed the levels of intracellular calcium ions under combination stimulus. We also found that G-CSF enhanced the expression of cell cycle proteins such as human cell division cycle protein 14A (hCdc14A), cyclinB and cyclinE, inhibited p53 activity, and facilitated cell cycle progression following combination stimulus. In addition, activation of extracellular signal-regulated kinase1/2 (ERK1/2) and Akt, and deactivation of c-Jun N terminal kinase (JNK) and p38 were proved to be required for the pro-survival effects of G-CSF on HBVECs exposed to combination stimulus. Overall, G-CSF is capable of alleviating HBVECs injury triggered by the combination administration with high glucose, FFA and hypoxia involving the mitogen-activated protein kinases (MAPK) and Akt signaling

  4. The permeation of dynorphin A 1-6 across the blood brain barrier and its effect on bovine brain microvessel endothelial cell monolayer permeability.

    Science.gov (United States)

    Sloan, Courtney D Kuhnline; Audus, Kenneth L; Aldrich, Jane V; Lunte, Susan M

    2012-12-01

    Dynorphin A 1-17 (Dyn A 1-17) is an endogenous neuropeptide known to act at the kappa opioid receptor; it has been implicated in a number of neurological disorders, including neuropathic pain, stress, depression, and Alzheimer's and Parkinson's diseases. The investigation of Dyn A 1-17 metabolism at the blood-brain barrier (BBB) is important since the metabolites exhibit unique biological functions compared to the parent compound. In this work, Dyn A 1-6 is identified as a metabolite of Dyn A 1-17 in the presence of bovine brain microvessel endhothelial cells (BBMECs), using LC-MS/MS. The transport of Dyn A 1-6 at the BBB was examined using this in vitro cell culture model of the BBB. Furthermore, the permeation of the BBB by the low molecular weight permeability marker fluorescein was characterized in the presence and absences of Dyn A 1-6.

  5. Induction of cyclooxygenase-2 expression during HIV-1-infected monocyte-derived macrophage and human brain microvascular endothelial cell interactions

    NARCIS (Netherlands)

    Pereira, CF; Boven, LA; Middel, J; Verhoef, J; Nottet, HSLM

    2000-01-01

    Human immunodeficiency virus type-1 (HIV-1)-associated dementia (HAD) is a neurodegenerative disease characterized by HIV infection and replication in brain tissue. HIV-1-infected monocytes overexpress inflammatory molecules that facilitate their entry into the brain. Prostanoids are lipid mediators

  6. Glutathione in Cerebral Microvascular Endothelial Biology and Pathobiology: Implications for Brain Homeostasis

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    Wei Li

    2012-01-01

    Full Text Available The integrity of the vascular endothelium of the blood-brain barrier (BBB is central to cerebrovascular homeostasis. Given the function of the BBB as a physical and metabolic barrier that buffers the systemic environment, oxidative damage to the endothelial monolayer will have significant deleterious impact on the metabolic, immunological, and neurological functions of the brain. Glutathione (GSH is a ubiquitous major thiol within mammalian cells that plays important roles in antioxidant defense, oxidation-reduction reactions in metabolic pathways, and redox signaling. The existence of distinct GSH pools within the subcellular organelles supports an elegant mode for independent redox regulation of metabolic processes, including those that control cell fate. GSH-dependent homeostatic control of neurovascular function is relatively unexplored. Significantly, GSH regulation of two aspects of endothelial function is paramount to barrier preservation, namely, GSH protection against oxidative endothelial cell injury and GSH control of postdamage cell proliferation in endothelial repair and/or wound healing. This paper highlights our current insights and hypotheses into the role of GSH in cerebral microvascular biology and pathobiology with special focus on endothelial GSH and vascular integrity, oxidative disruption of endothelial barrier function, GSH regulation of endothelial cell proliferation, and the pathological implications of GSH disruption in oxidative stress-associated neurovascular disorders, such as diabetes and stroke.

  7. Tanshinone IIA inhibits endothelin-1 production in TNF-α-induced brain microvascular endothelial cells through suppression of endothelin-converting enzyme-1 synthesis

    Institute of Scientific and Technical Information of China (English)

    Chao TANG; An-hua WU; Hong-li XUE; Yun-jie WANG

    2007-01-01

    Aim: To investigate the effects of tanshinone ⅡA (Tan ⅡA) on the regulation of the production of endothelin (ET)-1 (including large ET-1), mRNA levels of ET-1,endothelin-converting enzyme- 1 (ECE- 1), endothelin-A receptor (ETA) and endothelin-B receptor (ETB) induced by TNF-α in rat brain microvascular endo-thelial cells (BMVEC). Methods: The ET-1 release (including large ET-1) into the culture medium was determined by enzyme immunoassay. The levels of ET-1,ECE- 1, ETA, and ETB mRNA were measured by RT-PCR. Endothelin receptor bind-ing was also tested. Results: The induction of ET- 1 release by TNF-α from cul-tured BMVEC was dose-dependently reduced by Tan IIA, but large ET-1 levels progressively increased in response to Tan IIA; the mRNA expression of ET-1 was unaffected. Tan ⅡA also caused a decrease in ETA receptor mRNA and ECE-1expression in a dose-dependent manner. Endothelin receptor binding was unal-tered in BMVEC stimulated with TNF-α alone or a combination of TNF-α and Tan ⅡA. Conclusion: These findings suggest that Tan ⅡA may inhibit ET-1 produc-tion in TNF-α-induced BMVEC through the suppression of ECE-1 synthesis.

  8. Regulation of Toll-like receptor 2 interaction with Ecgp96 controls Escherichia coli K1 invasion of brain endothelial cells

    Science.gov (United States)

    Krishnan, Subramanian; Chen, Shuang; Turcatel, Gianluca; Arditi, Moshe; Prasadarao, Nemani V.

    2012-01-01

    SUMMARY 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 endothelial cells (HBMEC), whereas OmpA− E. coli upregulates TLR4 in these cells. Furthermore, infection with OmpA+ E. coli causes Ecgp96 and TLR2 translocate to the plasma membrane of HBMEC as a complex. Immunoprecipitation studies of the plasma membrane fractions from infected HBMEC reveal that the C-termini of Ecgp96 and TLR2 are critical for OmpA+ E. coli invasion. Knockdown of TLR2 using siRNA results in inefficient membrane translocation of Ecgp96 and significantly reduces invasion. In addition, the interaction of Ecgp96 and TLR2 induces a bipartite signal, one from Ecgp96 through PKC-α while the other from TLR2 through MyD88, ERK1/2 and NF-κB. This bipartite signal ultimately culminates in the efficient production of NO, which in turn promotes E. coli K1 invasion of HBMEC. PMID:22963587

  9. Citicoline induces angiogenesis improving survival of vascular/human brain microvessel endothelial cells through pathways involving ERK1/2 and insulin receptor substrate-1

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    Krupinski Jerzy

    2012-12-01

    Full Text Available Abstract Background Citicoline is one of the neuroprotective agents that have been used as a therapy in stroke patients. There is limited published data describing the mechanisms through which it acts. Methods We used in vitro angiogenesis assays: migration, proliferation, differentiation into tube-like structures in Matrigel™ and spheroid development assays in human brain microvessel endothelial cells (hCMEC/D3. Western blotting was performed on protein extraction from hCMEC/D3 stimulated with citicoline. An analysis of citicoline signalling pathways was previously studied using a Kinexus phospho-protein screening array. A staurosporin/calcium ionophore-induced apoptosis assay was performed by seeding hCMEC/D3 on to glass coverslips in serum poor medium. In a pilot in vivo study, transient MCAO in rats was carried out with and without citicoline treatment (1000 mg/Kg applied at the time of occlusion and subsequently every 3 days until euthanasia (21 days. Vascularity of the stroke-affected regions was examined by immunohistochemistry. Results Citicoline presented no mitogenic and chemotactic effects on hCMEC/D3; however, it significantly increased wound recovery, the formation of tube-like structures in Matrigel™ and enhanced spheroid development and sprouting. Citicoline induced the expression of phospho-extracellular-signal regulated kinase (ERK-1/2. Kinexus assays showed an over-expression of insulin receptor substrate-1 (IRS-1. Knock-down of IRS-1 with targeted siRNA in our hCMEC/D3 inhibited the pro-angiogenic effects of citicoline. The percentage of surviving cells was higher in the presence of citicoline. Citicoline treatment significantly increased the numbers of new, active CD105-positive microvessels following MCAO. Conclusions The findings demonstrate both a pro-angiogenic and protective effect of citicoline on hCMEC/D3 in vitro and following middle cerebral artery occlusion (MCAO in vivo.

  10. 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.

  11. Microvascular endothelial cells of the corpus luteum

    Directory of Open Access Journals (Sweden)

    Spanel-Borowski Katherina

    2003-11-01

    Full Text Available Abstract The cyclic nature of the capillary bed in the corpus luteum offers a unique experimental model to examine the life cycle of endothelial cells, involving discrete physiologically regulated steps of angiogenesis, blood vessel maturation and blood vessel regression. The granulosa cells and theca cells of the developing antral follicle and the steroidogenic cells of the corpus luteum produce and respond to angiogenic factors and vasoactive peptides. Following ovulation the neovascularization during the early stages of corpus luteum development has been compared to the rapid angiogenesis observed during tumor formation. On the other end of the spectrum, the microvascular endothelial cells are the first cells to undergo apoptosis at the onset of corpus luteum regression. Important insights on the morphology and function of luteal endothelial cells have been gained from a combination of in vitro and in vivo studies on endothelial cells. Endothelial cells communicate with cells comprising the functional unit of the corpus luteum, i.e., other vascular cells, steroidogenic cells, and immune cells. This review is designed to provide an overview of the types of endothelial cells present in the corpus luteum and their involvement in corpus luteum development and regression. Available evidence indicates that microvascular endothelial cells of the corpus luteum are not alike, and may differ during the process of angiogenesis and angioregression. The contributions of vasoactive peptides generated by the luteal endothelin-1 and the renin-angiotensin systems are discussed in context with the function of endothelial cells during corpus luteum formation and regression. The ability of two cytokines, tumor necrosis factor alpha and interferon gamma, are evaluated as paracrine mediators of endothelial cell function during angioregression. Finally, chemokines are discussed as a vital endothelial cell secretory products that contribute to the recruitment of

  12. Transferrin receptor expression and role in transendothelial transport of transferrin in cultured brain endothelial monolayers

    DEFF Research Database (Denmark)

    Hersom, Maria; Helms, Hans Christian; Pretzer, Natasia;

    2016-01-01

    across the endothelial cells by transcytosis. The aim of the present study was to investigate transferrin receptor expression and role in transendothelial transferrin transport in cultured bovine brain endothelial cell monolayers. Transferrin receptor mRNA and protein levels were investigated...... in endothelial mono-cultures and co-cultures with astrocytes, as well as in freshly isolated brain capillaries using qPCR, immunocytochemistry and Western blotting. Transendothelial transport and luminal association of holo-transferrin was investigated using [125I]holo-transferrin or [59Fe......]-transferrin. Transferrin receptor mRNA expression in all cell culture configurations was lower than in freshly isolated capillaries, but the expression slightly increased during six days of culture. The mRNA expression levels were similar in mono-cultures and co-cultures. Immunostaining demonstrated comparable transferrin...

  13. Development and Validation of an In-Cell Western for Quantifying P-Glycoprotein Expression in Human Brain Microvascular Endothelial (hCMEC/D3) Cells.

    Science.gov (United States)

    McInerney, Mitchell P; Pan, Yijun; Short, Jennifer L; Nicolazzo, Joseph A

    2017-01-05

    An in-cell western (ICW) protocol detecting the relative expression of P-glycoprotein (P-gp) in human cerebro-microvascular endothelial cells (hCMEC/D3) was developed and optimized, with the intention of improving throughput relative to western blotting (WB). For validation of the ICW protocol, hCMEC/D3 cells were incubated with known P-gp upregulators (10 μM rifampicin and 5 μM SR12813) and treated with siRNA targeted against MDR1, before measuring changes in P-gp expression, using both ICW and WB in parallel. To confirm a relationship between the detected P-gp expression and function, the uptake of the P-gp substrate rhodamine-123 was assessed following SR12813 treatment. Rifampicin and SR12813 significantly upregulated P-gp expression (1.5-fold and 1.9-fold, respectively) compared to control, as assessed by the ICW protocol. WB analysis of the same treatments revealed 1.4-fold and 1.5-fold upregulations. MDR1 siRNA reduced P-gp abundance by 20% and 35% when assessed by ICW and WB, respectively. SR12813 treatment reduced rhodamine-123 uptake by 18%, indicating that the observed changes in P-gp expression by ICW were associated with comparable functional changes. The correlation of P-gp upregulation by WB, rhodamine-123 uptake, and the ICW protocol provide validation of a new ICW method as an alternative method for quantification of P-gp in hCMEC/D3 cells.

  14. Inflammation- and tumor-induced anorexia and weight loss require MyD88 in hematopoietic/myeloid cells but not in brain endothelial or neural cells.

    Science.gov (United States)

    Ruud, Johan; Wilhelms, Daniel Björk; Nilsson, Anna; Eskilsson, Anna; Tang, Yan-Juan; Ströhle, Peter; Caesar, Robert; Schwaninger, Markus; Wunderlich, Thomas; Bäckhed, Fredrik; Engblom, David; Blomqvist, Anders

    2013-05-01

    Loss of appetite is a hallmark of inflammatory diseases. The underlying mechanisms remain undefined, but it is known that myeloid differentiation primary response gene 88 (MyD88), an adaptor protein critical for Toll-like and IL-1 receptor family signaling, is involved. Here we addressed the question of determining in which cells the MyD88 signaling that results in anorexia development occurs by using chimeric mice and animals with cell-specific deletions. We found that MyD88-knockout mice, which are resistant to bacterial lipopolysaccharide (LPS)-induced anorexia, displayed anorexia when transplanted with wild-type bone marrow cells. Furthermore, mice with a targeted deletion of MyD88 in hematopoietic or myeloid cells were largely protected against LPS-induced anorexia and displayed attenuated weight loss, whereas mice with MyD88 deletion in hepatocytes or in neural cells or the cerebrovascular endothelium developed anorexia and weight loss of similar magnitude as wild-type mice. Furthermore, in a model for cancer-induced anorexia-cachexia, deletion of MyD88 in hematopoietic cells attenuated the anorexia and protected against body weight loss. These findings demonstrate that MyD88-dependent signaling within the brain is not required for eliciting inflammation-induced anorexia. Instead, we identify MyD88 signaling in hematopoietic/myeloid cells as a critical component for acute inflammatory-driven anorexia, as well as for chronic anorexia and weight loss associated with malignant disease.

  15. 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.

  16. Do Neural Cells Communicate with Endothelial Cells via Secretory Exosomes and Microvesicles?

    Directory of Open Access Journals (Sweden)

    Neil R. Smalheiser

    2009-01-01

    Full Text Available Neurons, glial, cells, and brain tumor cells tissues release small vesicles (secretory exosomes and microvesicles, which may represent a novel mechanism by which neuronal activity could influence angiogenesis within the embryonic and mature brain. If CNS-derived vesicles can enter the bloodstream as well, they may communicate with endothelial cells in the peripheral circulation and with cells concerned with immune surveillance.

  17. Actin filament-associated protein 1 (AFAP-1) is a key mediator in inflammatory signaling-induced rapid attenuation of intrinsic P-gp function in human brain capillary endothelial cells.

    Science.gov (United States)

    Hoshi, Yutaro; Uchida, Yasuo; Tachikawa, Masanori; Ohtsuki, Sumio; Terasaki, Tetsuya

    2017-01-23

    The purpose of this study was to identify regulatory molecule(s) involved in the inflammatory signaling-induced decrease in P-glycoprotein (P-gp) efflux function at the blood-brain barrier (BBB) that may occur in brain diseases. We confirmed that in vivo P-gp efflux activity at the BBB was decreased without any change in P-gp protein expression level in a mouse model of acute inflammation induced by 3 mg/kg lipopolysaccharide. In a human BBB model cell line (human brain capillary endothelial cells; hCMEC/D3), 1-h treatment with 10 ng/mL tumor necrosis factor-α (TNF-α; an inflammatory mediator) rapidly reduced P-gp efflux activity, but had no effect on P-gp protein expression level. To clarify the non-transcriptional mechanism that causes the decrease in intrinsic efflux activity of P-gp in acute inflammation, we applied comprehensive quantitative phosphoproteomics to compare hCMEC/D3 cells treated with TNF-α and vehicle (control). Actin filament-associated protein-1 (AFAP-1), MAPK1, and transcription factor AP-1 (AP-1) were significantly phosphorylated in TNF-α-treated cells, and were selected as candidate proteins. In validation experiments, knockdown of AFAP-1 expression blocked the reduction in P-gp efflux activity by TNF-α treatment, whereas inhibition of MAPK function or knockdown of AP-1 expression did not. Quantitative targeted absolute proteomics revealed that the reduction in P-gp activity by TNF-α did not require any change in P-gp protein expression levels in the plasma membrane. Our results demonstrate that AFAP-1 is a key mediator in the inflammatory signaling-induced, translocation-independent rapid attenuation of P-gp efflux activity in human brain capillary endothelial cells.

  18. Endothelial progenitor cells in cardiovascular diseases

    Institute of Scientific and Technical Information of China (English)

    Poay; Sian; Sabrina; Lee; Kian; Keong; Poh

    2014-01-01

    Endothelial dysfunction has been associated with the development of atherosclerosis and cardiovascular diseases. Adult endothelial progenitor cells(EPCs) are derived from hematopoietic stem cells and are capable of forming new blood vessels through a process of vas-culogenesis. There are studies which report correlations between circulating EPCs and cardiovascular risk fac-tors. There are also studies on how pharmacotherapies may influence levels of circulating EPCs. In this review, we discuss the potential role of endothelial progenitor cells as both diagnostic and prognostic biomarkers. In addition, we look at the interaction between cardio-vascular pharmacotherapies and endothelial progenitor cells. We also discuss how EPCs can be used directly and indirectly as a therapeutic agent. Finally, we evalu-ate the challenges facing EPC research and how these may be overcome.

  19. Ammonia inhibits the C-type natriuretic peptide-dependent cyclic GMP synthesis and calcium accumulation in a rat brain endothelial cell line.

    Science.gov (United States)

    Konopacka, Agnieszka; Zielińska, Magdalena; Albrecht, Jan

    2008-05-01

    Recently we reported a decrease of C-type natriuretic peptide (CNP)-dependent, natriuretic peptide receptor 2 (NPR2)-mediated cyclic GMP (cGMP) synthesis in a non-neuronal compartment of cerebral cortical slices of hyperammonemic rats [Zielińska, M., Fresko, I., Konopacka, A., Felipo, V., Albrecht, J., 2007. Hyperammonemia inhibits the natriuretic peptide receptor 2 (NPR2)-mediated cyclic GMP synthesis in the astrocytic compartment of rat cerebral cortex slices. Neurotoxicology 28, 1260-1263]. Here we accounted for the possible involvement of cerebral capillary endothelial cells in this response by measuring the effect of ammonia on the CNP-mediated cGMP formation and intracellular calcium ([Ca2+]i) accumulation in a rat cerebral endothelial cell line (RBE-4). We first established that stimulation of cGMP synthesis in RBE-4 cells was coupled to protein kinase G (PKG)-mediated Ca2+ influx from the medium which was inhibited by an L-type channel blocker nimodipine. Ammonia treatment (1h, 5mM NH4Cl) evoked a substantial decrease of CNP-stimulated cGMP synthesis which was related to a decreased binding of CNP to NPR2 receptors, and depressed the CNP-dependent [Ca2+]i accumulation in these cells. Ammonia also abolished the CNP-dependent Ca2+ accumulation in the absence of Na+. In cells incubated with ammonia in the absence of Ca2+ a slight CNP-dependent increase of [Ca2+]i was observed, most likely representing Ca2+ release from intracellular stores. Depression of CNP-dependent cGMP-mediated [Ca2+]i accumulation may contribute to cerebral vascular endothelial dysfunction associated with hyperammonemia or hepatic encephalopathy.

  20. Endothelial cells derived from human embryonic stem cells

    Science.gov (United States)

    Levenberg, Shulamit; Golub, Justin S.; Amit, Michal; Itskovitz-Eldor, Joseph; Langer, Robert

    2002-04-01

    Human embryonic stem cells have the potential to differentiate into various cell types and, thus, may be useful as a source of cells for transplantation or tissue engineering. We describe here the differentiation steps of human embryonic stem cells into endothelial cells forming vascular-like structures. The human embryonic-derived endothelial cells were isolated by using platelet endothelial cell-adhesion molecule-1 (PECAM1) antibodies, their behavior was characterized in vitro and in vivo, and their potential in tissue engineering was examined. We show that the isolated embryonic PECAM1+ cells, grown in culture, display characteristics similar to vessel endothelium. The cells express endothelial cell markers in a pattern similar to human umbilical vein endothelial cells, their junctions are correctly organized, and they have high metabolism of acetylated low-density lipoprotein. In addition, the cells are able to differentiate and form tube-like structures when cultured on matrigel. In vivo, when transplanted into SCID mice, the cells appeared to form microvessels containing mouse blood cells. With further studies, these cells could provide a source of human endothelial cells that could be beneficial for potential applications such as engineering new blood vessels, endothelial cell transplantation into the heart for myocardial regeneration, and induction of angiogenesis for treatment of regional ischemia.

  1. THE RELATIONSHIP BETWEEN PERITUMORAL BRAIN EDEMA AND VASCULAR ENDOTHELIAL GROWTH FACTOR EXPRESSION IN PATIENTS WITH MENINGIOMA

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective To determine whether VEGF plays a role in the development of peritumoral brain edema. Methods 50 meningioma patients and their VEGF expression were studied. We took a mono- clonal antibody from mouse to VEGF to stain the tumor cells, the vascular endothelial cells and the interstitial cells. The severity of brain edema was evaluated according to CT or MR scans by the following equation: edema index = Vtumor+edema/Vtumor. The relationship between VEGF expression and edema index was analyzed statisti- cally. Results VEGF was expressed in meningioma tumor cells, which is usually concentrated at the pe- ripheral sites of the tumor. There was a positive linear correlation between the expression and the brain edema index. Conclusion VEGF may play a role in the development of peritumoral brain edema in meningioma patient.

  2. Salvianolic acid B improves the disruption of high glucose-mediated brain microvascular endothelial cells via the ROS/HIF-1α/VEGF and miR-200b/VEGF signaling pathways.

    Science.gov (United States)

    Yang, Ming-Chao; You, Fu-Li; Wang, Zhe; Liu, Xiang-Nan; Wang, Yan-Feng

    2016-09-06

    The study investigated the roles and mechanisms of Salvianolic acid B (Sal B) on permeability of rat brain microvascular endothelial cells (RBMECs) exposed to high glucose. The results demonstrated that Sal B greatly up-regulated the expression of tight junction (TJ) proteins and decreased the permeability of RBMECs compared with the control group. And the increase of reactive oxidative species (ROS) production, the upregulation of hypoxia-inducible factor-1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF) protein induced by high glucose were antagonized by Sal B. In addition, a great decrease of microRNA-200b (miR-200b) was observed in the RBMECs under high-glucose condition, which was significantly increased by Sal B pretreatment. And overexpression of miR-200b markedly attenuated the RBMECs permeability and inhibited the expression of VEGF protein by targeting with 3'-UTR of its mRNA. This led to the conclusion that Sal B-mediated improvement of blood-brain barrier dysfunction induced by high-glucose is related to the ROS/HIF-1α/VEGF and miR-200b/VEGF signaling pathways.

  3. Endothelial cell promotion of early liver and pancreas development.

    Science.gov (United States)

    Freedman, Deborah A; Kashima, Yasushige; Zaret, Kenneth S

    2007-01-01

    Different steps of embryonic pancreas and liver development require inductive signals from endothelial cells. During liver development, interactions between newly specified hepatic endoderm cells and nascent endothelial cells are crucial for the endoderm's subsequent growth and morphogenesis into a liver bud. Reconstitution of endothelial cell stimulation of hepatic cell growth with embryonic tissue explants demonstrated that endothelial signalling occurs independent of the blood supply. During pancreas development, midgut endoderm interactions with aortic endothelial cells induce Ptf1a, a crucial pancreatic determinant. Endothelial cells also have a later effect on pancreas development, by promoting survival of the dorsal mesenchyme, which in turn produces factors supporting pancreatic endoderm. A major goal of our laboratory is to determine the endothelial-derived molecules involved in these inductive events. Our data show that cultured endothelial cells induce Ptf1a in dorsal endoderm explants lacking an endogenous vasculature. We are purifying endothelial cell line product(s) responsible for this effect. We are also identifying endothelial-responsive regulatory elements in genes such as Ptf1a by genetic mapping and chromatin-based assays. These latter approaches will allow us to track endothelial-responsive signal pathways from DNA targets within progenitor cells. The diversity of organogenic steps dependent upon endothelial cell signalling suggests that cross-regulation of tissue development with its vasculature is a general phenomenon.

  4. EGb761 provides a protective effect against Aβ1-42 oligomer-induced cell damage and blood-brain barrier disruption in an in vitro bEnd.3 endothelial model.

    Directory of Open Access Journals (Sweden)

    Wen-bin Wan

    Full Text Available Alzheimer's disease (AD is the most common form of senile dementia which is characterized by abnormal amyloid beta (Aβ accumulation and deposition in brain parenchyma and cerebral capillaries, and leads to blood-brain barrier (BBB disruption. Despite great progress in understanding the etiology of AD, the underlying pathogenic mechanism of BBB damage is still unclear, and no effective treatment has been devised. The standard Ginkgo biloba extract EGb761 has been widely used as a potential cognitive enhancer for the treatment of AD. However, the cellular mechanism underlying the effect remain to be clarified. In this study, we employed an immortalized endothelial cell line (bEnd.3 and incubation of Aβ(1-42 oligomer, to mimic a monolayer BBB model under conditions found in the AD brain. We investigated the effect of EGb761 on BBB and found that Aβ1-42 oligomer-induced cell injury, apoptosis, and generation of intracellular reactive oxygen species (ROS, were attenuated by treatment with EGb761. Moreover, treatment of the cells with EGb761 decreased BBB permeability and increased tight junction scaffold protein levels including ZO-1, Claudin-5 and Occludin. We also found that the Aβ(1-42 oligomer-induced upregulation of the receptor for advanced glycation end-products (RAGE, which mediates Aβ cytotoxicity and plays an essential role in AD progression, was significantly decreased by treatment with EGb761. To our knowledge, we provide the first direct in vitro evidence of an effect of EGb761 on the brain endothelium exposed to Aβ(1-42 oligomer, and on the expression of tight junction (TJ scaffold proteins and RAGE. Our results provide a new insight into a possible mechanism of action of EGb761. This study provides a rational basis for the therapeutic application of EGb761 in the treatment of AD.

  5. 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

  6. Isolation, Characterization, and Transplantation of Cardiac Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Busadee Pratumvinit

    2013-01-01

    due to difficulties in isolation, cell heterogeneity, lack of specific markers to identify myocardial endothelial cells, and inadequate conditions to maintain long-term cultures. Herein, we developed a method for isolation, characterization, and expansion of cardiac endothelial cells applicable to study endothelial cell biology and clinical applications such as neoangiogenesis. First, we dissociated the cells from murine heart by mechanical disaggregation and enzymatic digestion. Then, we used flow cytometry coupled with specific markers to isolate endothelial cells from murine hearts. CD45+ cells were gated out to eliminate the hematopoietic cells. CD31+/Sca-1+ cells were isolated as endothelial cells. Cells isolated from atrium grew faster than those from ventricle. Cardiac endothelial cells maintain endothelial cell function such as vascular tube formation and acetylated-LDL uptake in vitro. Finally, cardiac endothelial cells formed microvessels in dorsal matrigel plug and engrafted in cardiac microvessels following intravenous and intra-arterial injections. In conclusion, our multicolor flow cytometry method is an effective method to analyze and purify endothelial cells from murine heart, which in turn can be ex vivo expanded to study the biology of endothelial cells or for clinical applications such as therapeutic angiogenesis.

  7. Effects of vascular endothelial growth factor on angiogenesis of the endothelial cells isolated from cavernous malformations

    Institute of Scientific and Technical Information of China (English)

    TAN YuZhen; ZHAO Yao; WANG HaiJie; ZHOU LiangFu; MAO Ying; LIU Rui; SHU Jia; WANG YongFei

    2008-01-01

    Human cerebral cavernous malformation (CM) is a common vascular malformation of the central nervous system. We have investigated the biological characteristics of CM endothelial cells and the cellular and molecular mechanisms of CM angiogenesis to offer new insights into exploring effective measures for treatment of this disease. The endothelial cells were isolated from CM tissue masses dissected during operation and expanded in vitro. Expression of VEGFR-1 and VEGFR-2 was examined with immunocytochemical staining. Proliferation, migration and tube formation of CM endothelial cells were determined using MTT, wounding and transmigration assays, and three-dimensional collagen type Ⅰ gel respectively. The endothelial cells were successfully isolated from the tissue specimens of 25 CMs dissected without dipolar electrocoagulation. The cells show the general characteristics of the vascular endothelial cells. Expression of VEGFR-1 and VEGFR-2 on the cells is higher than that on the normal cerebral microvascular endothelial cells. After treatment with VEGF, numbers of the proliferated and migrated cells, the maximal distance of cell migration and the length and area of capillary-like struc-tures formed in the three-dimensional collagen gel increase significantly. These results demonstrate that expression of VEGFR-1 and VEGFR-2 on CM endothelial cells is up-regulated. By binding to re-ceptors, VEGF may activate the downstream signaling pathways and promote proliferation, migration and tube formation of CM endothelial cells. VEGF/VEGFR signaling pathways play important regulating roles in CM angiogenesis.

  8. Transition of mesenchymal stem/stromal cells to endothelial cells

    NARCIS (Netherlands)

    M. Crisan (Mihaela)

    2013-01-01

    textabstractMesenchymal stem/stromal cells (MSCs) are heterogeneous. A fraction of these cells constitute multipotent cells that can self-renew and mainly give rise to mesodermal lineage cells such as adipocytes, osteocytes and chondrocytes. The ability of MSCs to differentiate into endothelial cell

  9. Amyloid β induces adhesion of erythrocytes to endothelial cells and affects endothelial viability and functionality.

    Science.gov (United States)

    Nakagawa, Kiyotaka; Kiko, Takehiro; Kuriwada, Satoko; Miyazawa, Taiki; Kimura, Fumiko; Miyazawa, Teruo

    2011-01-01

    It has been suggested that amyloid β-peptide (Aβ) might mediate the adhesion of erythrocytes to the endothelium which could disrupt the properties of endothelial cells. We provide evidence here that Aβ actually induced the binding of erythrocytes to endothelial cells and decreased endothelial viability, perhaps by the generation of oxidative and inflammatory stress. These changes are likely to contribute to the pathogenesis of Alzheimer's disease.

  10. Adhesion of endothelial cells and endothelial progenitor cells on peptide-linked polymers in shear flow.

    Science.gov (United States)

    Wang, Xin; Cooper, Stuart

    2013-05-01

    The initial adhesion of human umbilical vein endothelial cells (HUVECs), cord blood endothelial colony-forming cells (ECFCs), and human blood outgrowth endothelial cells (HBOECs) was studied under radial flow conditions. The surface of a variable shear-rate device was either coated with polymer films or covered by synthetic fibers. Spin-coating was applied to produce smooth polymer films, while fibrous scaffolds were generated by electrospinning. The polymer was composed of hexyl methacrylate, methyl methacrylate, poly(ethylene glycol) methacrylate (PEGMA), and CGRGDS peptide. The peptide was incorporated into the polymer system by coupling to an acrylate-PEG-N-hydroxysuccinimide comonomer. A shear-rate-dependent increase of the attached cells with time was observed with all cell types. The adhesion of ECs increased on RGD-linked polymer surfaces compared to polymers without adhesive peptides. The number of attached ECFCs and HBOECs are significantly higher than that of HUVECs within the entire shear-rate range and surfaces examined, especially on RGD-linked polymers at low shear rates. Their superior adhesion ability of endothelial progenitor cells under flow conditions suggests they are a promising source for in vivo seeding of vascular grafts and shows the potential to be used for self-endothelialized implants.

  11. 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

    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...... pg/mL) compared with HEX (77 ± 28 pg/mL; P = .02) and FFP (110 ± 28 pg/mL; P = .09), as was PF 1 + 2 in the brain when compared with FFP (PF 1 + 2, 89 ± 46 vs 37 ± 14 ng/mL; P = .035). Brain E-selectin was greater in the NS group compared with FFP (3.36 ± 0.02 vs 3.31 ± 0.01 ng/mL; P = .029...

  12. 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

  13. Fullerene derivatives protect endothelial cells against NO-induced damage

    Energy Technology Data Exchange (ETDEWEB)

    Lao Fang; Han Dong; Qu Ying; Liu Ying; Zhao Yuliang; Chen Chunying [CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology (NCNST), Beijing 100190 (China); Li Wei [CAS Key Laboratory for Nuclear Analytical Techniques, Institute of High Energy Physics (IHEP), Chinese Academy of Sciences, Beijing 100049 (China)], E-mail: chenchy@nanoctr.cn

    2009-06-03

    Functional fullerene derivatives have been demonstrated with potent antioxidation properties. Nitric oxide (NO) is a free radical that plays a part in leading to brain damage when it is accumulated to a high concentration. The possible scavenging activity of NO by the hydroxylated fullerene derivative C{sub 60}(OH){sub 22} and malonic acid derivative C{sub 60}(C(COOH){sub 2}){sub 2} was investigated using primary rat brain cerebral microvessel endothelial cells (CMECs). Results demonstrate that sodium nitroprusside (SNP), used as an NO donor, caused a marked decrease in cell viability and an increase in apoptosis. However, fullerene derivatives can remarkably protect against the apoptosis induced by NO assault. In addition, fullerene derivatives can also prevent NO-induced depolymerization of cytoskeleton and damage of the nucleus and accelerate endothelial cell repair. Further investigation shows that the sudden increase of the intercellular reactive oxygen species (ROS) induced by NO was significantly attenuated by post-treatment with fullerene derivatives. Our results suggest that functional fullerene derivatives are potential applications for NO-related disorders.

  14. Endothelial progenitor cells with Alzheimer's disease

    Institute of Scientific and Technical Information of China (English)

    KONG Xiao-dong; ZHANG Yun; LIU Li; SUN Ning; ZHANG Ming-yi; ZHANG Jian-ning

    2011-01-01

    Background Endothelial dysfunction is thought to be critical events in the pathogenesis of Alzheimer's disease (AD).Endothelial progenitor cells (EPCs) have provided insight into maintaining and repairing endothelial function. To study the relation between EPCs and AD, we explored the number of circulating EPCs in patients with AD.Methods A total of 104 patients were recruited from both the outpatients and inpatients of the geriatric neurology department at General Hospital, rianjin Medical University. Consecutive patients with newly diagnosed AD (n=30),patients with vascular dementia (VaD, n=34), and healthy elderly control subjects with normal cognition (n=40) were enrolled after matching for age, gender, body mass index, medical history, current medication and Mini Mental State Examination. Middle cerebral artery flow velocity was examined with transcranial Doppler. Endothelial function was evaluated according to the level of EPCs, and peripheral blood EPCs was counted by flow cytometry.Results There were no significant statistical differences of clinical data in AD, VaD and control groups (P >0.05). The patients with AD showed decreased CD34-positive (CD34+) or CD133-positive (CD133+) levels compared to the control subjects, but there were no significant statistical differences in patients with AD. The patients with AD had significantly lower CD34+CD133+ EPCs(CD34 and CD133 double positive endothelial progenitor cells) than the control subjects (P <0.05). In the patients with AD, a lower CD34+CD133+ EPCs count was independently associated with a lower Mini-Mental State Examination score (r=0.514, P=0.004). Patients with VaD also showed a significant decrease in CD34+CD133+ EPCs levels, but this was not evidently associated with the Mini-Mental State Examination score. The changes of middle cerebral artery flow velocity were similar between AD and VaD. Middle cerebral artery flow velocity was decreased in the AD and VaD groups and significantly lower than

  15. In vitro inhibition of protease-activated receptors 1, 2 and 4 demonstrates that these receptors are not involved in an Acanthamoeba castellanii keratitis isolate-mediated disruption of the human brain microvascular endothelial cells.

    Science.gov (United States)

    Iqbal, Junaid; Naeem, Komal; Siddiqui, Ruqaiyyah; Khan, Naveed Ahmed

    2014-11-01

    Granulomatous amoebic encephalitis is a rare but serious human disease leading almost always to death. The pathophysiology of amoebic encephalitis is better understood, while events leading to the constitution of brain infection are largely unknown. Traversal of the blood-brain barrier is a key step in amoebae invasion of the central nervous system and facilitated by amoebic extracellular proteases. By using specific inhibitors of protease-activated receptors 1, 2 and 4, here we studied the role of these host receptors in Acanthamoeba castellanii-mediated damage to human brain microvasculature endothelial cells (HBMEC), which constitute the blood-brain barrier. The primary HBMEC were incubated with A. castellanii-conditioned medium in the presence or absence of FR-171113 (selective inhibitor of protease-activated receptor 1), FSLLRY-NH2 (inhibitor of protease-activated receptor 2), and tcY-NH2 (inhibitor of protease-activated receptor 4). The HBMEC monolayer disruptions were assessed by microscopy using Eosin staining, while host cell cytotoxicity was determined by measuring the release of cytoplasmic lactate dehydrogenase. Zymographic assays were performed to determine the effects of inhibitors of protease-activated receptors on the extracellular proteolytic activities of A. castellanii. A. castellanii-conditioned medium produced severe HBMEC monolayer disruptions within 60 min. The selective inhibitors of protease-activated receptors tested did not affect HBMEC monolayer disruptions. On the contrary, pre-treatment of A. castellanii-conditioned medium with phenylmethylsulfonyl fluoride, a serine protease inhibitor, or heating for 10 min at 95°C abolished HBMEC monolayer disruptions. Additionally, inhibitors of protease-activated receptors tested, failed to block A. castellanii-mediated HBMEC cytotoxicity and did not affect extracellular proteolytic activities of A. castellanii. Protease-activated receptors 1, 2 and 4 do not appear to play a role in A. castellanii

  16. Stathmin expression in glioma-derived microvascular endothelial cells: a novel therapeutic target.

    Science.gov (United States)

    Dong, Baijing; Mu, Luyan; Qin, Xiangying; Qiao, Wanchen; Liu, Xiaodong; Yang, Liming; Xue, Li; Rainov, Nikolai G; Liu, Xiaoqian

    2012-03-01

    The purpose of this study was to investigate stathmin expression and its mechanisms of action in GDMEC. Microvascular endothelial cells were isolated from human gliomas (n=68) and normal brain specimans (n=20), and purified by magnetic beads coated with anti-CD105 antibody. The expression of stathmin mRNA and protein were detected by RT-PCR and western blotting, respectively. Stathmin expression was silenced by application of specific siRNA in high grade GDMEC. The proliferation, apoptosis and invasion behavior of GDMEC were investigated. The stathmin positive rate of endothelial cells in normal brain, grade I-II glioma and grade III-IV glioma was 20, 66 and 95.5%, respectively (Pstathmin, cell viability was reduced, the apoptosis rate increased and the migration of vascular endothelial cells was suppressed significantly (Pstathmin suppressed neoangiogenesis of glioma and provides a potential target for glioma treatment.

  17. Endothelial cells, tissue factor and infectious diseases

    Directory of Open Access Journals (Sweden)

    Lopes-Bezerra L.M.

    2003-01-01

    Full Text Available Tissue factor is a transmembrane procoagulant glycoprotein and a member of the cytokine receptor superfamily. It activates the extrinsic coagulation pathway, and induces the formation of a fibrin clot. Tissue factor is important for both normal homeostasis and the development of many thrombotic diseases. A wide variety of cells are able to synthesize and express tissue factor, including monocytes, granulocytes, platelets and endothelial cells. Tissue factor expression can be induced by cell surface components of pathogenic microorganisms, proinflammatory cytokines and membrane microparticles released from activated host cells. Tissue factor plays an important role in initiating thrombosis associated with inflammation during infection, sepsis, and organ transplant rejection. Recent findings suggest that tissue factor can also function as a receptor and thus may be important in cell signaling. The present minireview will focus on the role of tissue factor in the pathogenesis of septic shock, infectious endocarditis and invasive aspergillosis, as determined by both in vivo and in vitro models.

  18. Production of soluble Neprilysin by endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Kuruppu, Sanjaya, E-mail: Sanjaya.Kuruppu@monash.edu [Department of Biochemistry and Molecular Biology, Building 77, Monash University, Wellington Rd, Clayton, Vic 3800 (Australia); Rajapakse, Niwanthi W. [Department of Physiology, Building 13F, Monash University, Wellington Rd, Clayton, Vic 3800 (Australia); Minond, Dmitriy [Torrey Pines Institute for Molecular Studies, 11350 SW Village Parkway, Port Saint Lucie, FL 34987 (United States); Smith, A. Ian [Department of Biochemistry and Molecular Biology, Building 77, Monash University, Wellington Rd, Clayton, Vic 3800 (Australia)

    2014-04-04

    Highlights: • A soluble full-length form of Neprilysin exists in media of endothelial cells. • Exosomal release is the key mechanism for the production of soluble Neprilysin. • Inhibition of ADAM-17 by specific inhibitors reduce Neprilysin release. • Exosome mediated release of Neprilysin is dependent on ADAM-17 activity. - Abstract: A non-membrane bound form of Neprilysin (NEP) with catalytic activity has the potential to cleave substrates throughout the circulation, thus leading to systemic effects of NEP. We used the endothelial cell line Ea.hy926 to identify the possible role of exosomes and A Disintegrin and Metalloprotease 17 (ADAM-17) in the production of non-membrane bound NEP. Using a bradykinin based quenched fluorescent substrate (40 μM) assay, we determined the activity of recombinant human NEP (rhNEP; 12 ng), and NEP in the media of endothelial cells (10% v/v; after 24 h incubation with cells) to be 9.35 ± 0.70 and 6.54 ± 0.41 μmols of substrate cleaved over 3 h, respectively. The presence of NEP in the media was also confirmed by Western blotting. At present there are no commercially available inhibitors specific for ADAM-17. We therefore synthesised two inhibitors TPI2155-14 and TPI2155-17, specific for ADAM-17 with IC{sub 50} values of 5.36 and 4.32 μM, respectively. Treatment of cells with TPI2155-14 (15 μM) and TPI2155-17 (4.3 μM) resulted in a significant decrease in NEP activity in media (62.37 ± 1.43 and 38.30 ± 4.70, respectively as a % of control; P < 0.0001), implicating a possible role for ADAM-17 in NEP release. However, centrifuging media (100,000g for 1 h at 4 °C) removed all NEP activity from the supernatant indicating the likely role of exosomes in the release of NEP. Our data therefore indicated for the first time that NEP is released from endothelial cells via exosomes, and that this process is dependent on ADAM-17.

  19. Endothelial Progenitor Cells Enter the Aging Arena.

    Directory of Open Access Journals (Sweden)

    Kate eWilliamson

    2012-02-01

    Full Text Available Age is a significant risk factor for the development of vascular diseases, such as atherosclerosis. Although pharmacological treatments, including statins and anti-hypertensive drugs, have improved the prognosis for patients with cardiovascular disease, it remains a leading cause of mortality in those aged 65 years and over. Furthermore, given the increased life expectancy of the population in developed countries, there is a clear need for alternative treatment strategies. Consequently, the relationship between aging and progenitor cell-mediated repair is of great interest. Endothelial progenitor cells (EPCs play an integral role in the cellular repair mechanisms for endothelial regeneration and maintenance. However, EPCs are subject to age-associated changes that diminish their number in circulation and function, thereby enhancing vascular disease risk. A great deal of research is aimed at developing strategies to harness the regenerative capacity of these cells.In this review, we discuss the current understanding of the cells termed ‘EPCs’, examine the impact of age on EPC-mediated repair and identify therapeutic targets with potential for attenuating the age-related decline in vascular health via beneficial actions on EPCs.

  20. Differential Effects of Isoxazole-9 on Neural Stem/Progenitor Cells, Oligodendrocyte Precursor Cells, and Endothelial Progenitor Cells.

    Directory of Open Access Journals (Sweden)

    Seong-Ho Koh

    Full Text Available Adult mammalian brain can be plastic after injury and disease. Therefore, boosting endogenous repair mechanisms would be a useful therapeutic approach for neurological disorders. Isoxazole-9 (Isx-9 has been reported to enhance neurogenesis from neural stem/progenitor cells (NSPCs. However, the effects of Isx-9 on other types of progenitor/precursor cells remain mostly unknown. In this study, we investigated the effects of Isx-9 on the three major populations of progenitor/precursor cells in brain: NSPCs, oligodendrocyte precursor cells (OPCs, and endothelial progenitor cells (EPCs. Cultured primary NSPCs, OPCs, or EPCs were treated with various concentrations of Isx-9 (6.25, 12.5, 25, 50 μM, and their cell numbers were counted in a blinded manner. Isx-9 slightly increased the number of NSPCs and effectively induced neuronal differentiation of NSPCs. However, Isx-9 significantly decreased OPC number in a concentration-dependent manner, suggesting cytotoxicity. Isx-9 did not affect EPC cell number. But in a matrigel assay of angiogenesis, Isx-9 significantly inhibited tube formation in outgrowth endothelial cells derived from EPCs. This potential anti-tube-formation effect of Isx-9 was confirmed in a brain endothelial cell line. Taken together, our data suggest that mechanisms and targets for promoting stem/progenitor cells in the central nervous system may significantly differ between cell types.

  1. Enhancing endothelial progenitor cell for clinical use

    Institute of Scientific and Technical Information of China (English)

    2015-01-01

    Circulating endothelial progenitor cells (EPCs) havebeen demonstrated to correlate negatively with vascularendothelial dysfunction and cardiovascular risk factors.However, translation of basic research into the clinicalpractice has been limited by the lack of unambiguousand consistent definitions of EPCs and reduced EPCcell number and function in subjects requiring them forclinical use. This article critically reviews the definitionof EPCs based on commonly used protocols, their valueas a biomarker of cardiovascular risk factor in subjectswith cardiovascular disease, and strategies to enhanceEPCs for treatment of ischemic diseases.

  2. Differentiation state determines neural effects on microvascular endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Muffley, Lara A., E-mail: muffley@u.washington.edu [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States); Pan, Shin-Chen, E-mail: pansc@mail.ncku.edu.tw [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States); Smith, Andria N., E-mail: gnaunderwater@gmail.com [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States); Ga, Maricar, E-mail: marga16@uw.edu [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States); Hocking, Anne M., E-mail: ahocking@u.washington.edu [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States); Gibran, Nicole S., E-mail: nicoleg@u.washington.edu [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States)

    2012-10-01

    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: Black-Right-Pointing-Pointer Dorsal root ganglion neurons, not neural progenitor cells, regulate microvascular endothelial cell proliferation. Black-Right-Pointing-Pointer Neural progenitor cells, not dorsal root ganglion neurons, regulate microvascular endothelial cell migration. Black-Right-Pointing-Pointer Neural progenitor cells and dorsal root ganglion neurons do not effect microvascular endothelial tube formation. Black-Right-Pointing-Pointer Dorsal root ganglion neurons, not neural progenitor cells, regulate

  3. Asiaticoside Inhibits TNF-α-Induced Endothelial Hyperpermeability of Human Aortic Endothelial Cells.

    Science.gov (United States)

    Fong, Lai Yen; Ng, Chin Theng; Zakaria, Zainul Amiruddin; Baharuldin, Mohamad Taufik Hidayat; Arifah, Abdul Kadir; Hakim, Muhammad Nazrul; Zuraini, Ahmad

    2015-10-01

    The increase in endothelial permeability often promotes edema formation in various pathological conditions. Tumor necrosis factor-alpha (TNF-α), a pro-atherogenic cytokine, impairs endothelial barrier function and causes endothelial dysfunction in early stage of atherosclerosis. Asiaticoside, one of the triterpenoids derived from Centella asiatica, is known to possess antiinflammatory activity. In order to examine the role of asiaticoside in preserving the endothelial barrier, we assessed its effects on endothelial hyperpermeability and disruption of actin filaments evoked by TNF-α in human aortic endothelial cells (HAEC). TNF-α caused an increase in endothelial permeability to fluorescein isothiocyanate (FITC)-dextran. Asiaticoside pretreatment significantly suppressed TNF-α-induced increased permeability. Asiaticoside also prevented TNF-α-induced actin redistribution by suppressing stress fiber formation. However, the increased F to G actin ratio stimulated by TNF-α was not changed by asiaticoside. Cytochalasin D, an actin depolymerizing agent, was used to correlate the anti-hyperpermeability effect of asiaticoside with actin cytoskeleton. Surprisingly, asiaticoside failed to prevent cytochalasin D-induced increased permeability. These results suggest that asiaticoside protects against the disruption of endothelial barrier and actin rearrangement triggered by TNF-α without a significant change in total actin pool. However, asiaticoside seems to work by other mechanisms to maintain the integrity of endothelial barrier rather than stabilizing the F-actin organization.

  4. Ischemia-induced endothelial cell swelling and mitochondrial dysfunction are attenuated by dietary polyphenols in vitro

    Science.gov (United States)

    Polyphenols possess anti-oxidant and anti-inflammatory properties. Oxidative stress (OS) and inflammation have been implicated in the pathogenesis of cytotoxic brain edema in cerebral ischemia. In addition, OS and pro-inflammatory cytokines also damage the endothelial cells and the neurovascular uni...

  5. Development of Endothelial-Specific Single Inducible Lentiviral Vectors for Genetic Engineering of Endothelial Progenitor Cells.

    Science.gov (United States)

    Yang, Guanghua; Kramer, M Gabriela; Fernandez-Ruiz, Veronica; Kawa, Milosz P; Huang, Xin; Liu, Zhongmin; Prieto, Jesus; Qian, Cheng

    2015-11-27

    Endothelial progenitor cells (EPC) are able to migrate to tumor vasculature. These cells, if genetically modified, can be used as vehicles to deliver toxic material to, or express anticancer proteins in tumor. To test this hypothesis, we developed several single, endothelial-specific, and doxycycline-inducible self-inactivating (SIN) lentiviral vectors. Two distinct expression cassettes were inserted into a SIN-vector: one controlled by an endothelial lineage-specific, murine vascular endothelial cadherin (mVEcad) promoter for the expression of a transactivator, rtTA2S-M2; and the other driven by an inducible promoter, TREalb, for a firefly luciferase reporter gene. We compared the expression levels of luciferase in different vector constructs, containing either the same or opposite orientation with respect to the vector sequence. The results showed that the vector with these two expression cassettes placed in opposite directions was optimal, characterized by a robust induction of the transgene expression (17.7- to 73-fold) in the presence of doxycycline in several endothelial cell lines, but without leakiness when uninduced. In conclusion, an endothelial lineage-specific single inducible SIN lentiviral vector has been developed. Such a lentiviral vector can be used to endow endothelial progenitor cells with anti-tumor properties.

  6. Optical Investigations of Endothelial Cell Motility

    DEFF Research Database (Denmark)

    Rossen, Ninna Struck

    of tissues and holds great promises for treatments and regenerative therapies. It faces an important obstacle before such promises can be realized, the engineered tissues needs to be of a size large enough to function and to relieve the damaged bodily functions. The current state of the art in tissue......A monolayer of endothelial cells lines the entire circulatory system and create a barrier between the circulatory system and the tissues. To create and maintain an intact barrier, the individual cells have to connect tightly with their neighbors, which causes a highly correlated motion between...... are fascinating from a biophysical point of view. The vasculature also plays a signi cant role in many pathologies. In diabetic blindness or ischemic diseases the ow of blood is insucient to sustain certain tissues or whole limbs. The creation of new blood vessels can relieve or treat such diseases. In other...

  7. Endothelial progenitor cells and integrins: adhesive needs

    Directory of Open Access Journals (Sweden)

    Caiado Francisco

    2012-03-01

    Full Text Available Abstract In the last decade there have been multiple studies concerning the contribution of endothelial progenitor cells (EPCs to new vessel formation in different physiological and pathological settings. The process by which EPCs contribute to new vessel formation in adults is termed postnatal vasculogenesis and occurs via four inter-related steps. They must respond to chemoattractant signals and mobilize from the bone marrow to the peripheral blood; home in on sites of new vessel formation; invade and migrate at the same sites; and differentiate into mature endothelial cells (ECs and/or regulate pre-existing ECs via paracrine or juxtacrine signals. During these four steps, EPCs interact with different physiological compartments, namely bone marrow, peripheral blood, blood vessels and homing tissues. The success of each step depends on the ability of EPCs to interact, adapt and respond to multiple molecular cues. The present review summarizes the interactions between integrins expressed by EPCs and their ligands: extracellular matrix components and cell surface proteins present at sites of postnatal vasculogenesis. The data summarized here indicate that integrins represent a major molecular determinant of EPC function, with different integrin subunits regulating different steps of EPC biology. Specifically, integrin α4β1 is a key regulator of EPC retention and/or mobilization from the bone marrow, while integrins α5β1, α6β1, αvβ3 and αvβ5 are major determinants of EPC homing, invasion, differentiation and paracrine factor production. β2 integrins are the major regulators of EPC transendothelial migration. The relevance of integrins in EPC biology is also demonstrated by many studies that use extracellular matrix-based scaffolds as a clinical tool to improve the vasculogenic functions of EPCs. We propose that targeted and tissue-specific manipulation of EPC integrin-mediated interactions may be crucial to further improve the usage of

  8. Increased endothelial cell-leukocyte interaction in murine schistosomiasis: possible priming of endothelial cells by the disease.

    Directory of Open Access Journals (Sweden)

    Suellen D S Oliveira

    Full Text Available BACKGROUND AND AIMS: Schistosomiasis is an intravascular parasitic disease associated with inflammation. Endothelial cells control leukocyte transmigration and vascular permeability being modulated by pro-inflammatory mediators. Recent data have shown that endothelial cells primed in vivo in the course of a disease keep the information in culture. Herein, we evaluated the impact of schistosomiasis on endothelial cell-regulated events in vivo and in vitro. METHODOLOGY AND PRINCIPAL FINDINGS: The experimental groups consisted of Schistosoma mansoni-infected and age-matched control mice. In vivo infection caused a marked influx of leukocytes and an increased protein leakage in the peritoneal cavity, characterizing an inflamed vascular and cellular profile. In vitro leukocyte-mesenteric endothelial cell adhesion was higher in cultured cells from infected mice as compared to controls, either in the basal condition or after treatment with the pro-inflammatory cytokine tumor necrosis factor (TNF. Nitric oxide (NO donation reduced leukocyte adhesion to endothelial cells from control and infected groups; however, in the later group the effect was more pronounced, probably due to a reduced NO production. Inhibition of control endothelial NO synthase (eNOS increased leukocyte adhesion to a level similar to the one observed in the infected group. Besides, the adhesion of control leukocytes to endothelial cells from infected animals is similar to the result of infected animals, confirming that schistosomiasis alters endothelial cells function. Furthermore, NO production as well as the expression of eNOS were reduced in cultured endothelial cells from infected animals. On the other hand, the expression of its repressor protein, namely caveolin-1, was similar in both control and infected groups. CONCLUSION/SIGNIFICANCE: Schistosomiasis increases vascular permeability and endothelial cell-leukocyte interaction in vivo and in vitro. These effects are partially

  9. Human vascular smooth muscle cells both express and respond to heparin-binding growth factor I (endothelial cell growth factor)

    Energy Technology Data Exchange (ETDEWEB)

    Winkles, J.A.; Friesel, R.; Burgess, W.H.; Howk, R.; Mehlman, T.; Weinstein, R.; Maciag, T.

    1987-10-01

    The control of vascular endothelial and muscle cell proliferation is important in such processes as tumor angiogenesis, wound healing, and the pathogenesis of atherosclerosis. Class I heparin-binding growth factor (HBGF-I) is a potent mitogen and chemoattractant for human endothelial cells in vitro and will induce angiogenesis in vivo. RNA gel blot hybridization experiments demonstrate that cultured human vascular smooth muscle cells, but not human umbilical cells also synthesize an HBGF-I mRNA. Smooth muscle cells also synthesize an HBGF-I-like polypeptide since (i) extract prepared from smooth muscle cells will compete with /sup 125/I-labeled HBGF-I for binding to the HBGF-I cell surface receptor, and (ii) the competing ligand is eluted from heparin-Sepharose affinity resin at a NaCl concentration similar to that required by purified bovine brain HBGF-I and stimulates endothelial cell proliferation in vitro. Furthermore, like endothelial cells, smooth muscle cells possess cell-surface-associated HBGF-I receptors and respond to HBGF-I as a mitogen. These results indicate the potential for an additional autocrine component of vascular smooth muscle cell growth control and establish a vessel wall source of HBGF-I for endothelial cell division in vivo.

  10. Silencing of directional migration in roundabout4 knockdown endothelial cells

    Directory of Open Access Journals (Sweden)

    Roberts David D

    2008-11-01

    Full Text Available Abstract Background Roundabouts are axon guidance molecules that have recently been identified to play a role in vascular guidance as well. In this study, we have investigated gene knockdown analysis of endothelial Robos, in particular roundabout 4 (robo4, the predominant Robo in endothelial cells using small interfering RNA technology in vitro. Results Robo1 and Robo4 knockdown cells display distinct activity in endothelial cell migration assay. The knockdown of robo4 abrogated the chemotactic response of endothelial cells to serum but enhanced a chemokinetic response to Slit2, while robo1 knockdown cells do not display chemotactic response to serum or VEGF. Robo4 knockdown endothelial cells unexpectedly show up regulation of Rho GTPases. Zebrafish Robo4 rescues both Rho GTPase homeostasis and serum reduced chemotaxis in robo4 knockdown cells. Robo1 and Robo4 interact and share molecules such as Slit2, Mena and Vilse, a Cdc42-GAP. In addition, this study mechanistically implicates IRSp53 in the signaling nexus between activated Cdc42 and Mena, both of which have previously been shown to be involved with Robo4 signaling in endothelial cells. Conclusion This study identifies specific components of the Robo signaling apparatus that work together to guide directional migration of endothelial cells.

  11. Development of new therapeutic modalities for corneal endothelial disease focused on the proliferation of corneal endothelial cells using animal models.

    Science.gov (United States)

    Koizumi, Noriko; Okumura, Naoki; Kinoshita, Shigeru

    2012-02-01

    This review describes our recent attempts to develop new therapeutic modalities for corneal endothelial disease using animal models including non-human primate model in which the proliferative ability of corneal endothelial cells is severely limited, as is the case in humans. First, we describe our attempt to develop new surgical treatments using cultivated corneal endothelial cells for advanced corneal endothelial dysfunction. It includes two different approaches; a "corneal endothelial cell sheet transplantation" with cells grown on a type-I collagen carrier, and a "cell-injection therapy" combined with the application of Rho-kinase (ROCK) inhibitor. Recently, it was reported that the selective ROCK inhibitor, Y-27632, promotes cell adhesion and proliferation and inhibits the apoptosis of primate corneal endothelial cells in culture. When cultivated corneal endothelial cells were injected into the anterior chamber of animal eyes in the presence of ROCK inhibitor, endothelial cell adhesion was promoted and the cells achieved a high cell density and a morphology similar to corneal endothelial cells in vivo. We are also trying to develop a novel medical treatment for the early phase of corneal endothelial disease by the use of ROCK inhibitor eye drops. In rabbit and monkey experiments using partial endothelial dysfunction models, corneal endothelial wound healing was accelerated by the topical application of ROCK inhibitor to the ocular surface, and resulted in the regeneration of a corneal endothelial monolayer with a high endothelial cell density. We are now trying to advance the clinical application of these new therapies for patients with corneal endothelial dysfunction.

  12. 循环内皮细胞与放射性脑损伤相关性实验研究%Correlation between circulating endothelial cells and radiation-induced brain injury

    Institute of Scientific and Technical Information of China (English)

    马代远; 谭榜宪; 李祥攀; 阮林; 甘浪舸; 韦力

    2008-01-01

    Objective To investigate the correlation between circulating endothelial cells (CECs) and radiation-induced brain injury. Methods One hundred and eight SD rats were randomly divided into control group, single-dose 10 Gy 60Co irradiation group and single-dose 30 Gy group. The neurobehavioral changes were observed by Morris water labyrinth at 1 week, 1 month and 3 months after irradiation. The CECs in right ventricular blood were counted after Morrie water test. Hippocamp ultramicrostructure and GFAP positive cell were detected after perfusion of encephalon. Results Neuobehavior change: at 1 month and 3 months after irradiation the swim latency was significantly prolonged (30 Gy group>10 Gy group>control group, P 10 Gy group> 30 Gy group, P 10 Gy group>control group, P10 Gy group>control group, P<0.05). Good correlations between the numbers of CECs and the swim lantency (r10 Gy=0.97, P=0.034; r30 Gy=0.95, P=0.013),and the numbers of GFAP positive cells(r10 Gy=0.94, P=0.037; r30 Gy=0.96, P=0.027) were demonstrated.Conclusion The changes of the CECs numbers are definitely correlated to radiation-induced brain injury, which is more with irradiation dose and duration.%目的 探讨循环内皮细胞(circulating endothelial cegs,CECs)与放射性脑损伤相关性.方法 108只SD大鼠信封法随机分成对照组、实验组(10 Gy组、30 Gy组),分别于照射后1周、1月和3月每组随机抽取9只进行Morris水迷宫行为测试,心脏取血计数CECs,取脑观察海马形态和结构.结果 神经行为改变:照射1和3个月后平台潜伏期延长、穿越平台象限时间及次数明显减少.海马齿状回形态改变:照射1和3个月后实验组神经胶质酸性蛋白(glial fibfillary acidic protein,GFAP)阳性细胞数量明显高于对照组,30 Gy组高于10 Gy组;照射1和3个月后超微结构变化为毛细血管内皮细胞变薄、内皮细胞吞饮小泡明显增多、内皮细胞间紧密连接破坏,毛细血管基膜外星形胶质细胞

  13. Caspases and p38 MAPK regulate endothelial cell adhesiveness for mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Irina A Potapova

    Full Text Available Mesenchymal stem cells natively circulating or delivered into the blood stream home to sites of injury. The mechanism of mesenchymal stem cell homing to sites of injury is poorly understood. We have shown that the development of apoptosis in endothelial cells stimulates endothelial cell adhesiveness for mesenchymal stem cells. Adhesion of mesenchymal stem cells to apoptotic endothelial cells depends on the activation of endothelial caspases and p38 MAPK. Activation of p38 MAPK in endothelial cells has a primary effect while the activation of caspases potentiates the mesenchymal stem cell adhesion. Overall, our study of the mesenchymal stem cell interaction with endothelial cells indicates that mesenchymal stem cells recognize and specifically adhere to distressed/apoptotic endothelial cells.

  14. Genipin inhibits endothelial exocytosis via nitric oxide in cultured human umbilical vein endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Guang-fa WANG; Shao-yu WU; Jin-jun RAO; Lin L(U); Wei XU; Jian-xin PANG; Zhong-qiu LIU; Shu-guang WU; Jia-jie ZHANG

    2009-01-01

    Aim: Exocytosis of endothelial Weibel-Palade bodies, which contain von Willebrand factor (VWF), P-selectin and other modulators, plays an important role in both inflammation and thrombosis. The present study investigates whether genipin,an aglycon of geniposide, inhibits endothelial exocytosis.Methods: Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords and cultured. The concentration of VWF in cell supernatants was measured using an ELISA Kit. P-selectin translocation on the cell surface was analyzed by cell surface ELISA. Cell viability was measured using a Cell Counting Kit-8. Mouse bleeding times were measured by amputating the tail tip. Western blot analysis was used to determine the amount of endothelial nitric oxide synthase (eNOS) and phospho-eNOS present. Nitric oxide (NO) was measured in the cell supernatants as nitrite using an NO Colorimetric Assay.Results: Genipin inhibited thrombin-induced VWF release and P-selectin translocation in HUVECs in a dose- and time-dependent manner. The drug had no cytotoxic effect on the cells at the same doses that were able to inhibit exocytosis. The functional study that demonstrated that genipin inhibited exocytosis in vivo also showed that genipin prolonged the mouse bleeding time. Furthermore, genipin activated eNOS phosphorylation, promoted enzyme activation and increased NO production. L-NAME, an inhibitor of NOS, reversed the inhibitory effects of genipin on endothelial exocytosis.Conclusion: Genipin inhibits endothelial exocytosis in HUVECs. The mechanism by which this compound inhibits exocytosis may be related to its ability to stimulate eNOS activation and NO production. Our findings suggest a novel antiinflammatory mechanism for genipin. This compound may represent a new treatment for inflammation and/or thrombosis in which excess endothelial exocytosis plays a pathophysiological role.

  15. Variations in mass transfer to single endothelial cells.

    Science.gov (United States)

    Van Doormaal, Mark A; Zhang, Ji; Wada, Shigeo; Shaw, James E; Won, Doyon; Cybulsky, Myron I; Yip, Chris M; Ethier, C Ross

    2009-06-01

    Mass transfer between flowing blood and arterial mural cells (including vascular endothelial cells) may play an important role in atherogenesis. Endothelial cells are known to have an apical surface topography that is not flat, and hence mass transfer patterns to individual endothelial cells are likely affected by the local cellular topography. The purpose of this paper is to investigate the relationship between vascular endothelial cell surface topography and cellular level mass transfer. Confluent porcine endothelial monolayers were cultured under both shear and static conditions and atomic force microscopy was used to measure endothelial cell topography. Using finite element methods and the measured cell topography, flow and concentration fields were calculated for a typical, small, blood-borne solute. A relative Sherwood number was defined as the difference between the computed Sherwood number and that predicted by the Leveque solution for mass transfer over a flat surface: this eliminates the effects of axial location on mass transfer efficiency. The average intracellular relative Sherwood number range was found to be dependent on cell height and not dependent on cell elongation due to shear stress in culture. The mass flux to individual cells reached a maximum at the highest point on the endothelial cell surface, typically corresponding to the nucleus of the cell. Therefore, for small receptor-mediated solutes, increased solute uptake efficiency can be achieved by concentrating receptors near the nucleus. The main conclusion of the work is that although the rate of mass transfer varies greatly over an individual cell, the average mass transfer rate to a cell is close to that predicted for a flat cell. In comparison to other hemodynamic factors, the topography of endothelial cells therefore seems to have little effect on mass transfer rates and is likely physiologically insignificant.

  16. Angiogenic potential of endothelial progenitor cells and embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Rae Peter C

    2011-05-01

    Full Text Available Abstract Background Endothelial progenitor cells (EPCs are implicated in a range of pathological conditions, suggesting a natural therapeutic role for EPCs in angiogenesis. However, current angiogenic therapies involving EPC transplantation are inefficient due to rejection of donor EPCs. One solution is to derive an expanded population of EPCs from stem cells in vitro, to be re-introduced as a therapeutic transplant. To demonstrate the therapeutic potential of EPCs we performed in vitro transplantation of EPCs into endothelial cell (EC tubules using a gel-based tubule formation assay. We also described the production of highly angiogenic EPC-comparable cells from pluripotent embryonic stem cells (ESCs by direct differentiation using EC-conditioned medium (ECCM. Results The effect on tubule complexity and longevity varied with transplantation quantity: significant effects were observed when tubules were transplanted with a quantity of EPCs equivalent to 50% of the number of ECs originally seeded on to the assay gel but not with 10% EPC transplantation. Gene expression of the endothelial markers VEGFR2, VE-cadherin and CD31, determined by qPCR, also changed dynamically during transplantation. ECCM-treated ESC-derived progenitor cells exhibited angiogenic potential, demonstrated by in vitro tubule formation, and endothelial-specific gene expression equivalent to natural EPCs. Conclusions We concluded the effect of EPCs is cumulative and beneficial, relying on upregulation of the angiogenic activity of transplanted cells combined with an increase in proliferative cell number to produce significant effects upon transplantation. Furthermore, EPCs derived from ESCs may be developed for use as a rapidly-expandable alternative for angiogenic transplantation therapy.

  17. Enterococcus faecalis internalization in human umbilical vein endothelial cells (HUVEC).

    Science.gov (United States)

    Millán, Diana; Chiriboga, Carlos; Patarroyo, Manuel A; Fontanilla, Marta R

    2013-04-01

    Initial Enterococcus faecalis-endothelial cell molecular interactions which lead to enterococci associating in the host endothelial tissue, colonizing it and proliferating there can be assessed using in vitro models. Cultured human umbilical vein endothelial cells (HUVEC) have been used to study other Gram-positive bacteria-cell interactions; however, few studies have been aimed at establishing the relationship of E. faecalis with endothelial cells. The aggregation substance (AS) family of adhesins represents an E. faecalis virulence factor which has been implicated in endocarditis severity and bacterial persistence. The Asc10 protein (a member of this family) promotes bacterium-bacterium aggregation and bacterium-host cell binding. Evaluating Asc10 role in bacterial internalization by cultured enterocytes has shown that this adhesin facilitates E. faecalis endocytosis by HT-29 cells. A few eukaryotic cell structural components, such as cytoskeletal proteins, have been involved in E. faecalis entry into cell-lines; it is thus relevant to determine whether Asc10, as well as microtubules and actin microfilaments, play a role in E. faecalis internalization by cultured endothelial cells. The role of Asc10 and cytoskeleton proteins in E. faecalis ability to enter HUVEC was assessed in the present study, as well as cell apoptosis induction by enterococcal internalization by HUVEC; the data indicated increased cell apoptosis and that cytoskeleton components were partially involved in E. faecalis entry to endothelial cells, thereby suggesting that E. faecalis Asc10 protein would not be a critical factor for bacterial entry to cultured HUVEC.

  18. Vascular endothelial growth factor-dependent angiogenesis and dynamic vascular plasticity in the sensory circumventricular organs of adult mouse brain.

    Science.gov (United States)

    Morita, Shoko; Furube, Eriko; Mannari, Tetsuya; Okuda, Hiroaki; Tatsumi, Kouko; Wanaka, Akio; Miyata, Seiji

    2015-03-01

    The sensory circumventricular organs (CVOs), which comprise the organum vasculosum of the lamina terminalis (OVLT), the subfornical organ (SFO) and the area postrema (AP), lack a typical blood-brain barrier (BBB) and monitor directly blood-derived information to regulate body fluid homeostasis, inflammation, feeding and vomiting. Until now, almost nothing has been documented about vascular features of the sensory CVOs except fenestration of vascular endothelial cells. We therefore examine whether continuous angiogenesis occurs in the sensory CVOs of adult mouse. The angiogenesis-inducing factor vascular endothelial growth factor-A (VEGF-A) and the VEGF-A-regulating transcription factor hypoxia-inducible factor-1α were highly expressed in neurons of the OVLT and SFO and in both neurons and astrocytes of the AP. Expression of the pericyte-regulating factor platelet-derived growth factor B was high in astrocytes of the sensory CVOs. Immunohistochemistry of bromodeoxyuridine and Ki-67, a nuclear protein that is associated with cellular proliferation, revealed active proliferation of endothelial cells. Moreover, immunohistochemistry of caspase-3 and the basement membrane marker laminin showed the presence of apoptosis and sprouting of endothelial cells, respectively. Treatment with the VEGF receptor-associated tyrosine kinase inhibitor AZD2171 significantly reduced proliferation and filopodia sprouting of endothelial cells, as well as the area and diameter of microvessels. The mitotic inhibitor cytosine-b-D-arabinofuranoside reduced proliferation of endothelial cells and the vascular permeability of blood-derived low-molecular-weight molecules without changing vascular area and microvessel diameter. Thus, our data indicate that continuous angiogenesis is dependent on VEGF signaling and responsible for the dynamic plasticity of vascular structure and permeability.

  19. Sodium renders endothelial cells sticky for red blood cells

    Directory of Open Access Journals (Sweden)

    Hans eOberleithner

    2015-06-01

    Full Text Available Negative charges in the glycocalyx of red blood cells (RBC and vascular endothelial cells (EC facilitate frictionless blood flow through blood vessels. Na+ selectively shields these charges controlling surface electronegativity. The question was addressed whether the ambient Na+ concentration controls RBC-EC interaction. Using atomic force microscopy (AFM adhesion forces between RBC and endothelial glycocalyx were quantified. A single RBC, mounted on an AFM cantilever, was brought in physical contact with the endothelial surface and then pulled off. Adhesion forces were quantified (i after enzymatic removal of negative charges in the glycocalyx, (ii under different ambient Na+ and (iii after applying the intracellular aldosterone receptor antagonist spironolactone. Removal of negative surface charges increases RBC-EC interaction forces. A stepwise increase of ambient Na+ from 133 to 140 mM does not affect them. However, beyond 140 mM Na+ adhesion forces increase sharply (10% increase of adhesion force per 1 mM increase of Na+. Spironolactone prevents this response. It is concluded that negative charges reduce adhesion between RBC and EC. Ambient Na+ concentration determines the availability of free negative charges. Na+ concentrations in the low physiological range (below 140 mM allow sufficient amounts of vacant negative charges so that adhesion of RBC to the endothelial surface is small. In contrast, Na+ in the high physiological range (beyond 140 mM saturates the remaining negative surface charges thus increasing adhesion. Aldosterone receptor blockade by spironolactone prevents Na+ induced RBC adhesion to the endothelial glycocalyx. Extrapolation of in vitro experiments to in vivo conditions leads to the hypothesis that high sodium intake is likely to increase the incidence of thrombotic events.

  20. Sodium renders endothelial cells sticky for red blood cells.

    Science.gov (United States)

    Oberleithner, Hans; Wälte, Mike; Kusche-Vihrog, Kristina

    2015-01-01

    Negative charges in the glycocalyx of red blood cells (RBC) and vascular endothelial cells (EC) facilitate frictionless blood flow through blood vessels. Na(+) selectively shields these charges controlling surface electronegativity. The question was addressed whether the ambient Na(+) concentration controls RBC-EC interaction. Using atomic force microscopy (AFM) adhesion forces between RBC and endothelial glycocalyx were quantified. A single RBC, mounted on an AFM cantilever, was brought in physical contact with the endothelial surface and then pulled off. Adhesion forces were quantified (i) after enzymatic removal of negative charges in the glycocalyx, (ii) under different ambient Na(+) and (iii) after applying the intracellular aldosterone receptor antagonist spironolactone. Removal of negative surface charges increases RBC-EC interaction forces. A stepwise increase of ambient Na(+) from 133 to 140 mM does not affect them. However, beyond 140 mM Na(+) adhesion forces increase sharply (10% increase of adhesion force per 1 mM increase of Na(+)). Spironolactone prevents this response. It is concluded that negative charges reduce adhesion between RBC and EC. Ambient Na(+) concentration determines the availability of free negative charges. Na(+) concentrations in the low physiological range (below 140 mM) allow sufficient amounts of vacant negative charges so that adhesion of RBC to the endothelial surface is small. In contrast, Na(+) in the high physiological range (beyond 140 mM) saturates the remaining negative surface charges thus increasing adhesion. Aldosterone receptor blockade by spironolactone prevents Na(+) induced RBC adhesion to the endothelial glycocalyx. Extrapolation of in vitro experiments to in vivo conditions leads to the hypothesis that high sodium intake is likely to increase the incidence of thrombotic events.

  1. Biophysical Cueing and Vascular Endothelial Cell Behavior

    Directory of Open Access Journals (Sweden)

    Joshua A. Wood

    2010-03-01

    Full Text Available Human vascular endothelial cells (VEC line the vessels of the body and are critical for the maintenance of vessel integrity and trafficking of biochemical cues. They are fundamental structural elements and are central to the signaling environment. Alterations in the normal functioning of the VEC population are associated with a number of vascular disorders among which are some of the leading causes of death in both the United States and abroad. VECs attach to their underlying stromal elements through a specialization of the extracellular matrix, the basement membrane. The basement membrane provides signaling cues to the VEC through its chemical constituents, by serving as a reservoir for cytoactive factors and through its intrinsic biophysical properties. This specialized matrix is composed of a topographically rich 3D felt-like network of fibers and pores on the nano (1–100 nm and submicron (100–1,000 nm size scale. The basement membrane provides biophysical cues to the overlying VECs through its intrinsic topography as well as through its local compliance (relative stiffness. These biophysical cues modulate VEC adhesion, migration, proliferation, differentiation, and the cytoskeletal signaling network of the individual cells. This review focuses on the impact of biophysical cues on VEC behaviors and demonstrates the need for their consideration in future vascular studies and the design of improved prosthetics.

  2. Activation of Endothelial Nitric Oxide (eNOS Occurs through Different Membrane Domains in Endothelial Cells.

    Directory of Open Access Journals (Sweden)

    Jason Tran

    Full Text Available Endothelial cells respond to a large range of stimuli including circulating lipoproteins, growth factors and changes in haemodynamic mechanical forces to regulate the activity of endothelial nitric oxide synthase (eNOS and maintain blood pressure. While many signalling pathways have been mapped, the identities of membrane domains through which these signals are transmitted are less well characterized. Here, we manipulated bovine aortic endothelial cells (BAEC with cholesterol and the oxysterol 7-ketocholesterol (7KC. Using a range of microscopy techniques including confocal, 2-photon, super-resolution and electron microscopy, we found that sterol enrichment had differential effects on eNOS and caveolin-1 (Cav1 colocalisation, membrane order of the plasma membrane, caveolae numbers and Cav1 clustering. We found a correlation between cholesterol-induced condensation of the plasma membrane and enhanced high density lipoprotein (HDL-induced eNOS activity and phosphorylation suggesting that cholesterol domains, but not individual caveolae, mediate HDL stimulation of eNOS. Vascular endothelial growth factor (VEGF-induced and shear stress-induced eNOS activity was relatively independent of membrane order and may be predominantly controlled by the number of caveolae on the cell surface. Taken together, our data suggest that signals that activate and phosphorylate eNOS are transmitted through distinct membrane domains in endothelial cells.

  3. Endothelial cell tumor growth is Ape/ref-1 dependent.

    Science.gov (United States)

    Biswas, Ayan; Khanna, Savita; Roy, Sashwati; Pan, Xueliang; Sen, Chandan K; Gordillo, Gayle M

    2015-09-01

    Tumor-forming endothelial cells have highly elevated levels of Nox-4 that release H2O2 into the nucleus, which is generally not compatible with cell survival. We sought to identify compensatory mechanisms that enable tumor-forming endothelial cells to survive and proliferate under these conditions. Ape-1/ref-1 (Apex-1) is a multifunctional protein that promotes DNA binding of redox-sensitive transcription factors, such as AP-1, and repairs oxidative DNA damage. A validated mouse endothelial cell (EOMA) tumor model was used to demonstrate that Nox-4-derived H2O2 causes DNA oxidation that induces Apex-1 expression. Apex-1 functions as a chaperone to keep transcription factors in a reduced state. In EOMA cells Apex-1 enables AP-1 binding to the monocyte chemoattractant protein-1 (mcp-1) promoter and expression of that protein is required for endothelial cell tumor formation. Intraperitoneal injection of the small molecule inhibitor E3330, which specifically targets Apex-1 redox-sensitive functions, resulted in a 50% decrease in tumor volume compared with mice injected with vehicle control (n = 6 per group), indicating that endothelial cell tumor proliferation is dependent on Apex-1 expression. These are the first reported results to establish Nox-4 induction of Apex-1 as a mechanism promoting endothelial cell tumor formation.

  4. High-density lipoprotein endocytosis in endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Stefanie; Fruhwürth; Margit; Pavelka; Robert; Bittman; Werner; J; Kovacs; Katharina; M; Walter; Clemens; Rhrl; Herbert; Stangl

    2013-01-01

    AIM: To describe the way stations of high-density lipoprotein(HDL) uptake and its lipid exchange in endothelial cells in vitro and in vivo. METHODS: A combination of fluorescence microscopy using novel fluorescent cholesterol surrogates and electron microscopy was used to analyze HDL endocytosis in great detail in primary human endothelial cells. Further, HDL uptake was quantified using radio-labeled HDL particles. To validate the in vitro findings mice were injected with fluorescently labeled HDL and particle uptake in the liver was analyzed using fluorescencemicroscopy. RESULTS: HDL uptake occurred via clathrin-coated pits, tubular endosomes and multivesicular bodies in human umbilical vein endothelial cells. During uptake and resecretion, HDL-derived cholesterol was exchanged at a faster rate than cholesteryl oleate, resembling the HDL particle pathway seen in hepatic cells. In addition, lysosomes were not involved in this process and thus HDL degradation was not detectable. In vivo, we found HDL mainly localized in mouse hepatic endothelial cells. HDL was not detected in parenchymal liver cells, indicating that lipid transfer from HDL to hepatocytes occurs primarily via scavenger receptor, class B, type Ⅰ mediated selective uptake without concomitant HDL endocytosis. CONCLUSION: HDL endocytosis occurs via clathrincoated pits, tubular endosomes and multivesicular bodies in human endothelial cells. Mouse endothelial cells showed a similar HDL uptake pattern in vivo indicating that the endothelium is one major site of HDL endocytosis and transcytosis.

  5. 5-Hydroxytryptamine 4 Receptor in the Endothelial Cells

    DEFF Research Database (Denmark)

    Profirovic, Jasmina; Vardya, Irina; Voyno-Yasenetskaya, Tatyana

    2006-01-01

    in the CNS, none of the studies showed its expression and function in the endothelial cells. In the present study, we provide evidence for the first time that 5-HT4 receptor is expressed in the human umbilical vein endothelial cells (HUVECs). We demonstrate the transcription of 5-HT4 mRNA in the HUVECs using...... reverse transcription polimerase chain reaction. Additionally, we show 5- HT4 receptor expression in HUVECs by immunoblotting and immunofluorescent analysis with 5-HT4 specific antibody. Importantly, we determine that overexpression of 5-HT4 receptor leads to a pronounced cell rounding and intercellular...... gap formation in HUVECs. We are currently investigating the mechanism underlying 5-HT4 receptor-induced actin cytoskeleton changes in the endothelial cells. These data suggest that by activating 5-HT4 receptor, serotonin could be involved in regulation of actin cytoskeleton dynamics in the endothelial...

  6. Growth of fibroblasts and endothelial cells on wettability gradient surfaces

    NARCIS (Netherlands)

    Ruardy, TG; Moorlag, HE; Schakenraad, JM; VanderMei, HC; Busscher, HJ

    1997-01-01

    The growth, spreading, and shape of human skin fibroblasts (PK 84) and human umbilical cord endothelial cells on dichlorodimethylsilane (DDS) and dimethyloctadecylchlorosilane (DOGS) gradient surfaces were investigated in the presence of serum proteins. Gradient surfaces were prepared on glass using

  7. Tumor-derived circulating endothelial cell clusters in colorectal cancer.

    KAUST Repository

    Cima, Igor

    2016-06-29

    Clusters of tumor cells are often observed in the blood of cancer patients. These structures have been described as malignant entities for more than 50 years, although their comprehensive characterization is lacking. Contrary to current consensus, we demonstrate that a discrete population of circulating cell clusters isolated from the blood of colorectal cancer patients are not cancerous but consist of tumor-derived endothelial cells. These clusters express both epithelial and mesenchymal markers, consistent with previous reports on circulating tumor cell (CTC) phenotyping. However, unlike CTCs, they do not mirror the genetic variations of matched tumors. Transcriptomic analysis of single clusters revealed that these structures exhibit an endothelial phenotype and can be traced back to the tumor endothelium. Further results show that tumor-derived endothelial clusters do not form by coagulation or by outgrowth of single circulating endothelial cells, supporting a direct release of clusters from the tumor vasculature. The isolation and enumeration of these benign clusters distinguished healthy volunteers from treatment-naïve as well as pathological early-stage (≤IIA) colorectal cancer patients with high accuracy, suggesting that tumor-derived circulating endothelial cell clusters could be used as a means of noninvasive screening for colorectal cancer. In contrast to CTCs, tumor-derived endothelial cell clusters may also provide important information about the underlying tumor vasculature at the time of diagnosis, during treatment, and throughout the course of the disease.

  8. Endothelial cell proliferation in swine experimental aneurysm after coil embolization.

    Directory of Open Access Journals (Sweden)

    Yumiko Mitome-Mishima

    Full Text Available After coil embolization, recanalization in cerebral aneurysms adversely influences long-term prognosis. Proliferation of endothelial cells on the coil surface may reduce the incidence of recanalization and further improve outcomes after coil embolization. We aimed to map the expression of proliferating tissue over the aneurysmal orifice and define the temporal profile of tissue growth in a swine experimental aneurysm model. We compared the outcomes after spontaneous thrombosis with those of coil embolization using histological and morphological techniques. In aneurysms that we not coiled, spontaneous thrombosis was observed, and weak, easily detachable proliferating tissue was evident in the aneurysmal neck. In contrast, in the coil embolization group, histological analysis showed endothelial-like cells lining the aneurysmal opening. Moreover, immunohistochemical and morphological analysis suggested that these cells were immature endothelial cells. Our results indicated the existence of endothelial cell proliferation 1 week after coil embolization and showed immature endothelial cells in septal tissue between the systemic circulation and the aneurysm. These findings suggest that endothelial cells are lead to and proliferate in the former aneurysmal orifice. This is the first examination to evaluate the temporal change of proliferating tissue in a swine experimental aneurysm model.

  9. Cellular and molecular biology of aging endothelial cells.

    Science.gov (United States)

    Donato, Anthony J; Morgan, R Garrett; Walker, Ashley E; Lesniewski, Lisa A

    2015-12-01

    Cardiovascular disease (CVD) is the leading cause of death in the United States and aging is a major risk factor for CVD development. One of the major age-related arterial phenotypes thought to be responsible for the development of CVD in older adults is endothelial dysfunction. Endothelial function is modulated by traditional CVD risk factors in young adults, but advancing age is independently associated with the development of vascular endothelial dysfunction. This endothelial dysfunction results from a reduction in nitric oxide bioavailability downstream of endothelial oxidative stress and inflammation that can be further modulated by traditional CVD risk factors in older adults. Greater endothelial oxidative stress with aging is a result of augmented production from the intracellular enzymes NADPH oxidase and uncoupled eNOS, as well as from mitochondrial respiration in the absence of appropriate increases in antioxidant defenses as regulated by relevant transcription factors, such as FOXO. Interestingly, it appears that NFkB, a critical inflammatory transcription factor, is sensitive to this age-related endothelial redox change and its activation induces transcription of pro-inflammatory cytokines that can further suppress endothelial function, thus creating a vicious feed-forward cycle. This review will discuss the two macro-mechanistic processes, oxidative stress and inflammation, that contribute to endothelial dysfunction with advancing age as well as the cellular and molecular events that lead to the vicious cycle of inflammation and oxidative stress in the aged endothelium. Other potential mediators of this pro-inflammatory endothelial phenotype are increases in immune or senescent cells in the vasculature. Of note, genomic instability, telomere dysfunction or DNA damage has been shown to trigger cell senescence via the p53/p21 pathway and result in increased inflammatory signaling in arteries from older adults. This review will discuss the current state

  10. Uptake of gold nanoparticles in primary human endothelial cells

    DEFF Research Database (Denmark)

    Klingberg, Henrik; Oddershede, Lene B.; Löschner, Katrin

    2015-01-01

    Gold nanoparticles (AuNPs) are relevant in nanomedicine for drug delivery in the vascular system, where endothelial cells are the first point of contact. We investigated the uptake of 80 nm AuNPs in primary human umbilical vein endothelial cells (HUVECs) by flow cytometry, 3D confocal microscopy....... Uptake of AuNPs in HUVECs occurred mainly by clathrin-mediated endocytosis and trafficking to membrane enclosures in the form of single particles and agglomerates of 2–3 particles....

  11. Breast cancer cells stimulate osteoprotegerin (OPG production by endothelial cells through direct cell contact

    Directory of Open Access Journals (Sweden)

    Holen Ingunn

    2009-07-01

    Full Text Available Abstract Background Angiogenesis, the sprouting of capillaries from existing blood vessels, is central to tumour growth and progression, however the molecular regulation of this process remains to be fully elucidated. The secreted glycoprotein osteoprotegerin (OPG is one potential pro-angiogenic factor, and clinical studies have demonstrated endothelial cells within a number of tumour types to express high levels of OPG compared to those in normal tissue. Additionally, OPG can increase endothelial cell survival, proliferation and migration, as well as induce endothelial cell tube formation in vitro. This study aims to elucidate the processes involved in the pro-angiogenic effects of OPG in vitro, and also how OPG levels may be regulated within the tumour microenvironment. Results It has previously been demonstrated that OPG can induce tube formation on growth factor reduced matrigel. In this study, we demonstrate that OPG enhances the pro-angiogenic effects of VEGF and that OPG does not stimulate endothelial cell tube formation through activation of the VEGFR2 receptor. We also show that cell contact between HuDMECs and the T47D breast cancer cell line increases endothelial cell OPG mRNA and protein secretion levels in in vitro co-cultures. These increases in endothelial cell OPG secretion were dependent on ανβ3 ligation and NFκB activation. In contrast, the pro-angiogenic factors VEGF, bFGF and TGFβ had no effect on HuDMEC OPG levels. Conclusion These findings suggest that the VEGF signalling pathway is not involved in mediating the pro-angiogenic effects of OPG on endothelial cells in vitro. Additionally, we show that breast cancer cells cause increased levels of OPG expression by endothelial cells, and that direct contact between endothelial cells and tumour cells is required in order to increase endothelial OPG expression and secretion. Stimulation of OPG secretion was shown to involve ανβ3 ligation and NFκB activation.

  12. Traction Forces of Endothelial Cells under Slow Shear Flow

    Science.gov (United States)

    Perrault, Cecile M.; Brugues, Agusti; Bazellieres, Elsa; Ricco, Pierre; Lacroix, Damien; Trepat, Xavier

    2015-01-01

    Endothelial cells are constantly exposed to fluid shear stresses that regulate vascular morphogenesis, homeostasis, and disease. The mechanical responses of endothelial cells to relatively high shear flow such as that characteristic of arterial circulation has been extensively studied. Much less is known about the responses of endothelial cells to slow shear flow such as that characteristic of venous circulation, early angiogenesis, atherosclerosis, intracranial aneurysm, or interstitial flow. Here we used a novel, to our knowledge, microfluidic technique to measure traction forces exerted by confluent vascular endothelial cell monolayers under slow shear flow. We found that cells respond to flow with rapid and pronounced increases in traction forces and cell-cell stresses. These responses are reversible in time and do not involve reorientation of the cell body. Traction maps reveal that local cell responses to slow shear flow are highly heterogeneous in magnitude and sign. Our findings unveil a low-flow regime in which endothelial cell mechanics is acutely responsive to shear stress. PMID:26488643

  13. Mechanism of Corneal Endothelial Cells Lesion during Phacoemulsification and Aspiration

    Institute of Scientific and Technical Information of China (English)

    Songtao Yuan; Lina Xie; Qinghuai Liu; Nanrong Yuan

    2003-01-01

    Purpose: To evaluate the proportions of corneal endothelial lesion caused by differentfactors during phacoemulsification and aspiration.Methods: Fourteen cats (twenty eight eyes) were divided into four groups. The processedfactors were ultrasonic power, lens extraction by phacoemulsification or not, and lensextraction using different levels of ultrasonic power. The density of central cornealendothelial cells was measured before and after operation.Results: There is no statistic difference between pre-operation density and post-operationdensity for releasing ultrasonic power only without lens extraction group. But for the lensextraction group, there is difference in density of central corneal endothelial cells andthe higher level of ultrasonic power, the more the central corneal endothelial cells densitydecreased through operation.Conclusion: The primary factor that causes corneal endothelial lesion duringphacoemulsification and aspiration procedure is debris of lens nucleus, and the otherfactors cause the lesion of corneal endothelium in normal operations just in very smalldegree.

  14. 细胞饥饿及 TNF-α干预后内皮细胞膜微粒对人脑微血管内皮细胞的影响%Effects of cell starvation and TNF-αderived endothelial microvesicles on human brain microvascular endothelial cells

    Institute of Scientific and Technical Information of China (English)

    潘群文; 何彩霞; 刘雅静; 张惠婷; 王艳; 戴炳琰; 马晓瑭

    2015-01-01

    目的:探讨饥饿及TNF-α刺激条件下,内皮细胞膜微粒( EMVs )对人脑微血管内皮细胞( HBMECs )增殖、迁移及血管生成功能的影响。方法体外培养HBMECs,分为PBS组、饥饿组、TNF-α组。于饥饿24 h后提取饥饿后产生的EMVs( sHB-MVs),于TNF-α刺激24 h后提取TNF-α刺激下产生的EMVs(αHB-MVs)。将sHB-MVs与αHB-MVs按照1×108个/mL、每孔10μL分别与饥饿组及TNF-α组HBMECs共培养,PBS组给予10μL PBS处理。采用MTT法测定各组HBMECs增殖能力,倒置显微镜下测定HBMECs迁移距离,观察血管形成数。结果饥饿组增殖能力高于PBS组(P<0.01),TNF-α组HBMECs增殖能力低于PBS组(P<0.01)。饥饿组迁移距离长于PBS组(P<0.05),TNF-α组迁移距离短于PBS组(P<0.01)。饥饿组血管生成数多于PBS组(P<0.01),TNF-α组血管生成数少于PBS组(P<0.01)。结论饥饿刺激下产生的EMVs可促进HBMECs增殖、迁移及血管生成功能,TNF-α刺激下产生的EMVs可抑制HBMECs增殖、迁移及血管生成功能。%Objective To investigate the effects of cell starvation and TNF-αderived endothelial microvesicles (EMVs) on the proliferation, migration and angiogenesis of human brain microvascular endothelial cells (HBMECs) in vitro.Methods HBMECs were cultured and were divided into the PBS group, starvation group and TNF-αgroup.EMVs were extracted from HB-MECs cultured in a serum deprivation (SD) medium (starving stress, sHB-MVs) or SD medium containing tumor necrosis fac-tor-(TNF-α) (apoptotic stress,αHB-MVs).The HBMECs of the starvation group and TNF-αgroup were cultured with sHB-MVs andαHB-MVs (1 ×108/mL, 10μL each hole), respectively;and the PBS group was treated with 10μL PBS.The prolif-eration of HBMECs in each group was determined by MTT, the migration distance of HBMECs was measured by inverted micro-scope and the number of angiogenesis was observed under the

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

    Science.gov (United States)

    Eum, Sung Yong; Jaraki, Dima; András, Ibolya E.; Toborek, Michal

    2015-01-01

    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 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. PMID:26080028

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

    Science.gov (United States)

    Eum, Sung Yong; 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 1h. Moreover, lipid rafts modulated the reduction of occludin level through activation of matrix metalloproteinase 2 (MMP-2) after 24h 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.

  17. Endothelial Cells and Astrocytes: A Concerto en Duo in Ischemic Pathophysiology

    Directory of Open Access Journals (Sweden)

    Vincent Berezowski

    2012-01-01

    Full Text Available The neurovascular/gliovascular unit has recently gained increased attention in cerebral ischemic research, especially regarding the cellular and molecular changes that occur in astrocytes and endothelial cells. In this paper we summarize the recent knowledge of these changes in association with edema formation, interactions with the basal lamina, and blood-brain barrier dysfunctions. We also review the involvement of astrocytes and endothelial cells with recombinant tissue plasminogen activator, which is the only FDA-approved thrombolytic drug after stroke. However, it has a narrow therapeutic time window and serious clinical side effects. Lastly, we provide alternative therapeutic targets for future ischemia drug developments such as peroxisome proliferator- activated receptors and inhibitors of the c-Jun N-terminal kinase pathway. Targeting the neurovascular unit to protect the blood-brain barrier instead of a classical neuron-centric approach in the development of neuroprotective drugs may result in improved clinical outcomes after stroke.

  18. Triazole RGD antagonist reverts TGFβ1-induced endothelial-to-mesenchymal transition in endothelial precursor cells.

    Science.gov (United States)

    Bianchini, Francesca; Peppicelli, Silvia; Fabbrizzi, Pierangelo; Biagioni, Alessio; Mazzanti, Benedetta; Menchi, Gloria; Calorini, Lido; Pupi, Alberto; Trabocchi, Andrea

    2017-01-01

    Fibrosis is the dramatic consequence of a dysregulated reparative process in which activated fibroblasts (myofibroblasts) and Transforming Growth Factor β1 (TGFβ1) play a central role. When exposed to TGFβ1, fibroblast and epithelial cells differentiate in myofibroblasts; in addition, endothelial cells may undergo endothelial-to-mesenchymal transition (EndoMT) and actively participate to the progression of fibrosis. Recently, the role of αv integrins, which recognize the Arg-Gly-Asp (RGD) tripeptide, in the release and signal transduction activation of TGFβ1 became evident. In this study, we present a class of triazole-derived RGD antagonists that interact with αvβ3 integrin. Above different compounds, the RGD-2 specifically interferes with integrin-dependent TGFβ1 EndoMT in Endothelial Colony-Forming Cells (ECPCs) derived from circulating Endothelial Precursor Cells (ECPCs). The RGD-2 decreases the amount of membrane-associated TGFβ1, and reduces both ALK5/TGFβ1 type I receptor expression and Smad2 phosphorylation in ECPCs. We found that RGD-2 antagonist reverts EndoMT, reducing α-smooth muscle actin (α-SMA) and vimentin expression in differentiated ECPCs. Our results outline the critical role of integrin in fibrosis progression and account for the opportunity of using integrins as target for anti-fibrotic therapeutic treatment.

  19. Erythropoietin improves cardiac function through endothelial progenitor cell and vascular endothelial growth factor mediated neovascularization

    NARCIS (Netherlands)

    Westenbrink, B. Daan; Lipsic, Erik; van der Meer, Peter; van der Harst, Pirn; Oeseburg, Hisko; Sarvaas, Gideon J. Du Marchie; Koster, Johan; Voors, Adriaan A.; van Veldhuisen, Dirk J.; van Gilst, Wiek H.; Schoemaker, Regien G.

    2007-01-01

    Aims Erythropoietin (EPO) improves cardiac function and induces neovascutarization in chronic heart failure (CHF), although the exact mechanism has not been elucidated. We studied the effects of EPO on homing and incorporation of endothelial progenitor cells (EPC) into the myocardial microvasculatur

  20. Human iPSC-Derived Endothelial Cell Sprouting Assay in ...

    Science.gov (United States)

    Activation of vascular endothelial cells (ECs) by growth factors initiates a cascade of events in vivo consisting of EC tip cell selection, sprout formation, EC stalk cell proliferation, and ultimately vascular stabilization by support cells. Although EC functional assays can recapitulate one or more aspects of angiogenesis in vitro, they are often limited by a lack of definition to the substratum and lack of dependence on key angiogenic signaling axes. Here, we designed and characterized a chemically-defined model of endothelial sprouting behavior in vitro using human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs). Thiol-ene photopolymerization was used to rapidly encapsulate iPSC-ECs at high density in poly(ethylene glycol) (PEG) hydrogel spheres and subsequently to rapidly encapsulate iPSC-EC-containing hydrogel spheres in a cell-free over-layer. The hydrogel sprouting array here maintained pro-angiogenic phenotype of iPSC-ECs and supported growth factor-dependent proliferation and sprouting behavior. The sprouting model responded appropriately to several reference pharmacological angiogenesis inhibitors, which suggests the functional role of vascular endothelial growth factor, NF-κB, matrix metalloproteinase-2/9, protein kinase activity, and β-tubulin in endothelial sprouting. A blinded screen of 38 putative vascular disrupting compounds (pVDCs) from the US Environmental Protection Agency’s ToxCast library identified five compounds th

  1. Metformin improves endothelial function in aortic tissue and microvascular endothelial cells subjected to diabetic hyperglycaemic conditions.

    Science.gov (United States)

    Ghosh, Suparna; Lakshmanan, Arun P; Hwang, Mu Ji; Kubba, Haidar; Mushannen, Ahmed; Triggle, Chris R; Ding, Hong

    2015-12-01

    The cellular mechanisms whereby metformin, the first line drug for type 2 diabetes (T2DM), mediates its antidiabetic effects remain elusive, particularly as to whether metformin has a direct protective action on the vasculature. This study was designed to determine if a brief 3-h exposure to metformin protects endothelial function against the effects of hyperglycaemia. We investigated the protective effects of metformin on endothelial-dependent vasodilatation (EDV) in thoracic aortae from T2DM db/db mice and on high glucose (HG, 40 mM) induced changes in endothelial nitric oxide synthase (eNOS) signaling in mouse microvascular endothelial cells (MMECs) in culture. Exposure of aortae from db+/? non-diabetic control mice to high glucose (HG, 40 mM) containing Krebs for 3-h significantly (Pmetformin; metformin also improved ACh-induced EDV in aortae from diabetic db/db mice. Immunoblot analysis of MMECs cultured in HG versus NG revealed a significant reduction of the ratio of phosphorylated (p-eNOS)/eNOS and p-Akt/Akt, but not the expression of total eNOS or Akt. The 3-h exposure of MMECs to metformin significantly (Pmetformin can reverse/reduce the impact of HG on endothelial function, via mechanisms linked to increased phosphorylation of eNOS and Akt.

  2. Endothelial Progenitor Cells for Diagnosis and Prognosis in Cardiovascular Disease

    OpenAIRE

    2015-01-01

    Objective. To identify, evaluate, and synthesize evidence on the predictive power of circulating endothelial progenitor cells (EPCs) in cardiovascular disease, through a systematic review of quantitative studies. Data Sources. MEDLINE was searched using keywords related to “endothelial progenitor cells” and “endothelium” and, for the different categories, respectively, “smoking”; “blood pressure”; “diabetes mellitus” or “insulin resistance”; “dyslipidemia”; “aging” or “elderly”; “angina p...

  3. Characterization of vascular endothelial progenitor cells from chicken bone marrow

    Directory of Open Access Journals (Sweden)

    Bai Chunyu

    2012-05-01

    Full Text Available Abstract Background Endothelial progenitor cells (EPC are a type of stem cell used in the treatment of atherosclerosis, vascular injury and regeneration. At present, most of the EPCs studied are from human and mouse, whereas the study of poultry-derived EPCs has rarely been reported. In the present study, chicken bone marrow-derived EPCs were isolated and studied at the cellular level using immunofluorescence and RT-PCR. Results We found that the majority of chicken EPCs were spindle shaped. The growth-curves of chicken EPCs at passages (P 1, -5 and -9 were typically “S”-shaped. The viability of chicken EPCs, before and after cryopreservation was 92.2% and 81.1%, respectively. Thus, cryopreservation had no obvious effects on the viability of chicken EPCs. Dil-ac-LDL and FITC-UAE-1 uptake assays and immunofluorescent detection of the cell surface markers CD34, CD133, VEGFR-2 confirmed that the cells obtained in vitro were EPCs. Observation of endothelial-specific Weibel-Palade bodies using transmission electron microscopy further confirmed that the cells were of endothelial lineage. In addition, chicken EPCs differentiated into endothelial cells and smooth muscle cells upon induction with VEGF and PDGF-BB, respectively, suggesting that the chicken EPCs retained multipotency in vitro. Conclusions These results suggest that chicken EPCs not only have strong self-renewal capacity, but also the potential to differentiate into endothelial and smooth muscle cells. This research provides theoretical basis and experimental evidence for potential therapeutic application of endothelial progenitor cells in the treatment of atherosclerosis, vascular injury and diabetic complications.

  4. Fibroblast nemosis induces angiogenic responses of endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Enzerink, Anna, E-mail: anna.enzerink@helsinki.fi [Haartman Institute, University of Helsinki, P.O. BOX 21, FIN-00014 Helsinki (Finland); Rantanen, Ville, E-mail: ville.rantanen@helsinki.fi [Computational Systems Biology Laboratory, Institute of Biomedicine and Genome-Scale Biology Research Program, University of Helsinki, P.O. BOX 63, 00014 Helsinki (Finland); Vaheri, Antti, E-mail: antti.vaheri@helsinki.fi [Haartman Institute, University of Helsinki, P.O. BOX 21, FIN-00014 Helsinki (Finland)

    2010-03-10

    Increasing evidence points to a central link between inflammation and activation of the stroma, especially of fibroblasts therein. However, the mechanisms leading to such activation mostly remain undescribed. We have previously characterized a novel type of fibroblast activation (nemosis) where clustered fibroblasts upregulated the production of cyclooxygenase-2, secretion of prostaglandins, proteinases, chemotactic cytokines, and hepatocyte growth factor (HGF), and displayed activated nuclear factor-{kappa}B. Now we show that nemosis drives angiogenic responses of endothelial cells. In addition to HGF, nemotic fibroblasts secreted vascular endothelial growth factor (VEGF), and conditioned medium from spheroids promoted sprouting and networking of human umbilical venous endothelial cells (HUVEC). The response was partly inhibited by function-blocking antibodies against HGF and VEGF. Conditioned nemotic fibroblast medium promoted closure of HUVEC and human dermal microvascular endothelial cell monolayer wounds, by increasing the motility of the endothelial cells. Wound closure in HUVEC cells was partly inhibited by the antibodies against HGF. The stromal microenvironment regulates wound healing responses and often promotes tumorigenesis. Nemosis offers clues to the activation process of stromal fibroblasts and provides a model to study the part they play in angiogenesis-related conditions, as well as possibilities for therapeutical approaches desiring angiogenesis in tissue.

  5. Endothelial cells regulate neural crest and second heart field morphogenesis.

    Science.gov (United States)

    Milgrom-Hoffman, Michal; Michailovici, Inbal; Ferrara, Napoleone; Zelzer, Elazar; Tzahor, Eldad

    2014-07-04

    Cardiac and craniofacial developmental programs are intricately linked during early embryogenesis, which is also reflected by a high frequency of birth defects affecting both regions. The molecular nature of the crosstalk between mesoderm and neural crest progenitors and the involvement of endothelial cells within the cardio-craniofacial field are largely unclear. Here we show in the mouse that genetic ablation of vascular endothelial growth factor receptor 2 (Flk1) in the mesoderm results in early embryonic lethality, severe deformation of the cardio-craniofacial field, lack of endothelial cells and a poorly formed vascular system. We provide evidence that endothelial cells are required for migration and survival of cranial neural crest cells and consequently for the deployment of second heart field progenitors into the cardiac outflow tract. Insights into the molecular mechanisms reveal marked reduction in Transforming growth factor beta 1 (Tgfb1) along with changes in the extracellular matrix (ECM) composition. Our collective findings in both mouse and avian models suggest that endothelial cells coordinate cardio-craniofacial morphogenesis, in part via a conserved signaling circuit regulating ECM remodeling by Tgfb1.

  6. Endothelial cells regulate neural crest and second heart field morphogenesis

    Directory of Open Access Journals (Sweden)

    Michal Milgrom-Hoffman

    2014-07-01

    Full Text Available Cardiac and craniofacial developmental programs are intricately linked during early embryogenesis, which is also reflected by a high frequency of birth defects affecting both regions. The molecular nature of the crosstalk between mesoderm and neural crest progenitors and the involvement of endothelial cells within the cardio–craniofacial field are largely unclear. Here we show in the mouse that genetic ablation of vascular endothelial growth factor receptor 2 (Flk1 in the mesoderm results in early embryonic lethality, severe deformation of the cardio–craniofacial field, lack of endothelial cells and a poorly formed vascular system. We provide evidence that endothelial cells are required for migration and survival of cranial neural crest cells and consequently for the deployment of second heart field progenitors into the cardiac outflow tract. Insights into the molecular mechanisms reveal marked reduction in Transforming growth factor beta 1 (Tgfb1 along with changes in the extracellular matrix (ECM composition. Our collective findings in both mouse and avian models suggest that endothelial cells coordinate cardio–craniofacial morphogenesis, in part via a conserved signaling circuit regulating ECM remodeling by Tgfb1.

  7. Acrylamide induces accelerated endothelial aging in a human cell model.

    Science.gov (United States)

    Sellier, Cyril; Boulanger, Eric; Maladry, François; Tessier, Frédéric J; Lorenzi, Rodrigo; Nevière, Rémi; Desreumaux, Pierre; Beuscart, Jean-Baptiste; Puisieux, François; Grossin, Nicolas

    2015-09-01

    Acrylamide (AAM) has been recently discovered in food as a Maillard reaction product. AAM and glycidamide (GA), its metabolite, have been described as probably carcinogenic to humans. It is widely established that senescence and carcinogenicity are closely related. In vitro, endothelial aging is characterized by replicative senescence in which primary cells in culture lose their ability to divide. Our objective was to assess the effects of AAM and GA on human endothelial cell senescence. Human umbilical vein endothelial cells (HUVECs) cultured in vitro were used as model. HUVECs were cultured over 3 months with AAM or GA (1, 10 or 100 μM) until growth arrest. To analyze senescence, β-galactosidase activity and telomere length of HUVECs were measured by cytometry and semi-quantitative PCR, respectively. At all tested concentrations, AAM or GA reduced cell population doubling compared to the control condition (p < 0.001). β-galactosidase activity in endothelial cells was increased when exposed to AAM (≥10 μM) or GA (≥1 μM) (p < 0.05). AAM (≥10 μM) or GA (100 μM) accelerated telomere shortening in HUVECs (p < 0.05). In conclusion, in vitro chronic exposure to AAM or GA at low concentrations induces accelerated senescence. This result suggests that an exposure to AAM might contribute to endothelial aging.

  8. Characterization and comparison of embryonic stem cell-derived KDR+ cells with endothelial cells.

    Science.gov (United States)

    Sun, Xuan; Cheng, Lamei; Duan, Huaxin; Lin, Ge; Lu, Guangxiu

    2012-09-01

    Growing interest in utilizing endothelial cells (ECs) for therapeutic purposes has led to the exploration of human embryonic stem cells (hESCs) as a potential source for endothelial progenitors. In this study, ECs were induced from hESC lines and their biological characteristics were analyzed and compared with both cord blood endothelial progenitor cells (CBEPCs) and human umbilical vein endothelial cells (HUVECs) in vitro. The results showed that isolated embryonic KDR+ cells (EC-KDR+) display characteristics that were similar to CBEPCs and HUVECs. EC-KDR+, CBEPCs and HUVECs all expressed CD31 and CD144, incorporated DiI-Ac-LDL, bound UEA1 lectin, and were able to form tube-like structures on Matrigel. Compared with CBEPCs and HUVECs, the expression level of endothelial progenitor cell markers such as CD133 and KDR in EC-KDR+ was significantly higher, while the mature endothelial marker vWF was lowly expressed in EC-KDR+. In summary, the study showed that EC-KDR+ are primitive endothelial-like progenitors and might be a potential source for therapeutic vascular regeneration and tissue engineering.

  9. Infection of hepatitis B virus in extrahepatic endothelial tissues mediated by endothelial progenitor cells

    Directory of Open Access Journals (Sweden)

    Zhang Lili

    2007-04-01

    Full Text Available Abstract Background Hepatitis B virus (HBV replication has been reported to be involved in many extrahepatic viral disorders; however, the mechanism by which HBV is trans-infected into extrahepatic tissues such as HBV associated myocarditis remains largely unknown. Results In this study, we showed that human cord blood endothelial progenitor cells (EPCs, but not human umbilical vein endothelial cells (HUVECs could be effectively infected by uptake of HBV in vitro. Exposure of EPCs with HBV resulted in HBV DNA and viral particles were detected in EPCs at day 3 after HBV challenge, which were peaked around day 7 and declined in 3 weeks. Consistently, HBV envelope surface and core antigens were first detected in EPCs at day 3 after virus challenge and were retained to be detectable for 3 weeks. In contrast, HBV covalently closed circular DNA was not detected in EPCs at any time after virus challenge. Intravenous transplantation of HBV-treated EPCs into myocardial infarction and acute renal ischemia mouse model resulted in incorporation of HBV into injured heart, lung, and renal capillary endothelial tissues. Conclusion These results strongly support that EPCs serve as virus carrier mediating HBV trans-infection into the injured endothelial tissues. The findings might provide a novel mechanism for HBV-associated myocarditis and other HBV-related extrahepatic diseases as well.

  10. Isolation and culture of human umbilical vein endothelial cells (HUVEC).

    Science.gov (United States)

    Cheung, Ambrose L

    2007-02-01

    Human-derived endothelial cells can now be routinely harvested from human umbilical veins. Studies with human umbilical vein endothelial cells (HUVEC) have been conducted with cells from passage 2 to 5. It is now also possible to cryopreserve primary and early-passaged HUVEC for future propagation and for forwarding to an end user by express courier. Stored HUVEC have been stably retrieved even after several years. These retrieval techniques have facilitated the deployment of HUVEC for many studies, including those for homeostasis, inflammatory disorders, atherosclerosis, cancer, and microbial adhesion and invasion. In this unit, we will delineate the procedure for harvesting, propagation, and storage of HUVEC.

  11. Nanofiber density determines endothelial cell behavior on hydrogel matrix

    Energy Technology Data Exchange (ETDEWEB)

    Berti, Fernanda V., E-mail: fernanda@intelab.ufsc.br [Department of Chemical and Food Engineering, Federal University of Santa Catarina, 88040-900 Florianópolis, SC (Brazil); Rambo, Carlos R. [Department of Electrical Engineering, Federal University of Santa Catarina, 88040-900 Florianópolis, SC (Brazil); Dias, Paulo F. [Department of Cell Biology, Embryology and Genetics, Federal University of Santa Catarina, 88040-900 Florianópolis, SC (Brazil); Porto, Luismar M. [Department of Chemical and Food Engineering, Federal University of Santa Catarina, 88040-900 Florianópolis, SC (Brazil)

    2013-12-01

    When cultured under static conditions, bacterial cellulose pellicles, by the nature of the polymer synthesis that involves molecular oxygen, are characterized by two distinct surface sides. The upper surface is denser in fibers (entangled) than the lower surface that shows greater surface porosity. Human umbilical vein endothelial cells (HUVECs) were used to exploit how the microarchitecture (i.e., surface porosity, fiber network structure, surface topology, and fiber density) of bacterial cellulose pellicle surfaces influence cell–biomaterial interaction and therefore cell behavior. Adhesion, cell ingrowth, proliferation, viability and cell death mechanisms were evaluated on the two pellicle surface sides. Cell behavior, including secondary necrosis, is influenced only by the microarchitecture of the surface, since the biomaterial is extremely pure (constituted of cellulose and water only). Cell–cellulose fiber interaction is the determinant signal in the cell–biomaterial responses, isolated from other frequently present interferences such as protein and other chemical traces usually present in cell culture matrices. Our results suggest that microarchitecture of hydrogel materials might determine the performance of biomedical products, such as bacterial cellulose tissue engineering constructs (BCTECs). - Highlights: • Topography of BC pellicle is relevant to determine endothelial cells' fate. • Cell–biomaterial response is affected by the topography of BC-pellicle surface. • Endothelial cells exhibit different behavior depending on the BC topography. • Apoptosis and necrosis of endothelial cells were affected by the BC topography.

  12. In vitro behaviour of endothelial cells on a titanium surface

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    Oliveira-Filho Ricardo

    2008-07-01

    Full Text Available Abstract Background Endothelial cells play an important role in the delivery of cells to the inflammation site, chemotaxis, cell adhesion and extravasation. Implantation of a foreign material into the human body determines inflammatory and repair reactions, involving different cell types with a plethora of released chemical mediators. The evaluation of the interaction of endothelial cells and implanted materials must take into account other parameters in addition to the analysis of maintenance of cell viability. Methods In the present investigation, we examined the behavior of human umbilical vein endothelial cells (HUVECs harvested on titanium (Ti, using histological and immunohistochemical methods. The cells, after two passages, were seeded in a standard density on commercially plate-shaped titanium pieces, and maintained for 1, 7 or 14 days. Results After 14 days, we could observe a confluent monolayer of endothelial cells (ECs on the titanium surface. Upon one-day Ti/cell contact the expression of fibronectin was predominantly cytoplasmatic and stronger than on the control surface. It was observed strong and uniform cell expression along the time of α5β1 integrin on the cells in contact with titanium. Conclusion The attachment of ECs on titanium was found to be related to cellular-derived fibronectin and the binding to its specific receptor, the α5β1 integrin. It was observed that titanium effectively serves as a suitable substrate for endothelial cell attachment, growth and proliferation. However, upon a 7-day contact with Ti, the Weibel-Palade bodies appeared to be not fully processed and exhibited an anomalous morphology, with corresponding alterations of PECAM-1 localization.

  13. Endothelial Cells Stimulate Self-Renewal and Expand Neurogenesis of Neural Stem Cells

    Science.gov (United States)

    Shen, Qin; Goderie, Susan K.; Jin, Li; Karanth, Nithin; Sun, Yu; Abramova, Natalia; Vincent, Peter; Pumiglia, Kevin; Temple, Sally

    2004-05-01

    Neural stem cells are reported to lie in a vascular niche, but there is no direct evidence for a functional relationship between the stem cells and blood vessel component cells. We show that endothelial cells but not vascular smooth muscle cells release soluble factors that stimulate the self-renewal of neural stem cells, inhibit their differentiation, and enhance their neuron production. Both embryonic and adult neural stem cells respond, allowing extensive production of both projection neuron and interneuron types in vitro. Endothelial coculture stimulates neuroepithelial cell contact, activating Notch and Hes1 to promote self-renewal. These findings identify endothelial cells as a critical component of the neural stem cell niche.

  14. Human neural stem cell-induced endothelial morphogenesis requires autocrine/paracrine and juxtacrine signaling

    Science.gov (United States)

    Chou, Chung-Hsing; Modo, Michel

    2016-01-01

    Transplanted neural stem cells (NSC) interact with the host brain microenvironment. A neovascularization is commonly observed in the vicinity of the cell deposit, which is correlated with behavioral improvements. To elucidate the signaling mechanisms between human NSCs and endothelial cells (ECs), these were cocultured in an in vitro model in which NSC-induced endothelial morphogenesis produced a neurovascular environment. Soluble (autocrine/paracrine) and contact–mediated (juxtacrine) signaling molecules were evaluated for two conditionally immortalized fetal NSC lines derived from the cortical anlage (CTXOE03) and ganglionic eminence (STROC05), as well as an adult EC line (D3) derived from the cerebral microvasculature of a hippocampal biopsy. STROC05 were 4 times as efficient to induce endothelial morphogenesis compared to CTXOE03. The cascade of reciprocal interactions between NSCs and ECs in this process was determined by quantifying soluble factors, receptor mapping, and immunocytochemistry for extracellular matrix molecules. The mechanistic significance of these was further evaluated by pharmacological blockade. The sequential cell-specific regulation of autocrine/paracrine and juxtacrine signaling accounted for the differential efficiency of NSCs to induce endothelial morphogenesis. These in vitro studies shed new light on the reciprocal interactions between NSCs and ECs, which are pivotal for our mechanistic understanding of the efficacy of NSC transplantation. PMID:27374240

  15. Experiment Study of Effect of Perfiuorohexyloctane on Corneal Endothelial Cells

    Institute of Scientific and Technical Information of China (English)

    Xiaoyan Ding; Chunfang Li; Lin Lu; Guanguang Feng; Huling Zheng

    2001-01-01

    Purpose: To investigate the effect of Perfluorohexyloctane (F6H8)on corneal endothelial celIs(CEC) of rabbit eyes. Methods: Fifteen New Zealand white rabbits were devided into two groups:experimental group(F6H8) and control group(BSS) . All rabbits underwent anterior chamber injection of 0. 15ml F6H8 or BSS. Slit-lamp biomicroscopy and corneal endothelium photography were performed pre-operatively and postoperatively. Histopathological examination and Transmission electron microscopy(TEM) were done after the rabbits were sacrificed. Results: All the corneas were clear. Since 4 weeks after operation, the endothelial cells were markedly irregular in size and shape and the number of endothelial cells was markedly decreased. Multilayered retrocorneal membranes (RCM)grew gradually 2 weeks after surgery. Vacuolar degeneration was seen in some endothelial cells. Nuclear degeneration and edema of plasma were seen in TEM. Conclusion: Corneal endothelial cell degenerated after contacting with F6H8 for 2 ~4weeks. As a silicone solvent, it should be removed completely after injection. We don't recommend it to be used as a new intraocular temponade. Eye Science 2001: 17:21 ~ 26.

  16. Magnetizable stent-grafts enable endothelial cell capture

    Science.gov (United States)

    Tefft, Brandon J.; Uthamaraj, Susheil; Harburn, J. Jonathan; Hlinomaz, Ota; Lerman, Amir; Dragomir-Daescu, Dan; Sandhu, Gurpreet S.

    2017-04-01

    Emerging nanotechnologies have enabled the use of magnetic forces to guide the movement of magnetically-labeled cells, drugs, and other therapeutic agents. Endothelial cells labeled with superparamagnetic iron oxide nanoparticles (SPION) have previously been captured on the surface of magnetizable 2205 duplex stainless steel stents in a porcine coronary implantation model. Recently, we have coated these stents with electrospun polyurethane nanofibers to fabricate prototype stent-grafts. Facilitated endothelialization may help improve the healing of arteries treated with stent-grafts, reduce the risk of thrombosis and restenosis, and enable small-caliber applications. When placed in a SPION-labeled endothelial cell suspension in the presence of an external magnetic field, magnetized stent-grafts successfully captured cells to the surface regions adjacent to the stent struts. Implantation within the coronary circulation of pigs (n=13) followed immediately by SPION-labeled autologous endothelial cell delivery resulted in widely patent devices with a thin, uniform neointima and no signs of thrombosis or inflammation at 7 days. Furthermore, the magnetized stent-grafts successfully captured and retained SPION-labeled endothelial cells to select regions adjacent to stent struts and between stent struts, whereas the non-magnetized control stent-grafts did not. Early results with these prototype devices are encouraging and further refinements will be necessary in order to achieve more uniform cell capture and complete endothelialization. Once optimized, this approach may lead to more rapid and complete healing of vascular stent-grafts with a concomitant improvement in long-term device performance.

  17. Opioid-induced proliferation of vascular endothelial cells

    Directory of Open Access Journals (Sweden)

    Sandra Leo

    2009-05-01

    Full Text Available Sandra Leo1,2, Rony Nuydens1, Theo F Meert11Pain and Neurology, CNS Department, Johnson and Johnson Pharmaceutical Research and Development, a division of Janssen Pharmaceutica N.V, Beerse, Belgium; 2Laboratory of Biological Psychology, University of Leuven, Leuven, BelgiumAbstract: Angiogenesis is an important issue in cancer research and opioids are often used to treat pain in cancer patients. Therefore it is important to know if the use of opioids is associated with an aberrant stimulation of tumor growth triggered by the stimulation of angiogenesis in cancer patients. Some studies in the literature have suggested the presence of the μ3 opioid receptor, known as the receptor for many opioids, on endothelial cells, which are key players in the process of angiogenesis. In this study we used endothelial cells known to express the μ3 opioid receptor (MOR3, to evaluate the effects of morphine on angiogenesis. We first investigated the effect of morphine on the proliferation of endothelial cells. We showed that morphine is able to stimulate vascular endothelial cell proliferation in vitro. This effect of morphine is mediated by the mitogen-activated protein kinase (MAPK pathway as pre-treatment with PD98059 inhibited this excessive proliferation. Because previous studies indicated nitric oxide (NO as a downstream messenger we investigated the role of NO in the aberrant proliferation of endothelial cells. Our data could not confirm these findings using intracellular NO measurements and quantitative fluorescence microscopy. The potential use and pitfalls of opioids in cancer patients is discussed in light of these negative findings. Keywords: endothelial cells, morphine, cell proliferation, MAPK, nitric oxide, μ3 opioid receptor, angiogenesis

  18. Tumor endothelial cells express high pentraxin 3 levels.

    Science.gov (United States)

    Hida, Kyoko; Maishi, Nako; Kawamoto, Taisuke; Akiyama, Kosuke; Ohga, Noritaka; Hida, Yasuhiro; Yamada, Kenji; Hojo, Takayuki; Kikuchi, Hiroshi; Sato, Masumi; Torii, Chisaho; Shinohara, Nobuo; Shindoh, Masanobu

    2016-12-01

    It has been described that tumor progression has many similarities to inflammation and wound healing in terms of the signaling processes involved. Among biological responses, angiogenesis, which is necessary for tumor progression and metastasis, is a common hallmark; therefore, tumor blood vessels have been considered as important therapeutic targets in anticancer therapy. We focused on pentraxin 3 (PTX3), which is a marker of cancer-related inflammation, but we found no reports on its expression and function in tumor blood vessels. Here we showed that PTX3 is expressed in mouse and human tumor blood vessels based on immunohistochemical analysis. We found that PTX3 is upregulated in primary mouse and human tumor endothelial cells compared to normal endothelial cells. We also showed that PTX3 plays an important role in the proliferation of the tumor endothelial cells. These results suggest that PTX3 is an important target for antiangiogenic therapy.

  19. Neuroprotection via matrix-trophic coupling between cerebral endothelial cells and neurons.

    Science.gov (United States)

    Guo, Shuzhen; Kim, Woo Jean; Lok, Josephine; Lee, Sun-Ryung; Besancon, Elaine; Luo, Bing-Hao; Stins, Monique F; Wang, Xiaoying; Dedhar, Shoukat; Lo, Eng H

    2008-05-27

    The neurovascular unit is an emerging concept that emphasizes homeostatic interactions between endothelium and cerebral parenchyma. Here, we show that cerebral endothelium are not just inert tubes for delivering blood, but they also secrete trophic factors that can be directly neuroprotective. Conditioned media from cerebral endothelial cells broadly protects neurons against oxygen-glucose deprivation, oxidative damage, endoplasmic reticulum stress, hypoxia, and amyloid neurotoxicity. This phenomenon is largely mediated by endothelial-produced brain-derived neurotrophic factor (BDNF) because filtering endothelial-conditioned media with TrkB-Fc eliminates the neuroprotective effect. Endothelial production of BDNF is sustained by beta-1 integrin and integrin-linked kinase (ILK) signaling. Noncytotoxic levels of oxidative stress disrupts ILK signaling and reduces endothelial levels of neuroprotective BDNF. These data suggest that cerebral endothelium provides a critical source of homeostatic support for neurons. Targeting these signals of matrix and trophic coupling between endothelium and neurons may provide new therapeutic opportunities for stroke and other CNS disorders.

  20. Treponema pallidum Invades Intercellular Junctions of Endothelial Cell Monolayers

    Science.gov (United States)

    Thomas, D. Denee; Navab, Mahamad; Haake, David A.; Fogelman, Alan M.; Miller, James N.; Lovett, Michael A.

    1988-05-01

    The pathogenesis of syphilis reflects invasive properties of Treponema pallidum, but the actual mode of tissue invasion is unknown. We have found two in vitro parallels of treponemal invasiveness. We tested whether motile T. pallidum could invade host cells by determining the fate of radiolabeled motile organisms added to a HeLa cell monolayer; 26% of treponemes associated with the monolayer in a trypsin-resistant niche, presumably between the monolayer and the surface to which it adhered, but did not attain intracellularity. Attachment of T. pallidum to cultured human and rabbit aortic and human umbilical vein endothelial cells was 2-fold greater than to HeLa cells. We added T. pallidum to aortic endothelial cells grown on membrane filters under conditions in which tight intercellular junctions had formed. T. pallidum was able to pass through the endothelial cell monolayers without altering tight junctions, as measured by electrical resistance. In contrast, heat-killed T. pallidum and the nonpathogen Treponema phagedenis biotype Reiter failed to penetrate the monolayer. Transmission electron micrographs of sections of the monolayer showed T. pallidum in intercellular junctions. Our in vitro observations suggest that these highly motile spirochetes may leave the circulation by invading the junctions between endothelial cells.

  1. β2 integrin-mediated crawling on endothelial ICAM-1 and ICAM-2 is a prerequisite for transcellular neutrophil diapedesis across the inflamed blood-brain barrier.

    Science.gov (United States)

    Gorina, Roser; Lyck, Ruth; Vestweber, Dietmar; Engelhardt, Britta

    2014-01-01

    In acute neuroinflammatory states such as meningitis, neutrophils cross the blood-brain barrier (BBB) and contribute to pathological alterations of cerebral function. The mechanisms that govern neutrophil migration across the BBB are ill defined. Using live-cell imaging, we show that LPS-stimulated BBB endothelium supports neutrophil arrest, crawling, and diapedesis under physiological flow in vitro. Investigating the interactions of neutrophils from wild-type, CD11a(-/-), CD11b(-/-), and CD18(null) mice with wild-type, junctional adhesion molecule-A(-/-), ICAM-1(null), ICAM-2(-/-), or ICAM-1(null)/ICAM-2(-/-) primary mouse brain microvascular endothelial cells, we demonstrate that neutrophil arrest, polarization, and crawling required G-protein-coupled receptor-dependent activation of β2 integrins and binding to endothelial ICAM-1. LFA-1 was the prevailing ligand for endothelial ICAM-1 in mediating neutrophil shear resistant arrest, whereas Mac-1 was dominant over LFA-1 in mediating neutrophil polarization on the BBB in vitro. Neutrophil crawling was mediated by endothelial ICAM-1 and ICAM-2 and neutrophil LFA-1 and Mac-1. In the absence of crawling, few neutrophils maintained adhesive interactions with the BBB endothelium by remaining either stationary on endothelial junctions or displaying transient adhesive interactions characterized by a fast displacement on the endothelium along the direction of flow. Diapedesis of stationary neutrophils was unchanged by the lack of endothelial ICAM-1 and ICAM-2 and occurred exclusively via the paracellular pathway. Crawling neutrophils, although preferentially crossing the BBB through the endothelial junctions, could additionally breach the BBB via the transcellular route. Thus, β2 integrin-mediated neutrophil crawling on endothelial ICAM-1 and ICAM-2 is a prerequisite for transcellular neutrophil diapedesis across the inflamed BBB.

  2. Effect of Antioxidants on Endothelial Cell Reactive Oxygen Species (ROI) Generation and Adhesion of Leukocytes to Endothelial Cells

    Institute of Scientific and Technical Information of China (English)

    Huang Qian; Michael Grafe; Kristoph Graf; Hans Lehmkuhl; Eckart Fleck

    2000-01-01

    Objective To investigate whether antioxidants inhibit adhesion of leukocytes to endothelium and furthermore, whether all antioxidants regulate NF-κB activation through a redox sensitive mechanism. Methods The effect of the antioxidative substances pyrrolidin dithiocarbamat (PDTC),dichloroisocumarin (DCI), chrysin and probucol on the endothelial leukocyte adhesion were examined under near physiological flow conditions. The antioxidative activity of antioxidants was measured in a DCF fluorescence assay with flow cytometry. The activation of NF-κB in endothelial cells was investigated in a gel shift assay. Results PDTC and probucol did not show an inhibitory effect to the formation of intracellular H2O2 in TNFct activated human vascular endothelial cells (HUVEC) . Chrysin showed a moderate effect.DCI showed a strong antioxidative effect. In contrast,PDTC and chrysin inhibited the adhesion of HL 60 cells to TNFa-stimulated HUVEC. DCI and probucol did not have influence on the adhesion within the area of the examined shear stresses. Only PDTC inhibited the TNFα-induced activation of NF-kB in endothelial cells.Conclusion The inhibition of the endothelial leukocyte adhesion by antioxidative substances is not to be explained by its antioxidative characteristics only. The inhibitory effect of PDTC on NF-kB activation was probably not related to its antioxidative properties.

  3. Contractile proteins of endothelial cells, platelets and smooth muscle.

    Science.gov (United States)

    Becker, C G; Nachman, R L

    1973-04-01

    In experiments described herein it was observed, by direct and indirect immunofluorescence technics, that rabbit antisera to human platelet actomyosin (thrombosthenin) stained mature megakaryocytes, blood platelets, endothelial cells and smooth muscle cells of arteries and veins, endothelial cells of liver sinusoids and certain capillaries, uterine smooth muscle cells, myoepithelial cells, perineurial cells of peripheral nerves and "fibroblastic" cells of granulation tissue. The specificity of immunohistologic staining was confirmed by appropriate absorption and blocking studies and immunodiffusional analysis in agarose gel. It was also observed by immunodiffusional analysis in agarose gel, electrophoresis of actomyosin fragments in polyacrylamide gels, immune inhibition of actomyosin ATPase activity and immune aggregation of platelets that uterine and platelet actomyosin are partially, but not completely, identical.

  4. Effects of irradiated biodegradable polymer in endothelial cell monolayer formation

    Energy Technology Data Exchange (ETDEWEB)

    Arbeitman, Claudia R.; Grosso, Mariela F. del [CONICET – Consejo Nacional de Investigaciones Científicas y Técnicas (Argentina); Gerencia de Investigación y Aplicaciones, TANDAR-CNEA (Argentina); Behar, Moni [Instituto de Física, UFRGS, Porto Alegre, RS (Brazil); García Bermúdez, Gerardo, E-mail: ggb@tandar.cnea.gov.ar [CONICET – Consejo Nacional de Investigaciones Científicas y Técnicas (Argentina); Gerencia de Investigación y Aplicaciones, TANDAR-CNEA (Argentina); Escuela de Ciencia y Tecnología, UNSAM (Argentina)

    2013-11-01

    In this work we study cell adhesion, proliferation and cell morphology of endothelial cell cultured on poly-L-lactide acid (PLLA) modified by heavy ion irradiation. Thin films of PLLA samples were irradiated with sulfur (S) at energies of 75 MeV and gold (Au) at 18 MeV ion-beams. Ion beams were provided by the Tandar (Buenos Aires, Argentina) and Tandetron (Porto Alegre, Brazil) accelerators, respectively. The growth of a monolayer of bovine aortic endothelial cells (BAEC) onto unirradiated and irradiated surfaces has been studied by in vitro techniques in static culture. Cell viability and proliferation increased on modified substrates. But the results on unirradiated samples, indicate cell death (necrosis/apoptosis) with the consequent decrease in proliferation. We analyzed the correlation between irradiation parameters and cell metabolism and morphology.

  5. Endothelial cells create a stem cell niche in glioblastoma by providing NOTCH ligands that nurture self-renewal of cancer stem-like cells.

    Science.gov (United States)

    Zhu, Thant S; Costello, Mark A; Talsma, Caroline E; Flack, Callie G; Crowley, Jessica G; Hamm, Lisa L; He, Xiaobing; Hervey-Jumper, Shawn L; Heth, Jason A; Muraszko, Karin M; DiMeco, Francesco; Vescovi, Angelo L; Fan, Xing

    2011-09-15

    One important function of endothelial cells in glioblastoma multiforme (GBM) is to create a niche that helps promote self-renewal of cancer stem-like cells (CSLC). However, the underlying molecular mechanism for this endothelial function is not known. Since activation of NOTCH signaling has been found to be required for propagation of GBM CSLCs, we hypothesized that the GBM endothelium may provide the source of NOTCH ligands. Here, we report a corroboration of this concept with a demonstration that NOTCH ligands are expressed in endothelial cells adjacent to NESTIN and NOTCH receptor-positive cancer cells in primary GBMs. Coculturing human brain microvascular endothelial cells (hBMEC) or NOTCH ligand with GBM neurospheres promoted GBM cell growth and increased CSLC self-renewal. Notably, RNAi-mediated knockdown of NOTCH ligands in hBMECs abrogated their ability to induce CSLC self-renewal and GBM tumor growth, both in vitro and in vivo. Thus, our findings establish that NOTCH activation in GBM CSLCs is driven by juxtacrine signaling between tumor cells and their surrounding endothelial cells in the tumor microenvironment, suggesting that targeting both CSLCs and their niche may provide a novel strategy to deplete CSLCs and improve GBM treatment.

  6. 大鼠原代脑微血管内皮细胞体外分离与培养的实验研究%In Vitro Isolation and Culture of Primary Rat Brain Microvascular Endothelial Cells

    Institute of Scientific and Technical Information of China (English)

    李振; 刘云会; 薛一雪; 刘丽波; 王萍

    2012-01-01

    目的 探讨获取大鼠脑微血管内皮细胞(BMEC)的简单、有效方法,为构建体外血肿瘤屏障(BTB)模型提供材料.方法 采集出生3~5 d的Wistar胎鼠大脑皮质,应用酶消化法及葡聚糖离心法获得脑微血管段后,接种于培养皿中进行原代培养,采用倒置显微镜对所培养的细胞进行形态学观察;以Ⅷ因子相关抗原免疫组化染色法鉴定细胞;将BMEC与C6脑胶质瘤细胞共培养,构建体外BTB模型,并采用免疫组化法和免疫荧光法检测BMEC间紧密连接相关蛋白occludin的表达.结果 体外培养2h时脑微血管段贴壁,12~48 h见圆形生发中心形成,2~3d单层内皮细胞自生发中心长出,4~5 d见较大单层内皮细胞团,5~7 d可见融合成片的内皮细胞单层,外观呈“铺路石”样;第Ⅷ因子免疫组化结果显示“铺路石”样细胞胞质呈棕黄色染色;紧密连接相关蛋白occludin的免疫组化和荧光结果证明共培养的BMEC间表达BTB的特性.结论 本方法能成功地进行大鼠原代BMEC培养,构建大鼠体外BTB模型,进而应用于BTB的生理、生化及药理学研究.%Objective To explore a simple and reprodueable method lor the in vitro isolation and eulture of rat brain microvascular endothelial cells (BMEC), so as to provide materials for the construction of in vilro blood tumor barrier ( BTB) models. Methods Relative!* pure cerebral microvessel fragments were obtained from the cortex of 3-5 days old wistar rats through dissection,enzyme digestion,and dex-tran centrifugation. Then, these fragments were seeded on dishes for primary culture. The morphology of BMEC was observed under an invert microscope. BMEC were identified by immunohistoehemistry with factor VIII-associated antigen. In vitro BTB models were constructed by co-cultivation of BMEC with C6 glioma cells. The expression of tight junction-related protein oeeludin in BMEC of BTB was measured by immunohistoehemistry and

  7. Analysis of proteome response to the mobile phone radiation in two types of human primary endothelial cells

    Directory of Open Access Journals (Sweden)

    Kuster Niels

    2010-10-01

    Full Text Available Abstract Background Use of mobile phones has widely increased over the past decade. However, in spite of the extensive research, the question of potential health effects of the mobile phone radiation remains unanswered. We have earlier proposed, and applied, proteomics as a tool to study biological effects of the mobile phone radiation, using as a model human endothelial cell line EA.hy926. Exposure of EA.hy926 cells to 900 MHz GSM radiation has caused statistically significant changes in expression of numerous proteins. However, exposure of EA.hy926 cells to 1800 MHz GSM signal had only very small effect on cell proteome, as compared with 900 MHz GSM exposure. In the present study, using as model human primary endothelial cells, we have examined whether exposure to 1800 MHz GSM mobile phone radiation can affect cell proteome. Results Primary human umbilical vein endothelial cells and primary human brain microvascular endothelial cells were exposed for 1 hour to 1800 MHz GSM mobile phone radiation at an average specific absorption rate of 2.0 W/kg. The cells were harvested immediately after the exposure and the protein expression patterns of the sham-exposed and radiation-exposed cells were examined using two dimensional difference gel electrophoresis-based proteomics (2DE-DIGE. There were observed numerous differences between the proteomes of human umbilical vein endothelial cells and human brain microvascular endothelial cells (both sham-exposed. These differences are most likely representing physiological differences between endothelia in different vascular beds. However, the exposure of both types of primary endothelial cells to mobile phone radiation did not cause any statistically significant changes in protein expression. Conclusions Exposure of primary human endothelial cells to the mobile phone radiation, 1800 MHz GSM signal for 1 hour at an average specific absorption rate of 2.0 W/kg, does not affect protein expression, when the

  8. Endothelial progenitor cells induce a phenotype shift in differentiated endothelial cells towards PDGF/PDGFRβ axis-mediated angiogenesis.

    Directory of Open Access Journals (Sweden)

    Moritz Wyler von Ballmoos

    Full Text Available BACKGROUND: Endothelial Progenitor Cells (EPC support neovascularization and regeneration of injured endothelium both by providing a proliferative cell pool capable of differentiation into mature vascular endothelial cells and by secretion of angiogenic growth factors. OBJECTIVE: The aim of this study was to investigate the role of PDGF-BB and PDGFRβ in EPC-mediated angiogenesis of differentiated endothelial cells. METHODS AND RESULTS: Conditioned medium from human EPC (EPC-CM cultured in hypoxic conditions contained substantially higher levels of PDGF-BB as compared to normoxic conditions (P<0.01. EPC-CM increased proliferation (1.39-fold; P<0.001 and migration (2.13-fold; P<0.001 of isolated human umbilical vein endothelial cells (HUVEC, as well as sprouting of vascular structures from ex vivo cultured aortic rings (2.78-fold increase; P = 0.01. The capacity of EPC-CM to modulate the PDGFRβ expression in HUVEC was assessed by western blot and RT-PCR. All the pro-angiogenic effects of EPC-CM on HUVEC could be partially inhibited by inactivation of PDGFRβ (P<0.01. EPC-CM triggered a distinct up-regulation of PDGFRβ (2.5±0.5; P<0.05 and its phosphorylation (3.6±0.6; P<0.05 in HUVEC. This was not observed after exposure of HUVEC to recombinant human PDGF-BB alone. CONCLUSION: These data indicate that EPC-CM sensitize endothelial cells and induce a pro-angiogenic phenotype including the up-regulation of PDGFRβ, thereby turning the PDGF/PDGFRβ signaling-axis into a critical element of EPC-induced endothelial angiogenesis. This finding may be utilized to enhance EPC-based therapy of ischemic tissue in future.

  9. Growth-limiting role of endothelial cells in endoderm development.

    Science.gov (United States)

    Sand, Fredrik Wolfhagen; Hörnblad, Andreas; Johansson, Jenny K; Lorén, Christina; Edsbagge, Josefina; Ståhlberg, Anders; Magenheim, Judith; Ilovich, Ohad; Mishani, Eyal; Dor, Yuval; Ahlgren, Ulf; Semb, Henrik

    2011-04-15

    Endoderm development is dependent on inductive signals from different structures in close vicinity, including the notochord, lateral plate mesoderm and endothelial cells. Recently, we demonstrated that a functional vascular system is necessary for proper pancreas development, and that sphingosine-1-phosphate (S1P) exhibits the traits of a blood vessel-derived molecule involved in early pancreas morphogenesis. To examine whether S1P(1)-signaling plays a more general role in endoderm development, S1P(1)-deficient mice were analyzed. S1P(1) ablation results in compromised growth of several foregut-derived organs, including the stomach, dorsal and ventral pancreas and liver. Within the developing pancreas the reduction in organ size was due to deficient proliferation of Pdx1(+) pancreatic progenitors, whereas endocrine cell differentiation was unaffected. Ablation of endothelial cells in vitro did not mimic the S1P(1) phenotype, instead, increased organ size and hyperbranching were observed. Consistent with a negative role for endothelial cells in endoderm organ expansion, excessive vasculature was discovered in S1P(1)-deficient embryos. Altogether, our results show that endothelial cell hyperplasia negatively influences organ development in several foregut-derived organs.

  10. Ex Vivo Behaviour of Human Bone Tumor Endothelial Cells

    Energy Technology Data Exchange (ETDEWEB)

    Infante, Teresa [SDN-Foundation, Institute of Diagnostic and Nuclear Development, IRCCS, 80143 Naples (Italy); Cesario, Elena [Department of Biochemistry and Biophysics, Second University of Naples, 80138 Naples (Italy); Gallo, Michele; Fazioli, Flavio [Division of Skeletal Muscles Oncology Surgery, National Cancer Institute, Pascale Foundation, 80131 Naples (Italy); De Chiara, Annarosaria [Anatomic Pathology Unit, National Cancer Institute, Pascale Foundation, 80131 Naples (Italy); Tutucci, Cristina; Apice, Gaetano [Medical Oncology of Bone and Soft Sarcoma tissues Unit, National Cancer Institute, Pascale Foundation, 80131 Naples (Italy); Nigris, Filomena de, E-mail: filomena.denigris@unina2.it [Department of Biochemistry and Biophysics, Second University of Naples, 80138 Naples (Italy)

    2013-04-11

    Cooperation between endothelial cells and bone in bone remodelling is well established. In contrast, bone microvasculature supporting the growth of primary tumors and metastasis is poorly understood. Several antiangiogenic agents have recently been undergoing trials, although an extensive body of clinical data and experimental research have proved that angiogenic pathways differ in each tumor type and stage. Here, for the first time, we characterize at the molecular and functional level tumor endothelial cells from human bone sarcomas at different stages of disease and with different histotypes. We selected a CD31{sup +} subpopulation from biopsies that displayed the capability to grow as adherent cell lines without vascular endothelial growth factor (VEGF). Our findings show the existence in human primary bone sarcomas of highly proliferative endothelial cells expressing CD31, CD44, CD105, CD146 and CD90 markers. These cells are committed to develop capillary-like structures and colony formation units, and to produce nitric oxide. We believe that a better understanding of tumor vasculature could be a valid tool for the design of an efficacious antiangiogenic therapy as adjuvant treatment of sarcomas.

  11. Endothelial progenitor cell differentiation using cryopreserved, umbilical cord blood-derived mononuclear cells

    Institute of Scientific and Technical Information of China (English)

    Jun-ho JANG; Hugh C KIM; Sun-kyung KIM; Jeong-eun CHOI; Young-jin KIM; Hyun-woo LEE; Seok-yun KANG; Joon-seong PARK; Jin-hyuk CHOI; Ho-yeong LIM

    2007-01-01

    Aim: To investigate the endothelial differentiation potentiality of umbilical cord blood (UCB), we induced the differentiation of endothelial progenitor cells (EPC)from cryopreserved UCB-derived mononuclear cells (MNC). Methods: MNC from cryopreserved UCB and peripheral blood (PB) were cultured in M199 medium with endothelial cell growth supplements for 14 d. EPC were characterized by RT-PCR,flow cytometry, and immunocytochemistry analysis. The proliferation of differen-tiated EPC was studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTI') assay, and vascular endothelial growth factor (VEGF) concentra-tion was measured using an ELISA kit. Characteristics of UCB-derived EPC were compared with those of PB-derived EPC. Results: A number of round-shaped cells were loosely attached to the bottom after 24 h culture, and numerous spindle-shaped cells began to appear from the round-shaped ones on d 7. Those cells expressed endothelial markers such as, Fit-1/VEGFR-1, ecNOS, VE-cadherin, yon Willebrand factor, and secreted VEGF. The patterns of endothelial markers of EPC from PB and UCB did not show striking differences. The results of the prolifera-tion and secretion of VEGF were also similar. Conclusion: We successfully cul-tured UCB cells stored at -196 ℃ into cells with the quality of endothelial cells.Those EPC could be used for angiogenic therapeutics by activating adjacent endothelial cells and enhancing angiogenesis.

  12. Endothelial cells stimulate growth of normal and cancerous breast epithelial cells in 3D culture

    Directory of Open Access Journals (Sweden)

    Magnusson Magnus K

    2010-07-01

    Full Text Available Abstract Background Epithelial-stromal interaction provides regulatory signals that maintain correct histoarchitecture and homeostasis in the normal breast and facilitates tumor progression in breast cancer. However, research on the regulatory role of the endothelial component in the normal and malignant breast gland has largely been neglected. The aim of the study was to investigate the effects of endothelial cells on growth and differentiation of human breast epithelial cells in a three-dimensional (3D co-culture assay. Methods Breast luminal and myoepithelial cells and endothelial cells were isolated from reduction mammoplasties. Primary cells and established normal and malignant breast cell lines were embedded in reconstituted basement membrane in direct co-culture with endothelial cells and by separation of Transwell filters. Morphogenic and phenotypic profiles of co-cultures was evaluated by phase contrast microscopy, immunostaining and confocal microscopy. Results In co-culture, endothelial cells stimulate proliferation of both luminal- and myoepithelial cells. Furthermore, endothelial cells induce a subpopulation of luminal epithelial cells to form large acini/ducts with a large and clear lumen. Endothelial cells also stimulate growth and cloning efficiency of normal and malignant breast epithelial cell lines. Transwell and gradient co-culture studies show that endothelial derived effects are mediated - at least partially - by soluble factors. Conclusion Breast endothelial cells - beside their role in transporting nutrients and oxygen to tissues - are vital component of the epithelial microenvironment in the breast and provide proliferative signals to the normal and malignant breast epithelium. These growth promoting effects of endothelial cells should be taken into consideration in breast cancer biology.

  13. Biomechanical changes in endothelial cells result from an inflammatory response

    Science.gov (United States)

    Vaitkus, Janina; Stroka, Kimberly; Aranda-Espinoza, Helim

    2012-02-01

    During periods of infection and disease, the immune system induces the release of TNF-α, an inflammatory cytokine, from a variety of cell types, such as macrophages. TNF-α, while circulating in the vasculature, binds to the apical surface of endothelial cells and causes a wide range of biological and mechanical changes to the endothelium. While the biological changes have been widely studied, the biomechanical aspects have been largely unexplored. Here, we investigated the biomechanical changes of the endothelium as a function of TNF-α treatment. First, we studied the traction forces applied by the endothelium, an effect that is much less studied than others. Through the use of traction force microscopy, we found that TNF-α causes an increase in traction forces applied by the endothelial cells as compared to non-treated cells. Then, we investigated cell morphology, cell mechanics, migration, and cytoskeletal dynamics. We found that in addition to increasing applied traction forces, TNF-α causes an increase in cell area and aspect ratio on average, as well as a shift in the organization of F-actin filaments within the cell. Combining these findings together, our results show that an inflammatory response heavily impacts the morphology, cell mechanics, migration, cytoskeletal dynamics, and applied traction forces of endothelial cells.

  14. High glucose mediates endothelial-to-chondrocyte transition in human aortic endothelial cells

    Directory of Open Access Journals (Sweden)

    Tang Rining

    2012-09-01

    Full Text Available Abstract Background Vascular calcification is one of the common complications in diabetes mellitus. Many studies have shown that high glucose (HG caused cardiovascular calcification, but its underlying mechanism is not fully understood. Recently, medial calcification has been most commonly described in the vessels of patients with diabetes. Chondrocytes were involved in the medial calcification. Recent studies have shown that the conversion into mesenchymal stem cells (MSCs via the endothelial-to-mesenchymal transition (EndMT could be triggered in chondrocytes. Our previous research has indicated that HG induced EndMT in human aortic endothelial cells (HAECs. Therefore, we addressed the question of whether HG-induced EndMT could be transitioned into MSCs and differentiated into chondrocytes. Methods HAECs were divided into three groups: a normal glucose (NG group, HG group (30 mmol/L, and mannitol (5.5 mmol/L NG + 24.5 mmol/L group. Pathological changes were investigated using fluorescence microscopy and electron microscopy. Immunofluorescence staining was performed to detect the co-expression of endothelial markers, such as CD31, and fibroblast markers, such as fibroblast-specific protein 1 (FSP-1. The expression of FSP-1 was detected by real time-PCR and western blots. Endothelial-derived MSCs were grown in MSC medium for one week. The expression of the MSCs markers STRO-1, CD44, CD10 and the chondrocyte marker SOX9 was detected by immunofluorescence staining and western blots. Chondrocyte expression was detected by alcian blue staining. Calcium deposits were analyzed by alizarin red staining. Results The incubation of HAECs exposed to HG resulted in a fibroblast-like phenotype. Double staining of the HAECs indicated a co-localization of CD31 and FSP-1. The expression of FSP-1 was significantly increased in the HG group, and the cells undergoing EndMT also expressed STRO-1, CD44 and SOX9 compared with the controls (P  Conclusions Our

  15. 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

  16. Effect of propionyl-L-carnitine on human endothelial cells

    NARCIS (Netherlands)

    Hinsbergh, V.W.M. van; Scheffer, M.A.

    1991-01-01

    A possible protective effect of propionyl-L-carnitine on human endothelial cells was studied both under basal culture conditions and in the presence of agents capable of influencing oxidative damage, such as glucose/glucose oxidase and oxidized low-density lipoproteins. Propionyl-L-carnitine had no

  17. METABOLIC CAPACITY REGULATES IRON HOMEOSTATIS IN ENDOTHELIAL CELLS

    Science.gov (United States)

    The sensitivity of endothelial cells to oxidative stress and the high concentrations of iron in mitochondria led us to test the hypotheses that (1) changes in respiratory capacity alter iron homeostasis, and (2) lack of aerobic metabolism decreases labile iron stores and attenuat...

  18. Are endothelial cell bioeffects from acoustic droplet vaporization proximity dependent?

    Science.gov (United States)

    Seda, Robinson; Li, David; Fowlkes, J. Brian; Bull, Joseph

    2013-11-01

    Acoustic droplet vaporization (ADV) produces gas microbubbles that provide a means of selective occlusion in gas embolotherapy. Vaporization and subsequent occlusion occur inside blood vessels supplying the targeted tissue, such as tumors. Theoretical and computational studies showed that ADV within a vessel can impart high fluid mechanical stresses on the vessel wall. Previous in vitro studies have demonstrated that vaporization at an endothelial layer may affect cell attachment and viability. The current study is aimed at investigating the role of vaporization distance away from the endothelial layer. HUVECs were cultured in OptiCell™ chambers until reaching confluence. Dodecafluoropentane microdroplets were added, attaining a 10:1 droplet to cell ratio. A single ultrasound pulse (7.5 MHz) consisting of 16 cycles (~ 2 μs) and a 5 MPa peak rarefactional pressure was used to produce ADV while varying the vaporization distance from the endothelial layer (0 μm, 500 μm, 1000 μm). Results indicated that cell attachment and viability was significantly different if the distance was 0 μm (at the endothelial layer). Other distances were not significantly different from the control. ADV will significantly affect the endothelium if droplets are in direct contact with the cells. Droplet concentration and flow conditions inside blood vessels may play an important role. This work was supported by NIH grant R01EB006476.

  19. Cartographic system for spatial distribution analysis of corneal endothelial cells.

    Science.gov (United States)

    Corkidi, G; Márquez, J; García-Ruiz, M; Díaz-Cintra, S; Graue, E

    1994-07-01

    A combined cartographic and morphometric endothelium analyser has been developed by integrating the HISTO 2000 histological imaging and analysis system with a prototype human corneal endothelium analyser. The complete system allows the elaboration and analysis of cartographies of corneal endothelial tissue, and hence the in vitro study of the spatial distribution of corneal endothelial cells, according to their regional morphometric characteristics (cell size and polygonality). The global cartographic reconstruction is obtained by sequential integration of the data analysed for each microscopic field. Subsequently, the location of each microscopically analysed field is referred to its real position on the histologic preparation by means of X-Y co-ordinates; both are provided by micrometric optoelectronic sensors installed on the optical microscope stage. Some cartographies of an excised human corneal keratoconus button in vitro are also presented. These cartographic images allow a macroscopic view of endothelial cells analysed microscopically. Parametric colour images show the spatial distribution of endothelial cells, according to their specific morphometric parameters, and exhibit the variability in size and cellular shape which depend on the analysed area.

  20. Effects of hypergravity on the angiogenic potential of endothelial cells

    NARCIS (Netherlands)

    Costa-Almeida, R. (Raquel); Carvalho, D.T.O. (Daniel T.O.); Ferreira, M.J.S. (Miguel J.S.); Aresta, G. (Guilherme); Gomes, M.E. (Manuela E.); Van Loon, J.J.W.A. (Jack J.W.A.); K. van der Heiden (Kim); Granja, P.L. (Pedro L.)

    2016-01-01

    textabstractAngiogenesis, the formation of blood vessels from pre-existing ones, is a key event in pathology, including cancer progression, but also in homeostasis and regeneration. As the phenotype of endothelial cells (ECs) is continuously regulated by local biomechanical forces, studying endothel

  1. Endothelial progenitor cell-based neovascularization : implications for therapy

    NARCIS (Netherlands)

    Krenning, Guido; van Luyn, Marja J. A.; Harmsen, Martin C.

    2009-01-01

    Ischemic cardiovascular events are a major cause of death globally. Endothelial progenitor cell (EPC)-based approaches can result in improvement of vascular perfusion and might offer clinical benefit. However, although functional improvement is observed, the lack of long-term engraftment of EPCs int

  2. High glucose augments stress-induced apoptosis in endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Wenwen Zhong; Yang Liu; Hui Tian

    2009-01-01

    Hyperglycemia has been identified as one of the important factors involved in the microvascular complications of diabetes, and has been related to increased cardiovascular mortality. Endothelial damage and dysfunction result from diabetes; therefore, the aim of this study was to determine the response of endothelial cells to stressful stimuli, modelled in normal and high glucose concentrations in vitro. Eahy 926 endothelial cells were cultured in 5 mmol/L or 30 mmol/L glucose conditions for a 24 hour period and oxidative stress was induced by exposure to hydrogen peroxide (H2O2) or tumour necrosis factor- α (TNF- α ), following which the protective effect of the glucocorticoid dexamethasone was assessed. Apoptosis, necrosis and cell viability were determined using an ELISA for DNA fragmentation, an enzymatic lactate dehydrogenase assay and an MTT assay, respectively. High glucose significantly increased the susceptibility of Eahy 926 cells to apoptosis in the presence of 500 μmol/L H2O2, above that induced in normal glucose (P<0.02). A reduction of H2O2- and TNF- α -induced apoptosis occurred in both high and low glucose after treatment with dexametha-sone (P<0.05). Conclusion high glucose is effective in significantly augmenting stress caused by H2O2, but not in causing stress alone. These findings suggest a mechanism by which short term hyperglycemia may facilitate and augment endothelial damage.

  3. 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.

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  1. File list: DNS.CDV.50.AllAg.Primary_endothelial_cells [Chip-atlas[Archive

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  4. File list: His.CDV.10.AllAg.Primary_endothelial_cells [Chip-atlas[Archive

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  5. File list: DNS.CDV.10.AllAg.Primary_endothelial_cells [Chip-atlas[Archive

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  6. File list: Unc.CDV.20.AllAg.Primary_endothelial_cells [Chip-atlas[Archive

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  7. File list: Oth.CDV.10.AllAg.Primary_endothelial_cells [Chip-atlas[Archive

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  12. File list: ALL.CDV.05.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

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  20. File list: ALL.CDV.10.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

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  1. File list: His.CDV.10.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

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  3. File list: Pol.CDV.05.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

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  4. File list: Pol.CDV.50.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

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  5. File list: Unc.CDV.50.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

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  6. Vascular endothelial cells and dysfunctions: role of melatonin.

    Science.gov (United States)

    Rodella, Luigi Fabrizio; Favero, Gaia; Foglio, Eleonora; Rossini, Claudia; Castrezzati, Stefania; Lonati, Claudio; Rezzani, Rita

    2013-01-01

    Several pathological conditions, including hypertension, atherosclerosis, diabetes, ischemia/reperfusion injury and nicotine-induced vasculopathy, are associated with vascular endothelial dysfunction characterized by altered secretory output of endothelial cells. Therefore there is a search for molecules and interventions that could restore endothelial function, in particular augmenting NO production, reducing the generation of free radicals and vasoconstrictors and preventing undesired inflammation. The pineal hormone melatonin exhibits several endothelium protective properties: it scavenges free radicals, activates antioxidant defence enzymes, normalizes lipid and blood pressure profile and increases NO bioavailability. Melatonin improved vascular function in experimental hypertension, reducing intimal infiltration and restoring NO production. Melatonin improved the NO pathway also in animal models for the study of diabetes and prevented NO down-regulation and adhesive molecules up-regulation in nicotine-induced vasculopathy. The protection against endothelial damage, vasoconstriction, platelet aggregation and leukocyte infiltration might contribute to the beneficial effects against ischemia-reperfusion injury by melatonin. Therefore, melatonin administration has endothelium-protective potential in several pathological conditions. Nevertheless, it still needs to be established, whether melatonin is able to revert already established endothelial dysfunction in these conditions.

  7. Establishment of an in vitro blood-brain barrier model by co-culturing rat brain microvascular endothelial cells,pericytes and astrocytes%大鼠脑微血管内皮细胞与周细胞、星形胶质细胞共培养建立体外血脑屏障模型

    Institute of Scientific and Technical Information of China (English)

    查雨锋; 傅晓钟; 张顺; 罗敏; 欧瑜; 董永喜; 王爱民; 王永林

    2015-01-01

    目的:应用原代培养的大鼠脑微血管内皮细胞(brain-microvessel endothelial cells,BMECs )与脑微血管周细胞(brain-microvessel pericytes,BMPC )、星形胶质细胞(astro-cytes,AS)共培养建立可模拟在体状态的体外血脑屏障(blood-brain barrier,BBB)模型。方法原代分离、纯化和培养大鼠BMECs、BMPC和AS,通过细胞形态学和免疫细胞化学染色方法鉴定原代培养的细胞,应用Millicell细胞培养插(孔径0.4μm)建立5种不同类型的体外BBB模型,经跨内皮电阻值(transendothelial electrical resistance,TEER)、荧光素钠通透性(sodium fluorescent,Na-FLU )、碱性磷酸酶(AKP)和γ-谷氨酰转肽酶(γ-GT1)的表达测定以及阳性药在体内和体外BBB通透量的相似性,比较评价其屏障功能。结果原代培养的BMECs呈典型的铺路卵石样结构,BMPC胞体较大且呈分枝状,AS 有细长突触,胞质较浅;免疫细胞化学染色证实原代细胞为目标细胞;BMECs与BMPC、AS共培养后TEER值可达(478±25)Ω·cm2,Na-FLU 的表观渗透系数为[(8.23±0.78)×10-6]cm·s-1,AKP和γ-GT1表达分别为(6.90±0.27)金氏单位· g-1 Pro,(4.39±0.32)μg·g-1 Pro;阳性药在体外BBB的表观渗透系数(apparent permeability coefficient,Papp )与在体数据具有较好的相关性(R2=0.92)。结论原代培养的大鼠BMECs与BMPC、AS共培养建立的体外BBB模型在形态、结构及屏障功能方面具备BBB的基本特征,为研究BBB的生理学、病理学以及筛选化合物提供了一种有用工具。%Aim To establish in vitro blood-brain barrier (BBB) model with characteristics of simulation of in vivo BBB by primi-tive co-culture of brain-microvessel endothelial cells (BMECs) with brain-microvessel pericytes (BMPC)and astrocytes (AS). Methods BMECs,BMPC and AS from SD rats were primitively isolated,purified and cultured,and then

  8. Blood-Brain Barrier Deterioration and Hippocampal Gene Expression in Polymicrobial Sepsis: An Evaluation of Endothelial MyD88 and the Vagus Nerve.

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    Gerard Honig

    Full Text Available Systemic infection can initiate or exacerbate central nervous system (CNS pathology, even in the absence of overt invasion of bacteria into the CNS. Recent epidemiological studies have demonstrated that human survivors of sepsis have an increased risk of long-term neurocognitive decline. There is thus a need for improved understanding of the physiological mechanisms whereby acute sepsis affects the CNS. In particular, MyD88-dependent activation of brain microvascular endothelial cells and a resulting loss of blood-brain barrier integrity have been proposed to play an important role in the effects of systemic inflammation on the CNS. Signaling through the vagus nerve has also been considered to be an important component of CNS responses to systemic infection. Here, we demonstrate that blood-brain barrier permeabilization and hippocampal transcriptional responses during polymicrobial sepsis occur even in the absence of MyD88-dependent signaling in cerebrovascular endothelial cells. We further demonstrate that these transcriptional responses can occur without vagus nerve input. These results suggest that redundant signals mediate CNS responses in sepsis. Either endothelial or vagus nerve activation may be individually sufficient to transmit systemic inflammation to the central nervous system. Transcriptional activation in the forebrain in sepsis may be mediated by MyD88-independent endothelial mechanisms or by non-vagal neuronal pathways.

  9. Effect of Excessive Potassium Iodide on Rat Aorta Endothelial Cells.

    Science.gov (United States)

    Zhang, Man; Zou, Xiaoyan; Lin, Xinying; Bian, Jianchao; Meng, Huicui; Liu, Dan

    2015-08-01

    The aim of the current study was to investigate the effect of excess iodine on rat aorta endothelial cells and the potential underlying mechanisms. Rat aorta endothelial cells were cultured with iodide ion (3506, 4076, 4647, 5218, 5789, 6360, 6931, and 7512 mg/L) for 48 h. Morphological changes of cells were observed with microscope after Wright-Giemsa staining and acridine orange staining. Cell proliferation was determined with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and cell apoptosis was assessed with flow cytometry. The activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), endothelial nitric oxide synthase (eNOS), induced nitric oxide synthase (iNOS), and concentrations of malondialdehyde (MDA), glutathione (GSH), and protein carbonyl in culture medium were determined with colorimetric method. The expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was detected by enzyme linked immunosorbent assay. The results showed that excess iodine induced abnormal morphologic changes of cells, inhibited cell proliferation, and increased apoptosis rate. Iodine also reduced the activity of SOD, GSH-Px, and concentrations of GSH and increased the concentrations of MDA and protein carbonyl in a dose-dependent manner. Moreover, excess iodine decreased the activity of eNOS and increased the activity of iNOS and the expression of ICAM-1 and VCAM-1 in culture medium. Our results suggested that excess iodine exposure increased oxidative stress, caused damage of vascular endothelial cells, and altered the expression of adhesion factors and the activity of NOS. These changes may explain the mechanisms underlying excess iodine-induced vascular injury.

  10. Brown spider venom toxins interact with cell surface and are endocytosed by rabbit endothelial cells.

    Science.gov (United States)

    Nowatzki, Jenifer; de Sene, Reginaldo Vieira; Paludo, Katia Sabrina; Veiga, Silvio Sanches; Oliver, Constance; Jamur, Maria Célia; Nader, Helena Bonciani; Trindade, Edvaldo S; Franco, Célia Regina C

    2010-09-15

    Bites from the Loxosceles genus (brown spiders) cause severe clinical symptoms, including dermonecrotic injury, hemorrhage, hemolysis, platelet aggregation and renal failure. Histological findings of dermonecrotic lesions in animals exposed to Loxosceles intermedia venom show numerous vascular alterations. Study of the hemorrhagic consequences of the venom in endothelial cells has demonstrated that the degeneration of blood vessels results not only from degradation of the extracellular matrix molecule or massive leukocyte infiltration, but also from a direct and primary activity of the venom on endothelial cells. Exposure of an endothelial cell line in vitro to L. intermedia venom induce morphological alterations, such as cell retraction and disadhesion to the extracellular matrix. The aim of the present study was to investigate the interaction between the venom toxins and the endothelial cell surface and their possible internalization, in order to illuminate the information about the deleterious effect triggered by venom. After treating endothelial cells with venom toxins, we observed that the venom interacts with cell surface. Venom treatment also can cause a reduction of cell surface glycoconjugates. When cells were permeabilized, it was possible to verify that some venom toxins were internalized by the endothelial cells. The venom internalization involves endocytic vesicles and the venom was detected in the lysosomes. However, no damage to lysosomal integrity was observed, suggesting that the cytotoxic effect evoked by L. intermedia venom on endothelial cells is not mediated by venom internalization.

  11. The chemotactic activity of beta-carotene in endothelial cell progenitors and human umbilical vein endothelial cells: A microarray analysis

    NARCIS (Netherlands)

    Polus, A.; Kiec-wilk, B.; Hartwich, J.; Balwierz, A.; Stachura, J.; Dyduch, G.; Laidler, P.; Zagajewski, J.; Langman, T.; Schmitz, G.; Goralcsky, R.; Wertz, K.; Riss, G.; Keijer, J.; Dembinska-Kiec, A.

    2006-01-01

    Objectives: Endothelial cells and their progenitors play an important role in angiogenesis that is essential for organogenesis and tissue remodelling, as well as for inflammatory responses and carcinogenesis in all periods of life. In the present study, the authors concentrated on the direct effect

  12. ANTIBODIES DEFINING RAT ENDOTHELIAL-CELLS - RECA-1, A PAN-ENDOTHELIAL CELL-SPECIFIC MONOCLONAL-ANTIBODY

    NARCIS (Netherlands)

    DUIJVESTIJN, AM; VANGOOR, H; KLATTER, F; MAJOOR, GD; VANBUSSEL, E; VRIESMAN, PJCV

    1992-01-01

    We have been searching for antibodies reactive with rat endothelial cells. Two monoclonal antibodies (mAb), named RECA-1 and RECA-2 were produced and tested in immunoperoxidase staining on frozen sections of various rat tissues. Staining patterns were compared to those obtained with the mAbs OX-2, O

  13. The effects of TSH on human vascular endothelial cells and smooth muscle cells

    Institute of Scientific and Technical Information of China (English)

    田利民

    2014-01-01

    Objective To study the effect of thyroid-stimulating hormone(TSH)on human vascular endothelial cells and smooth muscle cells and to explore the roles of TSH in the development of atherosclerosis.Methods Human vascular endothelial cells and smooth muscle cells were cultured in vitro.MTT method was used to assay the effect of TSH on cell viability.Real-time PCR was used

  14. Characterization of Bioeffects on Endothelial Cells under Acoustic Droplet Vaporization.

    Science.gov (United States)

    Seda, Robinson; Li, David S; Fowlkes, J Brian; Bull, Joseph L

    2015-12-01

    Gas embolotherapy is achieved by locally vaporizing microdroplets through acoustic droplet vaporization, which results in bubbles that are large enough to occlude blood flow directed to tumors. Endothelial cells, lining blood vessels, can be affected by these vaporization events, resulting in cell injury and cell death. An idealized monolayer of endothelial cells was subjected to acoustic droplet vaporization using a 3.5-MHz transducer and dodecafluoropentane droplets. Treatments included insonation pressures that varied from 2 to 8 MPa (rarefactional) and pulse lengths that varied from 4 to 16 input cycles. The bubble cloud generated was directly dependent on pressure, but not on pulse length. Cellular damage increased with increasing bubble cloud size, but was limited to the bubble cloud area. These results suggest that vaporization near the endothelium may impact the vessel wall, an effect that could be either deleterious or beneficial depending on the intended overall therapeutic application.

  15. [Influence of n-hexane on vascular endothelial active substances in brain tissue in mice].

    Science.gov (United States)

    Lin, L; Zhang, Z Q; Zhang, C Z

    2017-01-20

    Objective: To investigate the influence of n-hexane on vascular endothelial active substances in brain tissue in mice and its significance. Methods: A total of 48 healthy Kunming mice were randomly divided into high-dose exposure group, middle-dose exposure group, low-dose exposure group, and control group, with 12 mice in each group. All groups except the control group were exposed to n-hexane via static inhalation (0.035 g/L, 0.018 g/L, and 0.009 g/L for the high-, middle-, and low-dose exposure groups, respectively) 4 hours a day for 21 days. the mice in the control groups were not exposed to n-hexane. After the exposure, the lev-els of endothelin-1 (ET-1) , nitric oxide (NO) , and angiotensin II (Ang II) in brain tissue were measured in all groups. Results: There were significant differences in the levels of ET-1, NO, and Ang II between the three ex-posure groups and the control group (PHexane can affect the vascular endothe-lial active substances in brain tissue in mice, and the changes and imbalance in vascular endothelial active sub-stances may be one of the reasons for central nervous system impairment caused by n-hexane.

  16. Nylon-3 polymers that enable selective culture of endothelial cells.

    Science.gov (United States)

    Liu, Runhui; Chen, Xinyu; Gellman, Samuel H; Masters, Kristyn S

    2013-11-06

    Substrates that selectively encourage the growth of specific cell types are valuable for the engineering of complex tissues. Some cell-selective peptides have been identified from extracellular matrix proteins; these peptides have proven useful for biomaterials-based approaches to tissue repair or regeneration. However, there are very few examples of synthetic materials that display selectivity in supporting cell growth. We describe nylon-3 polymers that support in vitro culture of endothelial cells but do not support the culture of smooth muscle cells or fibroblasts. These materials may be promising for vascular biomaterials applications.

  17. Glioblastoma-derived Leptin Induces Tube Formation and Growth of Endothelial Cells: Comparison with VEGF Effects

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    Otvos Laszlo

    2011-07-01

    Full Text Available Abstract Background Leptin is a pleiotropic hormone whose mitogenic and angiogenic activity has been implicated in the development and progression of several malignancies, including brain tumors. In human brain cancer, especially in glioblastoma multiforme (GBM, leptin and its receptor (ObR are overexpressed relative to normal tissue. Until present, the potential of intratumoral leptin to exert proangiogenic effects on endothelial cells has not been addressed. Using in vitro models, we investigated if GBM can express leptin, if leptin can affect angiogenic and mitogenic potential of endothelial cells, and if its action can be inhibited with specific ObR antagonists. Leptin effects were compared with that induced by the best-characterized angiogenic regulator, VEGF. Results We found that GBM cell lines LN18 and LN229 express leptin mRNA and LN18 cells secrete detectable amounts of leptin protein. Both lines also expressed and secreted VEGF. The conditioned medium (CM of LN18 and LN 229 cultures as well as 200 ng/mL pure leptin or 50 ng/mL pure VEGF stimulated proliferation of human umbilical vein endothelial cells (HUVEC at 24 h of treatment. Mitogenic effects of CM were ~2-fold greater than that of pure growth factors. Furthermore, CM treatment of HUVEC for 24 h increased tube formation by ~5.5-fold, while leptin increased tube formation by ~ 80% and VEGF by ~60% at 8 h. The mitogenic and angiogenic effects of both CM were blocked by Aca 1, a peptide ObR antagonist, and by SU1498, which inhibits the VEGF receptor. The best anti-angiogenic and cytostatic effects of Aca1 were obtained with 10 nM and 25 nM, respectively, while for SU1498, the best growth and angiogenic inhibition was observed at 5 μM. The combination of 5 μM SU1498 and Aca1 at 25 nM (growth inhibition or at 10 nM (reduction of tube formation produced superior effects compared with single agent treatments. Conclusions Our data provide the first evidence that LN18 and LN 229 human

  18. Propofol protects against high glucose-induced endothelial adhesion molecules expression in human umbilical vein endothelial cells

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    Zhu Minmin

    2013-01-01

    Full Text Available Abstract Background Hyperglycemia could induce oxidative stress, activate transcription factor nuclear factor kappa B (NF-κB, up-regulate expression of endothelial adhesion molecules, and lead to endothelial injury. Studies have indicated that propofol could attenuate oxidative stress and suppress NF-κB activation in some situations. In the present study, we examined whether and how propofol improved high glucose-induced up-regulation of endothelial adhesion molecules in human umbilical vein endothelial cells (HUVECs. Methods Protein expression of endothelial adhesion molecules, NF-κB, inhibitory subunit of NF-κBα (IκBα, protein kinase Cβ2 (PKCβ2, and phosphorylation of PKCβ2 (Ser660 were measured by Western blot. NF-κB activity was measured by electrophoretic mobility shift assay. PKC activity was measured with SignaTECT PKC assay system. Superoxide anion (O2.- accumulation was measured with the reduction of ferricytochrome c assay. Human peripheral mononuclear cells were prepared with Histopaque-1077 solution. Results High glucose induced the expression of endothelial selectin (E-selectin, intercellular adhesion molecule 1 (ICAM-1, vascular cell adhesion molecule 1 (VCAM-1, and increased mononuclear-endothelial adhesion. High glucose induced O2.- accumulation, PKCβ2 phosphorylation and PKC activation. Further, high glucose decreased IκBα expression in cytoplasm, increased the translocation of NF-κB from cytoplasm to nuclear, and induced NF-κB activation. Importantly, we found these high glucose-mediated effects were attenuated by propofol pretreatment. Moreover, CGP53353, a selective PKCβ2 inhibitor, decreased high glucose-induced NF-κB activation, adhesion molecules expression, and mononuclear-endothelial adhesion. Conclusion Propofol, via decreasing O2.- accumulation, down-regulating PKCβ2 Ser660 phosphorylation and PKC as well as NF-κB activity, attenuated high glucose-induced endothelial adhesion molecules expression

  19. Influence of mild hypothermia on vascular endothelial growth factor and infarct volume in brain tissues after cerebral ischemia in rats

    Institute of Scientific and Technical Information of China (English)

    Fei Ye; Gangming Xi; Biyong Qin; Shifeng Wang; Chengyan Li

    2006-01-01

    : All the rats were anesthetized by intraperitoneal injection overdose sodium pentobarbital 7 days postoperatively, and then the heads were cut down to harvest brain. The brain tissues were placed into -20 ℃ refrigerator for 20 minutes, coronal sections of 2 mm were prepared. The infarct sites were not stained, whereas normal brain tissues were stained as red. The infarct volumes were calculated by using MPLAS-500 multimedia color pathological image&word analytical system. ③ Counting positive cells of vascular endothelial growth factor protein: The brains were harvested by cutting heads, then coronal sections of 2 mm were prepared. Routine dehydration, hyalinization, wax immersion and embedding were performed, then the detected with SP immunohistochemistry, the kits were purchased from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd. The cells whose cytoplasm was yellow-brown were positive ones, a single sample as a unit, peri-ischemic site and ischemic core were selected, and the corresponding sites in controlateral hemisphere were taken as controls. Five visual fields were selected from each site to be observed under microscope, the cells were counted, and the average number of positive cells was calculated in each group. The numbers of positive cells were determined with the image analytical apparatus.MAIN OUTCOME MEASURES: Number of the positive cells of vascular endothelial growth factor protein; Infarct volume of rat brain tissue.RESULTS: All the 20 rats were involved in the analysis of results. ① Number of positive cells of vascular endothelial growth factor protein in brain tissue: It was obviously lower in the mild hypothermia group than in the normal temperature group [(24.02±5.05), (36.07±2.69) cells/high power visual field, P < 0.01]. ② Comparison of infarct volume of brain tissue: After MCAO, it was obviously smaller in the mild hypothermia group than in the normal temperature group [(153.25±23.14), (253.45±36.21) mm3, P< 0

  20. Arterial identity of endothelial cells is controlled by local cues.

    Science.gov (United States)

    Othman-Hassan, K; Patel, K; Papoutsi, M; Rodriguez-Niedenführ, M; Christ, B; Wilting, J

    2001-09-15

    The ephrins and their Eph receptors comprise the largest family of receptor tyrosine kinases. Studies on mice have revealed an important function of ephrin-B2 and Eph-B4 for the development of the arterial and venous vasculature, respectively, but the mechanisms regulating their expression have not been studied yet. We have cloned a chick ephrin-B2 cDNA probe. Expression was observed in endothelial cells of extra- and intraembryonic arteries and arterioles in all embryos studied from day 2 (stage 10 HH, before perfusion of the vessels) to day 16. Additionally, expression was found in the somites and neural tube in early stages, and later also in the smooth muscle cells of the aorta, parts of the Müllerian duct, dosal neural tube, and joints of the limbs. We isolated endothelial cells from the internal carotid artery and the vena cava of 14-day-old quail embryos and grafted them separately into day-3 chick embryos. Reincubation was performed until day 6 and the quail endothelial cells were identified with the QH1 antibody. The grafted arterial and venous endothelial cells expressed ephrin-B2 when they integrated into the lining of arteries. Cells that were not integrated into vessels, or into vessels other than arteries, were ephrin-B2-negative. The studies show that the expression of the arterial marker ephrin-B2 is controlled by local cues in arterial vessels of older embryos. Physical forces or the media smooth muscle cells may be involved in this process.

  1. CB2 Receptor Activation Inhibits Melanoma Cell Transmigration through the Blood-Brain Barrier

    Science.gov (United States)

    Haskó, János; Fazakas, Csilla; Molnár, Judit; Nyúl-Tóth, Ádám; Herman, Hildegard; Hermenean, Anca; Wilhelm, Imola; Persidsky, Yuri; Krizbai, István A.

    2014-01-01

    During parenchymal brain metastasis formation tumor cells need to migrate through cerebral endothelial cells, which form the morphological basis of the blood-brain barrier (BBB). The mechanisms of extravasation of tumor cells are highly uncharacterized, but in some aspects recapitulate the diapedesis of leukocytes. Extravasation of leukocytes through the BBB is decreased by the activation of type 2 cannabinoid receptors (CB2); therefore, in the present study we sought to investigate the role of CB2 receptors in the interaction of melanoma cells with the brain endothelium. First, we identified the presence of CB1, CB2(A), GPR18 (transcriptional variant 1) and GPR55 receptors in brain endothelial cells, while melanoma cells expressed CB1, CB2(A), GPR18 (transcriptional variants 1 and 2), GPR55 and GPR119. We observed that activation of CB2 receptors with JWH-133 reduced the adhesion of melanoma cells to the layer of brain endothelial cells. JWH-133 decreased the transendothelial migration rate of melanoma cells as well. Our results suggest that changes induced in endothelial cells are critical in the mediation of the effect of CB2 agonists. Our data identify CB2 as a potential target in reducing the number of brain metastastes originating from melanoma. PMID:24815068

  2. CB2 Receptor Activation Inhibits Melanoma Cell Transmigration through the Blood-Brain Barrier

    Directory of Open Access Journals (Sweden)

    János Haskó

    2014-05-01

    Full Text Available During parenchymal brain metastasis formation tumor cells need to migrate through cerebral endothelial cells, which form the morphological basis of the blood-brain barrier (BBB. The mechanisms of extravasation of tumor cells are highly uncharacterized, but in some aspects recapitulate the diapedesis of leukocytes. Extravasation of leukocytes through the BBB is decreased by the activation of type 2 cannabinoid receptors (CB2; therefore, in the present study we sought to investigate the role of CB2 receptors in the interaction of melanoma cells with the brain endothelium. First, we identified the presence of CB1, CB2(A, GPR18 (transcriptional variant 1 and GPR55 receptors in brain endothelial cells, while melanoma cells expressed CB1, CB2(A, GPR18 (transcriptional variants 1 and 2, GPR55 and GPR119. We observed that activation of CB2 receptors with JWH-133 reduced the adhesion of melanoma cells to the layer of brain endothelial cells. JWH-133 decreased the transendothelial migration rate of melanoma cells as well. Our results suggest that changes induced in endothelial cells are critical in the mediation of the effect of CB2 agonists. Our data identify CB2 as a potential target in reducing the number of brain metastastes originating from melanoma.

  3. Nitrones reverse hyperglycemia-induced endothelial dysfunction in bovine aortic endothelial cells.

    Science.gov (United States)

    Headley, Colwyn A; DiSilvestro, David; Bryant, Kelsey E; Hemann, Craig; Chen, Chun-An; Das, Amlan; Ziouzenkova, Ouliana; Durand, Grégory; Villamena, Frederick A

    2016-03-15

    Hyperglycemia has been implicated in the development of endothelial dysfunction through heightened ROS production. Since nitrones reverse endothelial nitric oxide synthase (eNOS) dysfunction, increase antioxidant enzyme activity, and suppress pro-apoptotic signaling pathway and mitochondrial dysfunction from ROS-induced toxicity, the objective of this study was to determine whether nitrone spin traps DMPO, PBN and PBN-LA were effective at duplicating these effects and improving glucose uptake in an in vitro model of hyperglycemia-induced dysfunction using bovine aortic endothelial cells (BAEC). BAEC were cultured in DMEM medium with low (5.5mM glucose, LG) or high glucose (50mM, HG) for 14 days to model in vivo hyperglycemia as experienced in humans with metabolic disease. Improvements in cell viability, intracellular oxidative stress, NO and tetrahydrobiopterin (BH4)​ levels, mitochondrial membrane potential, glucose transport, and activity of antioxidant enzymes were measured from single treatment of BAEC with nitrones for 24h after hyperglycemia. Chronic hyperglycemia significantly increased intracellular ROS by 50%, decreased cell viability by 25%, reduced NO bioavailability by 50%, and decreased (BH4) levels by 15% thereby decreasing NO production. Intracellular glucose transport and superoxide dismutase (SOD) activity were also decreased by 50% and 25% respectively. Nitrone (PBN and DMPO, 50 μM) treatment of BAEC grown in hyperglycemic conditions resulted in the normalization of outcome measures except for SOD and catalase activities. Our findings demonstrate that the nitrones reverse the deleterious effects of hyperglycemia in BAEC. We believe that in vivo testing of these nitrone compounds in models of cardiometabolic disease is warranted.

  4. Signal transduction pathways in mast cell granule-mediated endothelial cell activation

    Directory of Open Access Journals (Sweden)

    Luqi Chi

    2003-01-01

    Full Text Available Background: We have previously shown that incubation of human endothelial cells with mast cell granules results in potentiation of lipopolysaccharide-induced production of interleukin-6 and interleukin-8.

  5. Isolation of Endothelial Cells and Vascular Smooth Muscle Cells from Internal Mammary Artery Tissue

    Science.gov (United States)

    Moss, Stephanie C.; Bates, Michael; Parrino, Patrick E.; Woods, T. Cooper

    2007-01-01

    Analyses of vascular smooth muscle cell and endothelial cell function through tissue culture techniques are often employed to investigate the underlying mechanisms regulating cardiovascular disease. As diseases such as diabetes mellitus and chronic kidney disease increase a patient's risk of cardiovascular disease, the development of methods for examining the effects of these diseases on vascular smooth muscle cells and endothelial cells is needed. Commercial sources of endothelial cells and vascular smooth muscle cells generally provide minimal donor information and are in limited supply. This study was designed to determine if vascular smooth muscle cells and endothelial cells could be isolated from human internal mammary arteries obtained from donors undergoing coronary artery bypass graft surgery. As coronary artery bypass graft surgery is a commonly performed procedure, this method would provide a new source for these cells that when combined with the donor's medical history will greatly enhance our studies of the effects of complicating diseases on vascular biology. Internal mammary artery tissue was obtained from patients undergoing coronary artery bypass graft surgery. Through a simple method employing two separate tissue digestions, vascular smooth muscle cells and endothelial cells were isolated and characterized. The isolated vascular smooth muscle cells and endothelial cells exhibited the expected morphology and were able to be passaged for further analysis. The vascular smooth muscle cells exhibited positive staining for α-smooth muscle actin and the endothelial cells exhibited positive staining for CD31. The overall purity of the isolations was > 95%. This method allows for the isolation of endothelial cells and vascular smooth muscle cells from internal mammary arteries, providing a new tool for investigations into the interplay of vascular diseases and complicating diseases such as diabetes and kidney disease. PMID:21603530

  6. Leptin-induced transphosphorylation of vascular endothelial growth factor receptor increases Notch and stimulates endothelial cell angiogenic transformation.

    Science.gov (United States)

    Lanier, Viola; Gillespie, Corey; Leffers, Merle; Daley-Brown, Danielle; Milner, Joy; Lipsey, Crystal; Webb, Nia; Anderson, Leonard M; Newman, Gale; Waltenberger, Johannes; Gonzalez-Perez, Ruben Rene

    2016-10-01

    Leptin increases vascular endothelial growth factor (VEGF), VEGF receptor-2 (VEGFR-2), and Notch expression in cancer cells, and transphosphorylates VEGFR-2 in endothelial cells. However, the mechanisms involved in leptin's actions in endothelial cells are not completely known. Here we investigated whether a leptin-VEGFR-Notch axis is involved in these leptin's actions. To this end, human umbilical vein and porcine aortic endothelial cells (wild type and genetically modified to overexpress VEGFR-1 or -2) were cultured in the absence of VEGF and treated with leptin and inhibitors of Notch (gamma-secretase inhibitors: DAPT and S2188, and silencing RNA), VEGFR (kinase inhibitor: SU5416, and silencing RNA) and leptin receptor, OB-R (pegylated leptin peptide receptor antagonist 2: PEG-LPrA2). Interestingly, in the absence of VEGF, leptin induced the expression of several components of Notch signaling pathway in endothelial cells. Inhibition of VEGFR and Notch signaling significantly decreased leptin-induced S-phase progression, proliferation, and tube formation in endothelial cells. Moreover, leptin/OB-R induced transphosphorylation of VEGFR-1 and VEGFR-2 was essential for leptin's effects. These results unveil for the first time a novel mechanism by which leptin could induce angiogenic features via upregulation/trans-activation of VEGFR and downstream expression/activation of Notch in endothelial cells. Thus, high levels of leptin found in overweight and obese patients might lead to increased angiogenesis by activating VEGFR-Notch signaling crosstalk in endothelial cells. These observations might be highly relevant for obese patients with cancer, where leptin/VEGFR/Notch crosstalk could play an important role in cancer growth, and could be a new target for the control of tumor angiogenesis.

  7. Suprabasin as a novel tumor endothelial cell marker

    Science.gov (United States)

    Alam, Mohammad T; Nagao-Kitamoto, Hiroko; Ohga, Noritaka; Akiyama, Kosuke; Maishi, Nako; Kawamoto, Taisuke; Shinohara, Nobuo; Taketomi, Akinobu; Shindoh, Masanobu; Hida, Yasuhiro; Hida, Kyoko

    2014-01-01

    Recent studies have reported that stromal cells contribute to tumor progression. We previously demonstrated that tumor endothelial cells (TEC) characteristics were different from those of normal endothelial cells (NEC). Furthermore, we performed gene profile analysis in TEC and NEC, revealing that suprabasin (SBSN) was upregulated in TEC compared with NEC. However, its role in TEC is still unknown. Here we showed that SBSN expression was higher in isolated human and mouse TEC than in NEC. SBSN knockdown inhibited the migration and tube formation ability of TEC. We also showed that the AKT pathway was a downstream factor of SBSN. These findings suggest that SBSN is involved in the angiogenic potential of TEC and may be a novel TEC marker. PMID:25283635

  8. Antiproliferative Effects of Drugs on Endothelial and Osteoblastic Cells and Altered Release of Angioregulatory Mediators by Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Hilde Kvestad

    2014-01-01

    Full Text Available The combined use of the histone deacetylase inhibitor valproic acid (VPA, the retinoic acid receptor-α agonist all-trans retinoic acid (ATRA, and the deoxyribonucleic acid polymerase-α inhibitor cytarabine (Ara-C is now considered for disease-stabilizing treatment of acute myeloid leukemia (AML. Leukemogenesis and leukemia cell chemoresistance seem to be supported by neighbouring stromal cells in the bone marrow, and we have therefore investigated the effects of these drugs on primary human endothelial cells and the osteoblastic Cal72 cell line. The results show that VPA and Ara-C have antiproliferative effects, and the antiproliferative/cytotoxic effect of Ara-C was seen at low concentrations corresponding to serum levels found during low-dose in vivo treatment. Furthermore, in functional assays of endothelial migration and tube formation VPA elicited an antiangiogenic effect, whereas ATRA elicited a proangiogenic effect. Finally, VPA and ATRA altered the endothelial cell release of angiogenic mediators; ATRA increased levels of CXCL8, PDGF-AA, and VEGF-D, while VPA decreased VEGF-D and PDGF-AA/BB levels and both drugs reduced MMP-2 levels. Several of these mediators can enhance AML cell proliferation and/or are involved in AML-induced bone marrow angiogenesis, and direct pharmacological effects on stromal cells may thus indirectly contribute to the overall antileukemic activity of this triple drug combination.

  9. Coniferyl Aldehyde Ameliorates Radiation Intestine Injury via Endothelial Cell Survival

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Ye Ji; Jung, Myung Gu; Lee, Yoonjin; Lee, Haejune [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Lee, Yunsil [Ewha Woman' s Univ., Seoul (Korea, Republic of); Ko, Younggyu [Korea Univ., Seoul (Korea, Republic of)

    2014-05-15

    Cancer treatments related gastrointestinal toxicity has also been recognized as a significant economic burden. Especially, extensive apoptosis of microvascular endothelial cell of the lamina propria is the primary lesion initiating intestinal radiation damage after abdominal radiation therapy. Coniferyl aldehyde (CA) is phenolic compounds isolated from cork stoppers, and one of the major pyrolysis products of lignin. Shi H. was support for the empirical use of CA as a medicinal food for cardiovascular diseases. CA has positive effect in broad way but there is no consequence in radiation induced intestine damage. Here, we investigate effect of CA on small intestine after abdominal IR to mice in this study. In this study, CA increased the survival rate in C3H mice against 13.5 Gy abdominal IR. We found CA protects small intestine via preventing endothelial cell apoptosis and enhancing their angiogenic activity. CA also showed protective effect on crypt cell survival. Endothelial cell survival may affect crypt cell protection against IR. From this data, we concluded that CA is effective for protection against abdominal radiation injury. CA could ameliorate side-effect of radiation therapy.

  10. Pharmacologically active microcarriers for endothelial progenitor cell support and survival.

    Science.gov (United States)

    Musilli, Claudia; Karam, Jean-Pierre; Paccosi, Sara; Muscari, Claudio; Mugelli, Alessandro; Montero-Menei, Claudia N; Parenti, Astrid

    2012-08-01

    The regenerative potential of endothelial progenitor cell (EPC)-based therapies is limited due to poor cell viability and minimal retention following application. Neovascularization can be improved by means of scaffolds supporting EPCs. The aim of the present study was to investigate whether human early EPCs (eEPCs) could be efficiently cultured on pharmacologically active microcarriers (PAMs), made with poly(d,l-lactic-coglycolic acid) and coated with adhesion/extracellular matrix molecules. They may serve as a support for stem cells and may be used as cell carriers providing a controlled delivery of active protein such as the angiogenic factor, vascular endothelial growth factor-A (VEGF-A). eEPC adhesion to fibronectin-coated PAMs (FN-PAMs) was assessed by means of microscopic evaluation and by means of Alamar blue assay. Phospho ERK(1/2) and PARP-1 expression was measured by means of Western blot to assess the survival effects of FN-PAMs releasing VEGF-A (FN-VEGF-PAMs). The Alamar blue assay or a modified Boyden chamber assay was employed to assess proliferative or migratory capacity, respectively. Our data indicate that eEPCs were able to adhere to empty FN-PAMs within a few hours. FN-VEGF-PAMs increased the ability of eEPCs to adhere to them and strongly supported endothelial-like phenotype and cell survival. Moreover, the release of VEGF-A by FN-PAMs stimulated in vitro HUVEC migration and proliferation. These data strongly support the use of PAMs for supporting eEPC growth and survival and for stimulating resident mature human endothelial cells.

  11. Effect of Mitomycin-C augmented trabeculectomy on corneal endothelial cells

    Directory of Open Access Journals (Sweden)

    Reza Zarei

    2015-01-01

    Conclusion: MMC application in trabeculectomy seems to cause a small but significant corneal endothelial loss. Most of the damage occurs intraoperatively, or in the early postoperative period, however progressive endothelial cell loss is not a major concern.

  12. Protection of Candida parapsilosis from neutrophil killing through internalization by human endothelial cells.

    Science.gov (United States)

    Glass, Kyle A; Longley, Sarah J; Bliss, Joseph M; Shaw, Sunil K

    2015-01-01

    Candida parapsilosis is a fungal pathogen that is associated with hematogenously disseminated disease in premature neonates, acutely ill or immunocompromised patients. In cell culture, C. parapsilosis cells are actively and avidly endocytosed by endothelial cells via actin polymerization mediated by N-WASP. Here we present evidence that C. parapsilosis that were internalized by endothelial cells remained alive, and avoided being acidified or otherwise damaged via the host cell. Internalized fungal cells reproduced intracellularly and eventually burst out of the host endothelial cell. When neutrophils were added to endothelium and C. parapsilosis, they patrolled the endothelial surface and efficiently killed most adherent fungal cells prior to endocytosis. But after endocytosis by endothelial cells, internalized fungal cells evaded neutrophil killing. Silencing endothelial N-WASP blocked endocytosis of C. parapsilosis and left fungal cells stranded on the cell surface, where they were susceptible to neutrophil killing. These observations suggest that for C. parapsilosis to escape from the bloodstream, fungi may adhere to and be internalized by endothelial cells before being confronted and phagocytosed by a patrolling leukocyte. Once internalized by endothelial cells, C. parapsilosis may safely replicate to cause further rounds of infection. Immunosurveillance of the intravascular lumen by leukocytes crawling on the endothelial surface and rapid killing of adherent yeast may play a major role in controlling C. parapsilosis dissemination and infected endothelial cells may be a significant reservoir for fungal persistence.

  13. A microarray analysis of two distinct lymphatic endothelial cell populations

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    Bernhard Schweighofer

    2015-06-01

    Full Text Available We have recently identified lymphatic endothelial cells (LECs to form two morphologically different populations, exhibiting significantly different surface protein expression levels of podoplanin, a major surface marker for this cell type. In vitro shockwave treatment (IVSWT of LECs resulted in enrichment of the podoplaninhigh cell population and was accompanied by markedly increased cell proliferation, as well as 2D and 3D migration. Gene expression profiles of these distinct populations were established using Affymetrix microarray analyses. Here we provide additional details about our dataset (NCBI GEO accession number GSE62510 and describe how we analyzed the data to identify differently expressed genes in these two LEC populations.

  14. Morphological and protein profile comparison of large vessel and microvascular endothelial cells in culture

    Energy Technology Data Exchange (ETDEWEB)

    Beer, D.M.; Kim, J.S.; Carson, M.P.; Haudeuschild, C.C.; Patton, W.F.; Jacobson, B.S.

    1986-05-01

    Bovine adrenal medulla (AmMEC) and brain (BrMEC) microvessel endothelial cells, and bovine aortic (BAE) endothelial cells were isolated and cultured under identical conditions using a modification of a technique previously described for BrMEC. The cells were isolated and passaged under conditions minimizing cell surface alterations. Primary cultures were confluent in 4-6 days at a plating density in the region of 10/sup 4/ cells/cm/sup 2/. BAEs maintained a cobblestone morphology and a denser monolayer than MECs in primary and passaged cells whether the cells were passaged using Pancreatin, Trypsin-EDTA, or Collagenase-EDTA. MECs were initially elongate and became more like BAEs with passaging. BAEs and AmMECs were examined for differences in whole cell, Triton extracted cytoskeleton and plasma membrane (PM) protein profiles by two-dimensional gel electrophoresis. Cells were labeled with /sup 35/S-methionine and PM by lactoperoxidase catalyzed iodination. Though for the most part protein patterns were similar, several proteins in the PM and cytoskeletal preparations differed. A significant difference in the isoelectric forms of proteins with the same molecular weight was observed in the PM.

  15. Interleukin 3 stimulates proliferation and triggers endothelial-leukocyte adhesion molecule 1 gene activation of human endothelial cells.

    Science.gov (United States)

    Brizzi, M F; Garbarino, G; Rossi, P R; Pagliardi, G L; Arduino, C; Avanzi, G C; Pegoraro, L

    1993-06-01

    Proliferation and functional activation of endothelial cells within a tissue site of inflammation are regulated by humoral factors released by cells, such as T lymphocytes and monocytes, infiltrating the perivascular space. In the present study we investigated the effects of interleukin 3 (IL-3), an activated T lymphocyte-derived cytokine, on cultured human umbilical vein endothelial cells (HUVEC). Proliferative activity, evaluated both by estimation of the fraction of cells in the S phase and by direct cell count demonstrated that IL-3, at the dose of 25 ng/ml, enhances more than threefold both DNA synthesis and cell proliferation above baseline control conditions. Binding studies with radioiodinated ligand demonstrated that HUVEC constitutively express a smaller number of IL-3 binding sites (approximately 99 binding sites per cell, with an apparent Kd of 149 pM). Accordingly, molecular analysis showed the presence of transcripts for both alpha and beta subunits of the IL-3 receptor. Functional activation of endothelial cells was evaluated by the expression of the endothelial-leukocyte adhesion molecule 1 (ELAM-1) transcript and by leukocyte adhesion. The ELAM-1 gene transcript was clearly detectable 4 h after IL-3 addition and started to decrease after 12 h. Moreover, IL-3-induced ELAM-1 transcription was followed by enhanced adhesion of neutrophils and CD4+ T cells to HUVEC. The findings that IL-3 can stimulate both proliferation and functional activation of endothelial cells suggest that this cytokine can be involved in sustaining the process of chronic inflammation.

  16. Vascular Endothelial Growth Factor Increases during Blood-Brain Barrier-Enhanced Permeability Caused by Phoneutria nigriventer Spider Venom

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    Monique C. P. Mendonça

    2014-01-01

    Full Text Available Phoneutria nigriventer spider accidental envenomation provokes neurotoxic manifestations, which when critical, results in epileptic-like episodes. In rats, P. nigriventer venom (PNV causes blood-brain barrier breakdown (BBBb. The PNV-induced excitotoxicity results from disturbances on Na+, K+ and Ca2+ channels and glutamate handling. The vascular endothelial growth factor (VEGF, beyond its angiogenic effect, also, interferes on synaptic physiology by affecting the same ion channels and protects neurons from excitotoxicity. However, it is unknown whether VEGF expression is altered following PNV envenomation. We found that adult and neonates rats injected with PNV showed immediate neurotoxic manifestations which paralleled with endothelial occludin, β-catenin, and laminin downregulation indicative of BBBb. In neonate rats, VEGF, VEGF mRNA, and Flt-1 receptors, glutamate decarboxylase, and calbindin-D28k increased in Purkinje neurons, while, in adult rats, the BBBb paralleled with VEGF mRNA, Flk-1, and calbindin-D28k increases and Flt-1 decreases. Statistically, the variable age had a role in such differences, which might be due to age-related unequal maturation of blood-brain barrier (BBB and thus differential cross-signaling among components of the glial neurovascular unit. The concurrent increases in the VEGF/Flt-1/Flk-1 system in the cerebellar neuron cells and the BBBb following PNV exposure might imply a cytokine modulation of neuronal excitability consequent to homeostatic perturbations induced by ion channels-acting PNV neuropeptides. Whether such modulation represents neuroprotection needs further investigation.

  17. Mesangial cell integrin αvβ8 provides glomerular endothelial cell cytoprotection by sequestering TGF-β and regulating PECAM-1.

    Science.gov (United States)

    Khan, Shenaz; Lakhe-Reddy, Sujata; McCarty, Joseph H; Sorenson, Christine M; Sheibani, Nader; Reichardt, Louis F; Kim, Jane H; Wang, Bingcheng; Sedor, John R; Schelling, Jeffrey R

    2011-02-01

    Integrins are heterodimeric receptors that regulate cell adhesion, migration, and apoptosis. Integrin αvβ8 is most abundantly expressed in kidney and brain, and its major ligand is latent transforming growth factor-β (TGF-β). Kidney αvβ8 localizes to mesangial cells, which appose glomerular endothelial cells and maintain glomerular capillary structure by mechanical and poorly understood paracrine mechanisms. To establish kidney αvβ8 function, mice with homozygous Itgb8 deletion (Itgb8(-/-)) were generated on outbred and C57BL/6 congenic backgrounds. Most Itgb8(-/-) mice died in utero, and surviving Itgb8(-/-) mice failed to gain weight, and rarely survived beyond 6 weeks. A renal glomerular phenotype included azotemia and albuminuria, as well as increased platelet endothelial cell adhesion molecule-1 (PECAM-1) expression, which was surprisingly not associated with conventional functions, such as endothelial cell hyperplasia, hypertrophy, or perivascular inflammation. Itgb8(-/-) mesangial cells demonstrated reduced latent TGF-β binding, resulting in bioactive TGF-β release, which stimulated glomerular endothelial cell apoptosis. Using PECAM-1 gain and loss of function strategies, we show that PECAM-1 provides endothelial cytoprotection against mesangial cell TGF-β. These results clarify a singular mechanism of mesangial-to-endothelial cell cross-talk, whereby mesangial cell αvβ8 homeostatically arbitrates glomerular microvascular integrity by sequestering TGF-β in its latent conformation. Under pathological conditions associated with decreased mesangial cell αvβ8 expression and TGF-β secretion, compensatory PECAM-1 modulation facilitates glomerular endothelial cell survival.

  18. The Glycoprofile Patterns of Endothelial Cells in Usual Interstitial Pneumonia

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    A Barkhordari

    2014-09-01

    Full Text Available [THIS ARTICLE HAS BEEN RETRACTED FOR DUPLICATE PUBLICATION] Background: The pathological classification of cryptogenic fibrosing alveolitis has been a matter of debate and controversy for histopathologists.Objective: To identify and specify the glycotypes of capillary endothelial cells in usual interstitial pneumonia (UIP compared to those found in normal tissue.Methods: Sections of formalin-fixed, paraffin-embedded blocks from 16 cases of UIP were studied by lectin histochemistry with a panel of 27 biotinylated lectins and an avidin-peroxidase revealing system.Results: High expression of several classes of glycan was seen de novo in capillary endothelial cells from patients with UIP including small complex and bi/tri-antennary bisected complex N-linked sequences bolund by Concanavalin A and erythro-phytohemagglutinin, respectively, GalNAca1 residues bound by Helix pomatia and Maclura pomifera agglutinins, and L-fucosylated derivatives of type II glycan chains recognized by Ulex europaeus agglutinin-I. Glycans bound by agglutinins from Lycopersicon esculentum (β1,4GlcNAc and Wisteria floribunda (GalNAc as well as GlcNAc oligomers bound by Phytolacca americana and succinylated Wheat Germ agglutinin were also seen in the capillary endothelial cells of UIP. In contrast, L-fucosylated derivatives of type I glycan chains were absent in cells from cases of UIP when Anguilla anguilla agglutinin was applied, unlike the situation in normal tissue.Conclusion: These results may indicate existence of two distinct populations of endothelial cell in UIP with markedly different patterns of glycosylation, reflecting a pattern of differentiation and angiogenesis, which is not detectable morphologically.

  19. Antiproliferative effect of elevated glucose in human microvascular endothelial cells

    Science.gov (United States)

    Kamal, K.; Du, W.; Mills, I.; Sumpio, B. E.

    1998-01-01

    Diabetic microangiopathy has been implicated as a fundamental feature of the pathological complications of diabetes including retinopathy, neuropathy, and diabetic foot ulceration. However, previous studies devoted to examining the deleterious effects of elevated glucose on the endothelium have been performed largely in primary cultured cells of macrovessel origin. Difficulty in the harvesting and maintenance of microvascular endothelial cells in culture have hindered the study of this relevant population. Therefore, the objective of this study was to characterize the effect of elevated glucose on the proliferation and involved signaling pathways of an immortalized human dermal microvascular endothelial cell line (HMEC-1) that possess similar characteristics to their in vivo counterparts. Human dermal microvascular endothelial cells (HMEC-1) were grown in the presence of normal (5 mM) or high D-glucose (20 mM) for 14 days. The proliferative response of HMEC-1 was compared under these conditions as well as the cAMP and PKC pathways by in vitro assays. Elevated glucose significantly inhibited (P diabetic microangiopathy.

  20. Differences in Cell Activation by Chlamydophila pneumoniae and Chlamydia trachomatis Infection in Human Endothelial Cells

    Science.gov (United States)

    Krüll, M.; Kramp, J.; Petrov, T.; Klucken, A. C.; Hocke, A. C.; Walter, C.; Schmeck, B.; Seybold, J.; Maass, M.; Ludwig, S.; Kuipers, Jens G.; Suttorp, N.; Hippenstiel, S.

    2004-01-01

    Seroepidemiological studies and demonstration of viable bacteria in atherosclerotic plaques have linked Chlamydophila pneumoniae infection to the development of chronic vascular lesions and coronary heart disease. In this study, we characterized C. pneumoniae-mediated effects on human endothelial cells and demonstrated enhanced phosphorylation and activation of the endothelial mitogen-activated protein kinase (MAPK) family members extracellular receptor kinase (ERK1/2), p38-MAPK, and c-Jun-NH2 kinase (JNK). Subsequent interleukin-8 (IL-8) expression was dependent on p38-MAPK and ERK1/2 activation as demonstrated by preincubation of endothelial cells with specific inhibitors for the p38-MAPK (SB202190) or ERK (U0126) pathway. Inhibition of either MAPK had almost no effect on intercellular cell adhesion molecule 1 (ICAM-1) expression. While Chlamydia trachomatis was also able to infect endothelial cells, it did not induce the expression of endothelial IL-8 or ICAM-1. These effects were specific for a direct stimulation with viable C. pneumoniae and independent of paracrine release of endothelial cell-derived mediators like platelet-activating factor, NO, prostaglandins, or leukotrienes. Thus, C. pneumoniae triggers an early signal transduction cascade in target cells that could lead to endothelial cell activation, inflammation, and thrombosis, which in turn may result in or promote atherosclerosis. PMID:15501794

  1. Histone Deacetylase (HDAC Inhibitors Down-Regulate Endothelial Lineage Commitment of Umbilical Cord Blood Derived Endothelial Progenitor Cells

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    Horia Maniu

    2012-11-01

    Full Text Available To test the involvement of histone deacetylases (HDACs activity in endothelial lineage progression, we investigated the effects of HDAC inhibitors on endothelial progenitors cells (EPCs derived from umbilical cord blood (UCB. Adherent EPCs, that expressed the endothelial marker proteins (PCAM-1, CD105, CD133, and VEGFR2 revealed by flow cytometry were treated with three HDAC inhibitors: Butyrate (BuA, Trichostatin A (TSA, and Valproic acid (VPA. RT-PCR assay showed that HDAC inhibitors down-regulated the expression of endothelial genes such as VE-cadherin, CD133, CXCR4 and Tie-2. Furthermore, flow cytometry analysis illustrated that HDAC inhibitors selectively reduce the expression of VEGFR2, CD117, VE-cadherin, and ICAM-1, whereas the expression of CD34 and CD45 remained unchanged, demonstrating that HDAC is involved in endothelial differentiation of progenitor cells. Real-Time PCR demonstrated that TSA down-regulated telomerase activity probably via suppression of hTERT expression, suggesting that HDAC inhibitor decreased cell proliferation. Cell motility was also decreased after treatment with HDAC inhibitors as shown by wound-healing assay. The balance of acethylation/deacethylation kept in control by the activity of HAT (histone acetyltransferases/HDAC enzymes play an important role in differentiation of stem cells by regulating proliferation and endothelial lineage commitment.

  2. A role for activated endothelial cells in red blood cell clearance: implications for vasopathology

    DEFF Research Database (Denmark)

    Fens, Marcel H A M; van Wijk, Richard; Andringa, Grietje

    2012-01-01

    Background Phosphatidylserine exposure by red blood cells is acknowledged as a signal that initiates phagocytic removal of the cells from the circulation. Several disorders and conditions are known to induce phosphatidylserine exposure. Removal of phosphatidylserine-exposing red blood cells gener...... cells play a role in red blood cell clearance in vivo. Significant erythrophagocytosis can induce endothelial cell loss, which may contribute to vasopathological effects as seen, for instance, in sickle cell disease.......Background Phosphatidylserine exposure by red blood cells is acknowledged as a signal that initiates phagocytic removal of the cells from the circulation. Several disorders and conditions are known to induce phosphatidylserine exposure. Removal of phosphatidylserine-exposing red blood cells...... generally occurs by macrophages in the spleen and liver. Previously, however, we have shown that endothelial cells are also capable of erythrophagocytosis. Key players in the erythrophagocytosis by endothelial cells appeared to be lactadherin and αv-integrin. Phagocytosis via the phosphatidylserine...

  3. Endothelial cells of intramuscular (infantile) hemangioma express glut1.

    Science.gov (United States)

    Drut, Ricardo; Altamirano, Eugenia

    2007-04-01

    Glut1 is a marker of infantile hemangioma, and its positivity has resulted in defining this tumor at several sites (eg, skin, breast, salivary glands, liver, and placenta). We herein report on the presence of Glut1 positivity in the endothelial cells of 2 examples of intramuscular hemangioma, a peculiar tumor considered to be most probably congenital. The finding expands the sites where infantile hemangioma may be recognized and suggests that this intramuscular variety should be renamed intramuscular infantile hemangioma. An additional previously unreported finding was the presence of a strong membranous pattern of staining for Glut1 in the intralesional fat cells, a known component of the tumor, which parallels that of another endothelial marker, namely CD34. These findings could prove useful for diagnostic purposes in small biopsies.

  4. Endothelial progenitor cells: Exploring the pleiotropic effects of statins

    Science.gov (United States)

    Sandhu, Kully; Mamas, Mamas; Butler, Robert

    2017-01-01

    Statins have become a cornerstone of risk modification for ischaemic heart disease patients. A number of studies have shown that they are effective and safe. However studies have observed an early benefit in terms of a reduction in recurrent infarct and or death after a myocardial infarction, prior to any significant change in lipid profile. Therefore, pleiotropic mechanisms, other than lowering lipid profile alone, must account for this effect. One such proposed pleiotropic mechanism is the ability of statins to augment both number and function of endothelial progenitor cells. The ability to augment repair and maintenance of a functioning endothelium may have profound beneficial effect on vascular repair and potentially a positive impact on clinical outcomes in patients with cardiovascular disease. The following literature review will discuss issues surrounding endothelial progenitor cell (EPC) identification, role in vascular repair, factors affecting EPC numbers, the role of statins in current medical practice and their effects on EPC number. PMID:28163831

  5. “Decoding” angiogenesis: new facets controlling endothelial cell behavior

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    Massimo Mattia Santoro

    2016-07-01

    Full Text Available Angiogenesis, the formation of new blood vessels, is a unique and crucial biological process occurring during both development and adulthood. A better understanding of the mechanisms that regulates such process is mandatory to intervene in pathophysiological conditions. Here we highlight some recent argument on new players that are critical in endothelial cells, by summarizing novel discoveries that regulate notorious vascular pathways such as Vascular Endothelial Growth Factor (VEGF, Notch and Planar Cell Polarity, and by discussing more recent findings that put metabolism, redox signaling and hemodynamic forces as novel unforeseen facets in angiogenesis. These new aspects, that critically regulate angiogenesis and vascular homeostasis in health and diseased, represent unforeseen new ground to develop anti-angiogenic therapies.

  6. Platelet Endothelial Cell Adhesion Molecule 1 (PECAM-1/CD31): A Multifunctional Vascular Cell Adhesion Molecule.

    Science.gov (United States)

    Delisser, H M; Baldwin, H S; Albelda, S M

    1997-08-01

    PECAM-1/CD31 is a member of the immunoglobulin gene superfamily found on platelets, leukocytes, and endothelial cells, where it concentrates at cell-cell borders. It has been shown to both mediate cell-cell adhesion through homophilic and heterophilic interactions and to transduce intracellular signals that upregulate the function of integrins on leukocytes. Its cellular distribution and ability to mediate adhesive and signaling phenomena suggested that PECAM-1 was a multifunctional vascular cell adhesion molecule involved in leukocyte-endothelial and endothelial-endothelial interactions. These initial suggestions have been largely confirmed as recent studies have implicated PECAM-1 in the inflammatory process and in the formation of blood vessels. As our understanding of the molecular and functional properties of PECAM-1 grows, new insights will be gained that may have therapeutic implications for cardiovascular development and disease. (Trends Cardiovasc Med 1997;7:203-210). © 1997, Elsevier Science Inc.

  7. Stem cell-derived vascular endothelial cells and their potential application in regenerative medicine

    Science.gov (United States)

    Although a 'vascular stem cell' population has not been identified or generated, vascular endothelial and mural cells (smooth muscle cells and pericytes) can be derived from currently known pluripotent stem cell sources, including human embryonic stem cells and induced pluripotent stem cells. We rev...

  8. ENDOTHELIAL PROGENITOR CELLS AS SHUTTLE OF ANTICANCER AGENTS.

    Science.gov (United States)

    Laurenzana, Anna; Margheri, Francesca; Chilla', Anastasia; Biagioni, Alessio; Margheri, Giancarlo; Calorini, Lido; Fibbi, Gabriella; Del Rosso, Mario

    2016-08-08

    Cell therapies are treatments in which stem or progenitor cells are induced to differentiate into the specific cell type required to repair damaged or destroyed tissues. Following their discovery, endothelial progenitor cells (EPCs) have stimulated a worldwide interest as possible vehicles to perform an autologous cell-therapy of tumors. Taking into account the tumor-homing properties of EPCs, two different approaches to control cancer progression have been pursued by combining the cell-based therapy with gene therapy or with nanomedicine. The first one is based on the possibility to engineer EPCs to express different transgenes, the second one on the capacity of EPCs to uptake nanomaterials. Here we will review the most important progresses covering the following issues: the characterization of bona fide endothelial progenitor cells, their role in tumor vascularisation and metastasis, and preclinical data about their use in cell-based tumor therapy, considering anti-angiogenic, suicide, immune-stimulating and oncolytic virus gene-therapy. The mixed approach of EPC cell therapy and nanomedicine will be discussed in terms of plasmonic-dependent thermoablation and molecular imaging.

  9. Adherence of human basophils to cultured umbilical vein endothelial cells.

    OpenAIRE

    1988-01-01

    The mechanism by which circulating human basophils adhere to vascular endothelium and migrate to sites of allergic reactions is unknown. Agents have been identified which stimulate the adherence of purified basophils to cultured human umbilical vein vascular endothelial cells (HuVEC). Treatment of HuVEC with interleukin 1, tumor necrosis factor (TNF), bacterial endotoxin, and 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in time and dose-dependent increases of adhesiveness for basophils...

  10. Directionally Solidified Biopolymer Scaffolds: Mechanical Properties and Endothelial Cell Responses

    OpenAIRE

    Meghri, Nichols W.; Donius, Amalie E.; Riblett, Benjamin W.; Martin, Elizabeth J.; Clyne, Alisa Morss; Wegst, Ulrike G.K.

    2010-01-01

    Vascularization is a primary challenge in tissue engineering. To achieve it in a tissue scaffold, an environment with the appropriate structural, mechanical, and biochemical cues must be provided enabling endothelial cells to direct blood vessel growth. While biochemical stimuli such as growth factors can be added through the scaffold material, the culture medium, or both, a well-designed tissue engineering scaffold is required to provide the necessary local structural and mechanical cues. As...

  11. Corneal endothelial cell density and morphology in Phramongkutklao Hospital

    Directory of Open Access Journals (Sweden)

    Narumon Sopapornamorn

    2008-03-01

    Full Text Available Narumon Sopapornamorn1, Manapon Lekskul1, Suthee Panichkul21Department of Ophthalmology, Phramongkutklao Hospital, Bangkok, Thailand; 2Department of Obstetrics and Gynecology, Phramongkutklao College of Medicine, Bangkok, ThailandObjective: To describe the corneal endothelial density and morphology in patients of Phramongkutklao Hospital and the relationship between endothelial cell parameters and other factors.Methods: Four hundred and four eyes of 202 volunteers were included. Noncontact specular microscopy was performed after taking a history and testing the visual acuity, intraocular pressure measurement, Schirmer’s test and routine eye examination by slit lamp microscope. The studied parameters included mean endothelial cell density (MCD, coefficient of variation (CV, and percentage of hexagonality.Results: The mean age of volunteers was 45.73 years; the range being 20 to 80 years old. Their MCD (SD, mean percentage of CV (SD and mean (SD percentage of hexagonality were 2623.49(325 cell/mm2, 39.43(8.23% and 51.50(10.99%, respectively. Statistically, MCD decreased significantly with age (p < 0.01. There was a significant difference in the percentage of CV between genders. There was no statistical significance between parameters and other factors.Conclusion: The normative data of the corneal endothelium of Thai eyes indicated that, statistically, MCD decreased significantly with age. Previous studies have reported no difference in MCD, percentage of CV, and percentage of hexagonality between gender. Nevertheless, significantly different percentages of CV between genders were presented in this study.Keywords: Corneal endothelial cell, parameters, age, gender, smoking, Thailand

  12. Mechanism of neuronal versus endothelial cell uptake of Alzheimer's disease amyloid beta protein.

    Directory of Open Access Journals (Sweden)

    Karunya K Kandimalla

    Full Text Available Alzheimer's disease (AD is characterized by significant neurodegeneration in the cortex and hippocampus; intraneuronal tangles of hyperphosphorylated tau protein; and accumulation of beta-amyloid (Abeta proteins 40 and 42 in the brain parenchyma as well as in the cerebral vasculature. The current understanding that AD is initiated by the neuronal accumulation of Abeta proteins due to their inefficient clearance at the blood-brain-barrier (BBB, places the neurovascular unit at the epicenter of AD pathophysiology. The objective of this study is to investigate cellular mechanisms mediating the internalization of Abeta proteins in the principle constituents of the neurovascular unit, neurons and BBB endothelial cells. Laser confocal micrographs of wild type (WT mouse brain slices treated with fluorescein labeled Abeta40 (F-Abeta40 demonstrated selective accumulation of the protein in a subpopulation of cortical and hippocampal neurons via nonsaturable, energy independent, and nonendocytotic pathways. This groundbreaking finding, which challenges the conventional belief that Abeta proteins are internalized by neurons via receptor mediated endocytosis, was verified in differentiated PC12 cells and rat primary hippocampal (RPH neurons through laser confocal microscopy and flow cytometry studies. Microscopy studies have demonstrated that a significant proportion of F-Abeta40 or F-Abeta42 internalized by differentiated PC12 cells or RPH neurons is located outside of the endosomal or lysosomal compartments, which may accumulate without degradation. In contrast, BBME cells exhibit energy dependent uptake of F-Abeta40, and accumulate the protein in acidic cell organelle, indicative of endocytotic uptake. Such a phenomenal difference in the internalization of Abeta40 between neurons and BBB endothelial cells may provide essential clues to understanding how various cells can differentially regulate Abeta proteins and help explain the vulnerability of cortical

  13. Direct evidence of endothelial injury in acute myocardial infarction and unstable angina by demonstration of circulating endothelial cells.

    Science.gov (United States)

    Mutin, M; Canavy, I; Blann, A; Bory, M; Sampol, J; Dignat-George, F

    1999-05-01

    Circulating endothelial cells (CECs) have been detected in association with endothelial injury and therefore represent proof of serious damage to the vascular tree. Our aim was to investigate, using the technique of immunomagnetic separation, whether the pathological events in unstable angina (UA) or acute myocardial infarction (AMI) could cause desquamation of endothelial cells in circulating blood compared with effort angina (EA) and noncoronary chest pain. A high CEC count was found in AMI (median, 7.5 cells/mL; interquartile range, 4.1 to 43.5, P chest pain as compared with controls (0; 0 to 0 cells/mL) and stable angina (0; 0 to 0 cells/mL). CEC levels in serial samples peaked at 15.5 (2.7 to 39) cells/mL 18 to 24 hours after AMI (P angina, confirming that these diseases have different etiopathogenic mechanisms.

  14. The soyabean isoflavone genistein modulates endothelial cell behaviour.

    Science.gov (United States)

    Sandoval, Marisa J; Cutini, Pablo H; Rauschemberger, María Belén; Massheimer, Virginia L

    2010-07-01

    The aim of the present study was to investigate the direct action of the phyto-oestrogen genistein (Gen) on vascular endothelial behaviour, either in the presence or absence of proinflammatory agents. In rat aortic endothelial cell (EC) cultures, 24 h of treatment with Gen significantly increased cell proliferation in a wide range of concentration (0.001-10 nm). This mitogenic action was prevented by the oestrogen receptor (ER) antagonist ICI 182780 or by the presence of the specific NO synthase inhibitor l-nitro-arginine methyl ester. When monocytes adhesion to EC was measured, Gen partially attenuated leucocyte adhesion not only under basal conditions, but also in the presence of bacterial lipopolysaccharides (LPS). The effect of the phyto-oestrogen on the expression of EC adhesion molecules was evaluated. Gen down-regulated the enhancement in mRNA levels of E-selectin, vascular cell adhesion molecule-1 and P-selectin elicited by the proinflammatory agent bacterial LPS. The regulation of EC programmed death induced by the isoflavone was also demonstrated. Incubation with 10 nm Gen prevented DNA fragmentation induced by the apoptosis inductor H2O2. The results presented suggest that Gen would exert a protective effect on vascular endothelium, due to its regulatory action on endothelial proliferation, apoptosis and leucocyte adhesion, events that play a critical role in vascular diseases. The molecular mechanism displayed by the phyto-oestrogen involved the participation of the ER and the activation of the NO pathway.

  15. Proteomic profiling of endorepellin angiostatic activity on human endothelial cells

    Directory of Open Access Journals (Sweden)

    Iozzo Renato V

    2008-02-01

    Full Text Available Abstract Background Endorepellin, the C-terminal domain V of the heparan sulfate proteoglycan perlecan, exhibits powerful and targeted anti-angiogenic activity on endothelial cells. To identify proteins involved with endorepellin anti-angiogenic action, we performed an extensive comparative proteomic analysis between vehicle- and endorepellin-treated human endothelial cells. Results Proteomic analysis of endorepellin influence on human umbilical vein endothelial cells identified five differentially expressed proteins, three of which (β-actin, calreticulin, and chaperonin/Hsp60 were down-regulated and two of which (vimentin and the β subunit of prolyl 4-hydroxylase also known as protein disulfide isomerase were up-regulated in response to endorepellin treatment—and associated with a fold change (endorepellin/control ≤ 0.75 and ≥ 2.00, and a statistically significant p-value as determined by Student's t test. Conclusion The proteins identified represent potential target areas involved with endorepellin anti-angiogenic mechanism of action. Further elucidation as such will ultimately provide useful in utilizing endorepellin as an anti-angiogenic therapy in humans.

  16. Interaction of recombinant octameric hemoglobin with endothelial cells.

    Science.gov (United States)

    Gaucher, Caroline; Domingues-Hamdi, Élisa; Prin-Mathieu, Christine; Menu, Patrick; Baudin-Creuza, Véronique

    2015-02-01

    Hemoglobin-based oxygen carriers (HBOCs) may generate oxidative stress, vasoconstriction and inflammation. To reduce these undesirable vasoactive properties, we increased hemoglobin (Hb) molecular size by genetic engineering with octameric Hb, recombinant (r) HbβG83C. We investigate the potential side effects of rHbβG83C on endothelial cells. The rHbβG83C has no impact on cell viability, and induces a huge repression of endothelial nitric oxide synthase gene transcription, a marker of vasomotion. No induction of Intermolecular-Adhesion Molecule 1 and E-selectin (inflammatory markers) transcription was seen. In the presence of rHbβG83C, the transcription of heme oxygenase-1 (oxidative stress marker) is weakly increased compared to the two other HBOCs (references) or Voluven (control). This genetically engineered octameric Hb, based on a human Hb βG83C mutant, leads to little impact at the level of endothelial cell inflammatory response and thus appears as an interesting molecule for HBOC development.

  17. Endothelial cells downregulate apolipoprotein D expression in mural cells through paracrine secretion and Notch signaling.

    Science.gov (United States)

    Pajaniappan, Mohanasundari; Glober, Nancy K; Kennard, Simone; Liu, Hua; Zhao, Ning; Lilly, Brenda

    2011-09-01

    Endothelial and mural cell interactions are vitally important for proper formation and function of blood vessels. These two cell types communicate to regulate multiple aspects of vessel function. In studying genes regulated by this interaction, we identified apolipoprotein D (APOD) as one gene that is downregulated in mural cells by coculture with endothelial cells. APOD is a secreted glycoprotein that has been implicated in governing stress response, lipid metabolism, and aging. Moreover, APOD is known to regulate smooth muscle cells and is found in abundance within atherosclerotic lesions. Our data show that the regulation of APOD in mural cells is bimodal. Paracrine secretion by endothelial cells causes partial downregulation of APOD expression. Additionally, cell contact-dependent Notch signaling plays a role. NOTCH3 on mural cells promotes the downregulation of APOD, possibly through interaction with the JAGGED-1 ligand on endothelial cells. Our results show that NOTCH3 contributes to the downregulation of APOD and by itself is sufficient to attenuate APOD transcript expression. In examining the consequence of decreased APOD expression in mural cells, we show that APOD negatively regulates cell adhesion. APOD attenuates adhesion by reducing focal contacts; however, it has no effect on stress fiber formation. These data reveal a novel mechanism in which endothelial cells control neighboring mural cells through the downregulation of APOD, which, in turn, influences mural cell function by modulating adhesion.

  18. Regulation and function of TRPM7 in human endothelial cells: TRPM7 as a potential novel regulator of endothelial function.

    Directory of Open Access Journals (Sweden)

    Erika Baldoli

    Full Text Available TRPM7, a cation channel of the transient receptor potential channel family, has been identified as a ubiquitous magnesium transporter. We here show that TRPM7 is expressed in endothelial cells isolated from the umbilical vein (HUVEC, widely used as a model of macrovascular endothelium. Quiescence and senescence do not modulate TRPM7 amounts, whereas oxidative stress generated by the addition of hydrogen peroxide increases TRPM7 levels. Moreover, high extracellular magnesium decreases the levels of TRPM7 by activating calpains, while low extracellular magnesium, known to promote endothelial dysfunction, stimulates TRPM7 accumulation partly through the action of free radicals. Indeed, the antioxidant trolox prevents TRPM7 increase by low magnesium. We also demonstrate the unique behaviour of HUVEC in responding to pharmacological and genetic inhibition of TRPM7 with an increase of cell growth and migration. Our results indicate that TRPM7 modulates endothelial behavior and that any condition leading to TRPM7 upregulation might impair endothelial function.

  19. Oxidized extracellular DNA suppresses nitric oxide production by endothelial NO synthase (eNOS) in human endothelial cells (HUVEC).

    Science.gov (United States)

    Kostyuk, S V; Alekseeva, A Yu; Kon'kova, M S; Glebova, K V; Smirnova, T D; Kameneva, L V; Izhevskaya, V L; Veiko, N N

    2014-06-01

    Circulating DNA from patients with cardiovascular diseases reduce the synthesis of NO in endothelial cells, which is probably related to oxidative modification of DNA. To test this hypothesis, HUVEC cells were cultured in the presence of DNA containing ~1 (nonoxidized DNA), 700, or 2100 8-oxodG/10(6) nucleosides. Nonoxidized DNA stimulated the synthesis of NO, which was associated with an increase in the expression of endothelial NO synthase. Oxidized NO decreased the amount of mRNA and protein for endothelial NO synthase, but increased the relative content of its low active form. These changes were accompanied by reduction of NO production. These findings suggest that oxidative modification of circulating extracellular DNA contributes to endothelial dysfunction manifested in suppression of NO production.

  20. Effect of endothelial progenitor cells in neovascularization and their application in tumor therapy

    Institute of Scientific and Technical Information of China (English)

    DONG Fang; HA Xiao-qin

    2010-01-01

    Objective To review the effect of endothelial progenitor cells in neovascularization as well as their application to the therapy of tumors.Data sources The data used in this review were mainly from PubMed for relevant English language articles published from 1997 to 2009. The search term was "endothelial progenitor cells".Study selection Articles regarding the role of endothelial progenitor cells in neovascularization and their application to the therapy of tumors were selected.Results Endothelial progenitor cells isolated from bone marrow, umbilical cord blood and peripheral blood can proliferate, mobilize and differentiate into mature endothelial cells. Experiments suggest endothelial progenitor cells take part in forming the tumor vascular through a variety of mechanisms related to vascular endothelial growth factor, matrix metalloproteinases, chemokine stromal cell-derived factor 1 and its receptor C-X-C receptor-4, erythropoietin, Notchsignal pathway and so on. Evidence demonstrates that the number and function change of endothelial progenitor cells in peripheral blood can be used as a biomarker of the response of cancer patients to anti-tumor therapy and predict the prognosis and recurrence. In addition, irradiation temporarily increased endothelial cells number and decreased the endothelial progenitor cell counts in animal models. Meanwhile, in preclinical experiments, therapeutic gene-modified endothelial progenitor cells have been approved to attenuate tumor growth and offer a novel strategy for cell therapy and gene therapy of cancer.Conclusions Endothelial progenitor cells play a particular role in neovascularization and have attractively potential prognostic and therapeutic applications to malignant tumors. However, a series of problems, such as the definitive biomarkers of endothelial progenitor cells, their interrelationship with radiotherapy and their application in cell therapy and gene therapy of tumors, need further investigation.

  1. Endothelial Cell Toxicity of Vancomycin Infusion Combined with Other Antibiotics.

    Science.gov (United States)

    Drouet, Maryline; Chai, Feng; Barthélémy, Christine; Lebuffe, Gilles; Debaene, Bertrand; Décaudin, Bertrand; Odou, Pascal

    2015-08-01

    French guidelines recommend central intravenous (i.v.) infusion for high concentrations of vancomycin, but peripheral intravenous (p.i.v.) infusion is often preferred in intensive care units. Vancomycin infusion has been implicated in cases of phlebitis, with endothelial toxicity depending on the drug concentration and the duration of the infusion. Vancomycin is frequently infused in combination with other i.v. antibiotics through the same administrative Y site, but the local toxicity of such combinations has been poorly evaluated. Such an assessment could improve vancomycin infusion procedures in hospitals. Human umbilical vein endothelial cells (HUVEC) were challenged with clinical doses of vancomycin over 24 h with or without other i.v. antibiotics. Cell death was measured with the alamarBlue test. We observed an excess cellular death rate without any synergistic effect but dependent on the numbers of combined infusions when vancomycin and erythromycin or gentamicin were infused through the same Y site. Incompatibility between vancomycin and piperacillin-tazobactam was not observed in our study, and rinsing the cells between the two antibiotic infusions did not reduce endothelial toxicity. No endothelial toxicity of imipenem-cilastatin was observed when combined with vancomycin. p.i.v. vancomycin infusion in combination with other medications requires new recommendations to prevent phlebitis, including limiting coinfusion on the same line, reducing the infusion rate, and choosing an intermittent infusion method. Further studies need to be carried out to explore other drug combinations in long-term vancomycin p.i.v. therapy so as to gain insight into the mechanisms of drug incompatibility under multidrug infusion conditions.

  2. Hypoxia-induced reactive oxygen species cause chromosomal abnormalities in endothelial cells in the tumor microenvironment.

    Directory of Open Access Journals (Sweden)

    Miyako Kondoh

    Full Text Available There is much evidence that hypoxia in the tumor microenvironment enhances tumor progression. In an earlier study, we reported abnormal phenotypes of tumor-associated endothelial cells such as those resistant to chemotherapy and chromosomal instability. Here we investigated the role of hypoxia in the acquisition of chromosomal abnormalities in endothelial cells. Tumor-associated endothelial cells isolated from human tumor xenografts showed chromosomal abnormalities, >30% of which were aneuploidy. Aneuploidy of the tumor-associated endothelial cells was also shown by simultaneous in-situ hybridization for chromosome 17 and by immunohistochemistry with anti-CD31 antibody for endothelial staining. The aneuploid cells were surrounded by a pimonidazole-positive area, indicating hypoxia. Human microvascular endothelial cells expressed hypoxia-inducible factor 1 and vascular endothelial growth factor A in response to either hypoxia or hypoxia-reoxygenation, and in these conditions, they acquired aneuploidy in 7 days. Induction of aneuploidy was inhibited by either inhibition of vascular endothelial growth factor signaling with vascular endothelial growth factor receptor 2 inhibitor or by inhibition of reactive oxygen species by N-acetyl-L-cysteine. These results indicate that hypoxia induces chromosomal abnormalities in endothelial cells through the induction of reactive oxygen species and excess signaling of vascular endothelial growth factor in the tumor microenvironment.

  3. Hypoxia-Induced Reactive Oxygen Species Cause Chromosomal Abnormalities in Endothelial Cells in the Tumor Microenvironment

    Science.gov (United States)

    Hida, Yasuhiro; Maishi, Nako; Towfik, Alam Mohammad; Inoue, Nobuo; Shindoh, Masanobu; Hida, Kyoko

    2013-01-01

    There is much evidence that hypoxia in the tumor microenvironment enhances tumor progression. In an earlier study, we reported abnormal phenotypes of tumor-associated endothelial cells such as those resistant to chemotherapy and chromosomal instability. Here we investigated the role of hypoxia in the acquisition of chromosomal abnormalities in endothelial cells. Tumor-associated endothelial cells isolated from human tumor xenografts showed chromosomal abnormalities, >30% of which were aneuploidy. Aneuploidy of the tumor-associated endothelial cells was also shown by simultaneous in-situ hybridization for chromosome 17 and by immunohistochemistry with anti-CD31 antibody for endothelial staining. The aneuploid cells were surrounded by a pimonidazole-positive area, indicating hypoxia. Human microvascular endothelial cells expressed hypoxia-inducible factor 1 and vascular endothelial growth factor A in response to either hypoxia or hypoxia-reoxygenation, and in these conditions, they acquired aneuploidy in 7 days. Induction of aneuploidy was inhibited by either inhibition of vascular endothelial growth factor signaling with vascular endothelial growth factor receptor 2 inhibitor or by inhibition of reactive oxygen species by N-acetyl-L-cysteine. These results indicate that hypoxia induces chromosomal abnormalities in endothelial cells through the induction of reactive oxygen species and excess signaling of vascular endothelial growth factor in the tumor microenvironment. PMID:24260373

  4. 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.

  5. Overexpression of Ref-1 Inhibits Lead-induced Endothelial Cell Death via the Upregulation of Catalase.

    Science.gov (United States)

    Lee, Kwon Ho; Lee, Sang Ki; Kim, Hyo Shin; Cho, Eun Jung; Joo, Hee Kyoung; Lee, Eun Ji; Lee, Ji Young; Park, Myoung Soo; Chang, Seok Jong; Cho, Chung-Hyun; Park, Jin Bong; Jeon, Byeong Hwa

    2009-12-01

    The role of apurinic/apyrimidinic endonuclease1/redox factor-1 (Ref-1) on the lead (Pb)-induced cellular response was investigated in the cultured endothelial cells. Pb caused progressive cellular death in endothelial cells, which occurred in a concentration- and time-dependent manner. However, Ref-1 overexpression with AdRef-1 significantly inhibited Pb-induced cell death in the endothelial cells. Also the overexpression of Ref-1 significantly suppressed Pb-induced superoxide and hydrogen peroxide elevation in the endothelial cells. Pb exposure induced the downregulation of catalase, it was inhibited by the Ref-1 overexpression in the endothelial cells. Taken together, our data suggests that the overexpression of Ref-1 inhibited Pb-induced cell death via the upregulation of catalase in the cultured endothelial cells.

  6. Atrial natriuretic peptide prevents cancer metastasis through vascular endothelial cells.

    Science.gov (United States)

    Nojiri, Takashi; Hosoda, Hiroshi; Tokudome, Takeshi; Miura, Koichi; Ishikane, Shin; Otani, Kentaro; Kishimoto, Ichiro; Shintani, Yasushi; Inoue, Masayoshi; Kimura, Toru; Sawabata, Noriyoshi; Minami, Masato; Nakagiri, Tomoyuki; Funaki, Soichiro; Takeuchi, Yukiyasu; Maeda, Hajime; Kidoya, Hiroyasu; Kiyonari, Hiroshi; Shioi, Go; Arai, Yuji; Hasegawa, Takeshi; Takakura, Nobuyuki; Hori, Megumi; Ohno, Yuko; Miyazato, Mikiya; Mochizuki, Naoki; Okumura, Meinoshin; Kangawa, Kenji

    2015-03-31

    Most patients suffering from cancer die of metastatic disease. Surgical removal of solid tumors is performed as an initial attempt to cure patients; however, surgery is often accompanied with trauma, which can promote early recurrence by provoking detachment of tumor cells into the blood stream or inducing systemic inflammation or both. We have previously reported that administration of atrial natriuretic peptide (ANP) during the perioperative period reduces inflammatory response and has a prophylactic effect on postoperative cardiopulmonary complications in lung cancer surgery. Here we demonstrate that cancer recurrence after curative surgery was significantly lower in ANP-treated patients than in control patients (surgery alone). ANP is known to bind specifically to NPR1 [also called guanylyl cyclase-A (GC-A) receptor]. In mouse models, we found that metastasis of GC-A-nonexpressing tumor cells (i.e., B16 mouse melanoma cells) to the lung was increased in vascular endothelium-specific GC-A knockout mice and decreased in vascular endothelium-specific GC-A transgenic mice compared with control mice. We examined the effect of ANP on tumor metastasis in mice treated with lipopolysaccharide, which mimics systemic inflammation induced by surgical stress. ANP inhibited the adhesion of cancer cells to pulmonary arterial and micro-vascular endothelial cells by suppressing the E-selectin expression that is promoted by inflammation. These results suggest that ANP prevents cancer metastasis by inhibiting the adhesion of tumor cells to inflamed endothelial cells.

  7. An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics.

    Science.gov (United States)

    Martinelli, Roberta; Carman, Christopher V

    2015-12-24

    Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells ('APCs') referred to as 'immunological synapses'. Within these intimate cell-cell interfaces discrete sub-cellular clusters of MHC/Ag-TCR, F-actin, adhesion and signaling molecules form and remodel rapidly. These dynamics are thought to be critical determinants of both the efficiency and quality of the immune responses that develop and therefore of protective versus pathologic immunity. Current understanding of immunological synapses with physiologic APCs is limited by the inadequacy of the obtainable imaging resolution. Though artificial substrate models (e.g., planar lipid bilayers) offer excellent resolution and have been extremely valuable tools, they are inherently non-physiologic and oversimplified. Vascular and lymphatic endothelial cells have emerged as an important peripheral tissue (or stromal) compartment of 'semi-professional APCs'. These APCs (which express most of the molecular machinery of professional APCs) have the unique feature of forming virtually planar cell surface and are readily transfectable (e.g., with fluorescent protein reporters). Herein a basic approach to implement endothelial cells as a novel and physiologic 'planar cellular APC model' for improved imaging and interrogation of fundamental antigenic signaling processes will be described.

  8. Antioxidant Effects of Sheep Whey Protein on Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Efthalia Kerasioti

    2016-01-01

    Full Text Available Excessive production of reactive oxygen species (ROS may cause endothelial dysfunction and consequently vascular disease. In the present study, the possible protective effects of sheep whey protein (SWP from tert-butyl hydroperoxide- (tBHP- induced oxidative stress in endothelial cells (EA.hy926 were assessed using oxidative stress biomarkers. These oxidative stress biomarkers were glutathione (GSH and ROS levels determined by flow cytometry. Moreover, thiobarbituric acid-reactive substances (TBARS, protein carbonyls (CARB, and oxidized glutathione (GSSG were determined spectrophotometrically. The results showed that SWP at 0.78, 1.56, 3.12, and 6.24 mg of protein mL−1 increased GSH up to 141%, while it decreased GSSG to 46.7%, ROS to 58.5%, TBARS to 52.5%, and CARB to 49.0%. In conclusion, the present study demonstrated for the first time that SWP protected endothelial cells from oxidative stress. Thus, SWP may be used for developing food supplements or biofunctional foods to attenuate vascular disturbances associated with oxidative stress.

  9. Tumor Necrosis Factor Type α , a Potent Inhibitor of Endothelial Cell Growth in vitro, is Angiogenic in vivo

    Science.gov (United States)

    Frater-Schroder, Marijke; Risau, Werner; Hallmann, Rupert; Gautschi, Peter; Bohlen, Peter

    1987-08-01

    Tumor necrosis factor type α (TNF-α ) inhibits endothelial cell proliferation in vitro. Basal cell growth (in the absence of exogenously added growth factor) and fibroblast growth factor (FGF)-stimulated cell proliferation are inhibited in a dose-dependent manner from 0.1 to 10 ng/ml with half-maximal inhibition occurring at 0.5-1.0 ng of TNF-α per ml. Bovine aortic and brain capillary endothelial and smooth muscle cells are similarly affected. TNF-α is a noncompetitive antagonist of FGF-stimulated cell proliferation. Its action on endothelial cells is reversible and noncytotoxic. Surprisingly, TNF-α does not seem to inhibit endothelial cell proliferation in vivo. In the rabbit cornea, even a high dose of TNF-α (10 μ g) does not suppress angiogenesis induced by basic FGF. On the contrary, in this model system TNF-α stimulates neovascularization. The inflammatory response that is seen in the cornea after TNF-α implantation suggests that the angiogenic properties of this agent may be a consequence of leukocyte infiltration.

  10. Salvianolic acid B improves vascular endothelial function in diabetic rats with blood glucose fluctuations via suppression of endothelial cell apoptosis.

    Science.gov (United States)

    Ren, Younan; Tao, Shanjun; Zheng, Shuguo; Zhao, Mengqiu; Zhu, Yuanmei; Yang, Jieren; Wu, Yuanjie

    2016-11-15

    Vascular endothelial cell injury is an initial event in atherosclerosis. Salvianolic acid B (Sal B), a main bioactive component in the root of Salvia miltiorrhiza, has vascular protective effect in diabetes, but the underlying mechanisms remain unclear. The present study investigated the effect of Sal B on vascular endothelial function in diabetic rats with blood glucose fluctuations and the possible mechanisms implicated. The results showed that diabetic rats developed marked endothelial dysfunction as exhibited by impaired acetylcholine induced vasodilation. Supplementation with Sal B resulted in an evident improvement of endothelial function. Phosphorylation (Ser 1177) of endothelial nitric oxide synthase (eNOS) was significantly restored in Sal B treated diabetic rats, accompanied by an evident recovery of NO metabolites. Sal B effectively reduced vascular endothelial cell apoptosis, with Bcl-2 protein up-regulated and Bax protein down-regulated markedly. Treatment with Sal B led to an evident amelioration of oxidative stress in diabetic rats as manifested by enhanced antioxidant capacity and decreased contents of malondialdehyde in aortas. Protein levels of NOX2 and NOX4, two main isoforms of NADPH oxidase known as the major source of reactive oxygen species in the vasculature, were markedly decreased in Sal B treated groups. In addition, treatment with Sal B led to an evident decrease of serum lipids. Taken together, this study indicates that Sal B is capable of improving endothelial function in diabetic rats with blood glucose fluctuations, of which the underlying mechanisms might be related to suppression of endothelial cell apoptosis and stimulation of eNOS phosphorylation (Ser 1177).

  11. In vitro differentiation of human adipose-derived mesenchymal stem cells into endothelial-like cells

    Institute of Scientific and Technical Information of China (English)

    GUAN Lidong; SHI Shuangshuang; PEI Xuetao; LI Shaoqing; WANG Yunfang; YUE Huimin; LIU Daqing; HE Lijuan; BAI Cixian; YAN Fang; NAN Xue

    2006-01-01

    The neovascularization of ischemic tissue is a crucial initial step for the functional rehabilitation and wound healing. However, the short of seed cell candidate for the foundation of vascular network is still a big issue. Human adipose tissue derived mesenchymal stem cells (hADSCs), which possess multilineage potential, are capable of adipogenic, osteogenic, and chondrogenic differentiation. We examined whether this kind of stem cells could differentiate into endothelial-like cells and participate in blood vessel formation, and whether they could be used as an ideal cell source for therapeutic angiogenesis in ischemic diseases or vascularization of tissue constructs. The results showed that hADSCs, grown under appropriately induced conditions, displayed characteristics similar to those of vessel endothelium. The differentiated cells expressed endothelial cell markers CD34 and vWF, and had high metabolism of acetylated low-density lipoprotein and prostacyclin. In addition, the induced cells were able to form tube-like structures when cultured on matrigel. Our data indicated that induced hADSCs could exhibit characteristics of endothelial cells. Therefore, these cells, as a source of human endothelial cells, may find many applications in such realms as engineering blood vessels, endothelial cell transplantation for myocardial regeneration, and induction of angiogenesis for treatment of regional ischemia.

  12. Endothelial cell compatibility of trovafloxacin and levofloxacin for intravenous use.

    Science.gov (United States)

    Armbruster, C; Robibaro, B; Griesmacher, A; Vorbach, H

    2000-04-01

    Levofloxacin and trovafloxacin have excellent activity against a variety of Gram-positive and Gram-negative organisms resistant to the established agents. One local side-effect closely related to the use of parenteral fluoroquinolones is phlebitis. To evaluate the effect of trovafloxacin and levofloxacin on endothelial cell viability, intracellular levels of adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), guanosine 5'-triphosphate (GTP) and guanosine 5'-diphosphate (GDP) levels were measured using high-performance liquid chromatography. Trovafloxacin at concentrations of 2 and 1 mg/mL reduced the intracellular ATP content from 12.5 +/- 1.7 to 1.9 +/- 0.3 nmol/10(6) cells and 9.3 +/- 0.8 nmol/10(6) cells, respectively, within 60 min. In addition, ADP, GTP and GDP levels were extensively depleted. Levofloxacin at concentrations of 5 and 2.5 mg/mL led to a significant ATP decline from 12.5 +/- 1.7 to 2.3 +/- 0.2 nmol/10(6) cells and 10.3 +/- 0.9 nmol/10(6) cells, respectively, within 60 min. These data indicate that infusions of high doses of trovafloxacin or levofloxacin are not compatible with maintenance of endothelial cell function. Commercial preparations have to be diluted and should be administered into large veins.

  13. SECs (Sinusoidal Endothelial Cells), Liver Microenvironment, and Fibrosis

    Science.gov (United States)

    Natarajan, Vaishaali; Harris, Edward N.

    2017-01-01

    Liver fibrosis is a wound-healing response to chronic liver injury such as alcoholic/nonalcoholic fatty liver disease and viral hepatitis with no FDA-approved treatments. Liver fibrosis results in a continual accumulation of extracellular matrix (ECM) proteins and paves the way for replacement of parenchyma with nonfunctional scar tissue. The fibrotic condition results in drastic changes in the local mechanical, chemical, and biological microenvironment of the tissue. Liver parenchyma is supported by an efficient network of vasculature lined by liver sinusoidal endothelial cells (LSECs). These nonparenchymal cells are highly specialized resident endothelial cell type with characteristic morphological and functional features. Alterations in LSECs phenotype including lack of LSEC fenestration, capillarization, and formation of an organized basement membrane have been shown to precede fibrosis and promote hepatic stellate cell activation. Here, we review the interplay of LSECs with the dynamic changes in the fibrotic liver microenvironment such as matrix rigidity, altered ECM protein profile, and cell-cell interactions to provide insight into the pivotal changes in LSEC physiology and the extent to which it mediates the progression of liver fibrosis. Establishing the molecular aspects of LSECs in the light of fibrotic microenvironment is valuable towards development of novel therapeutic and diagnostic targets of liver fibrosis. PMID:28293634

  14. In-vivo cell tracking to quantify endothelial cell migration during zebrafish angiogenesis

    Science.gov (United States)

    Menon, Prahlad G.; Rochon, Elizabeth R.; Roman, Beth L.

    2016-03-01

    The mechanism of endothelial cell migration as individual cells or collectively while remaining an integral component of a functional blood vessel has not been well characterized. In this study, our overarching goal is to define an image processing workflow to facilitate quantification of how endothelial cells within the first aortic arch and are proximal to the zebrafish heart behave in response to the onset of flow (i.e. onset of heart beating). Endothelial cell imaging was conducted at this developmental time-point i.e. ~24-28 hours post fertilization (hpf) when flow first begins, using 3D+time two-photon confocal microscopy of a live, wild-type, transgenic, zebrafish expressing green fluorescent protein (GFP) in endothelial cell nuclei. An image processing pipeline comprised of image signal enhancement, median filtering for speckle noise reduction, automated identification of the nuclei positions, extraction of the relative movement of nuclei between consecutive time instances, and finally tracking of nuclei, was designed for achieving the tracking of endothelial cell nuclei and the identification of their movement towards or away from the heart. Pilot results lead to a hypothesis that upon the onset of heart beat and blood flow, endothelial cells migrate collectively towards the heart (by 21.51+/-10.35 μm) in opposition to blood flow (i.e. subtending 142.170+/-21.170 with the flow direction).

  15. Flow-induced Expression and Phosphorylation of VASPin Endothelial Cells

    Institute of Scientific and Technical Information of China (English)

    Muller; SYLYAINE; Jean-FranoisSYOLTZ

    2005-01-01

    1 Introduction It is well known that mechanical forces have important influence on endothelial cells, in particular, on cytoskeleton reorganization. VASP (vasodilator stimulated phosphoprotein) is a 46 KD actin associated protein. It is a member of Ena/VASP protein family and composed of EVH1, proline-rich and EVH2 domains. It is considered as an important component of the sub-cellular regions where remodelling of the actin cytoskeleton takes place, such as the front of spreading lamellipodia in motile cell...

  16. Bone marrow-derived cells serve as proangiogenic macrophages but not endothelial cells in wound healing

    OpenAIRE

    Okuno, Yuji; Nakamura-Ishizu, Ayako; Kishi,Kazuo; Suda, Toshio; Kubota, Yoshiaki

    2011-01-01

    Bone marrow-derived cells (BMDCs) contribute to postnatal vascular growth by differentiating into endothelial cells or secreting angiogenic factors. However, the extent of their endothelial differentiation highly varies according to the angiogenic models used. Wound healing is an intricate process in which the skin repairs itself after injury. As a process also observed in cancer progression, neoangiogenesis into wound tissues is profoundly involved in this healing process, suggesting the con...

  17. Galectin-3 induces pulmonary artery endothelial cell morphogenesis and angiogenesis

    Institute of Scientific and Technical Information of China (English)

    ZHANG Li; LI Yu-mei; WANG Xiao-yan; ZHU Da-ling

    2016-01-01

    AIM:Increasing evidence suggests that carbohydrate-binding proteins play an essential role in tumor growth and metastasis .Ga-lectin-3, a multifunctional protein of an expanding family of β-galactoside-binding animal lectins , is the major nonintegrin cellular laminin-binding protein , and is implicated in a variety of biologic events , such as inflammation and angiogenesis .Because galectin-3 expression was shown to participate in mediating tumor angiogenesis and initiate signaling cascades in several diseases .We hypothe-sized that galectin-3 may promote pulmonary vascular endothelial neovascularization .METHODS:Hypoxic and MCT rat model of pul-monary artery remodeling was used .The mRNA and protein levels of galectin-3 in rats were measured by in situ hybrization and West-ern blot analysis.Endothelial cell (EC) proliferation, migration and tube formation were measured using MTT , cell scratch and Matri-gel assays, respectively.Protein expression was quantitated by Western blot analysis .LC 3A/B staining was detected with cellular im-munofluorescence staining .RESULTS:We found that galectin-3 was localized on the intima and adventitial wall .Galectin-3 was in-creased after rat hypoxia and MCT administration .Galectin-3 promoted EC proliferation , migration and tube formation , while its roles were reversed by RNA interference.Galectin-3 induced Atg 5, Beclin-1, LAMP-2, and LC 3A/B expression increases.Galectin-3 al-so increased LC 3A/B staining in ECs.Akt/mTOR and GSK-3βsignaling pathways were activated after galectin-3 treated ECs using its specific phosphorylation antibodies , while blocked it with LY294002 inhibited cell autophagy and EC dynamic alterations induced by galectin-3.CONCLUSION:These findings demonstrate that galectin-3 can induce an Akt signaling cascade leading to cell autoph-agy, and then the differentiation and angiogenesis of pulmonary artery endothelial cells .

  18. Microvascular endothelial cell heterogeneity : general concepts and pharmacological consequences for anti-angiogenic therapy of cancer

    NARCIS (Netherlands)

    Langenkamp, Elise; Molema, Grietje

    2009-01-01

    Microvascular endothelial cells display a large degree of heterogeneity in function depending on their location in the vascular tree. The existence of organ-specific, microvascular-bed-specific, and even intravascular variations in endothelial cell gene expression emphasizes their high cell-to-cell

  19. Tie-1-directed expression of Cre recombinase in endothelial cells of embryoid bodies and transgenic mice

    DEFF Research Database (Denmark)

    Gustafsson, E; Brakebusch, C; Hietanen, K

    2001-01-01

    the production and screening of multiple transgenic lines we used embryonic stem cell and embryoid body technology to identify recombinant embryonic stem cell clones with high, endothelial-specific Cre activity. One embryonic stem cell clone that showed high Cre activity in endothelial cells was used to generate...

  20. Differences in the primary culture, purification and biological characteristics between endothelial cells and smooth muscle cells from rat aorta

    Institute of Scientific and Technical Information of China (English)

    Shaobo Hu; Zifang Song; Qichang Zheng; Jun Nie

    2009-01-01

    Objective: To investigate the differences of primary culture, purification and biological characteristics between endothelial cells and smooth muscle cells from rat aorta. Methods: Endothelial cells were obtained using the vascular ring adherence, collagenase digestion method and an improved vascular ring adherence method, while smooth muscle cells were separated from tissue sections of rat aorta. Clones of endothelial cells were selected by limiting dilution assay. Both cell types were identified using specific cell immunofluorescent markers,and phase contrast microscopy was used to observe the morphological disparity between endothelial cells and smooth muscle cells at the single cell and colony level. Cell proliferation was determined by the cell counting kit-8. Differences between endothelial cells and smooth muscle cells were evaluated in trypsin digestion 6me, attachment time and recovery after cryopreservation. Results: Endothelial cells were obtained by all three methods. The improved vascular ring method provided the most reproducible results. Cells were in good condition, and of high purity. Smooth muscle cells were cultured successfully by the tissue fragment culture method. Clonal expansion of singleendothelial cells was attained. The two cell types expressed their respective specific markers, and the rate of proliferation of smooth muscle cells exceeded that of endothelial cells. Endothelial cells were more sensitive to trypsin digestion than smooth muscle cells. In addition, they had a shorter adherence time and better recovery following cryopreservation than smooth muscle cells. Conclusion: The improved vascular ring method was optimal for yielding endothelial cells. Limiting dilution is a novel and valid method for purifying primary endothelial cells from rat aorta. Primary rat endothelial cell and vascular smooth muscle cell cultures exhibited different morphological characteristics, proliferation rate, adherence time, susceptibility to trypsin

  1. Recombinant Treponema pallidum protein Tp0965 activates endothelial cells and increases the permeability of endothelial cell monolayer.

    Directory of Open Access Journals (Sweden)

    Rui-Li Zhang

    Full Text Available The recombinant Treponema pallidum protein Tp0965 (rTp0965, one of the many proteins derived from the genome of T. pallidum subsp. pallidum, shows strong immunogenicity and immunoreactivity. In this study, we investigated the effects of rTp0965 on the endothelial barrier. Treatment of human umbilical vein endothelial cells (HUVECs with rTp0965 resulted in increased levels of ICAM-1, E-selectin, and MCP-1 mRNA and protein expression. These increases contributed to the adhesion and chemataxis of monocytes (THP-1 cells to HUVECs preincubated with rTp0965. In addition, rTp0965 induced reorganization of F-actin and decreased expression of claudin-1 in HUVECs. Interestingly, inhibition of the RhoA/ROCK signal pathway protected against rTp0965-induced higher endothelial permeability as well as transendothelial migration of monocytes. These data indicate that Tp0965 protein may play an important role in the immunopathogenesis of syphilis.

  2. Prune melanoidins protect against oxidative stress and endothelial cell death.

    Science.gov (United States)

    Posadino, Anna Maria; Cossu, Annalisa; Piga, Antonio; Madrau, Monica Assunta; Del Caro, Alessandra; Colombino, Maria; Paglietti, Bianca; Rubino, Salvatore; Iaccarino, Ciro; Crosio, Claudia; Sanna, Bastiano; Pintus, Gianfranco

    2011-06-01

    The health-promoting effects of fruit and vegetable consumption are thought to be due to phytochemicals contained in fresh plant material. Whether processed plant foods provide the same benefits as unprocessed ones is an open question. Melanoidins from heat-processed plums (prunes) were isolated and their presence confirmed by hydroxymethylfurfural content and browning index. Oxidative-induced endothelial cell (EC) damage is the trigger for the development of cardiovascular diseases (CVD); therefore the potential protective effect of prune melanoidins on hydrogen peroxide-induced oxidative cell damage was investigated on human endothelial ECV304 cells. Cytoplasmic and mitochondrial redox status was assessed by using the novel, redox-sensitive, ratiometric fluorescent protein sensor (roGFP), while mitochondrial membrane potential (MMP) was investigated with the fluorescent dye, JC-1. Treatment of ECV304 cells with hydrogen peroxide dose-dependently induced both mitochondrial and cytoplasmic oxidation, in addition to MMP dissipation, with ensuing cell death. Pretreatment of ECV304 with prune melanoidins, significantly counteracted and ultimately abolished hydrogen peroxide elicited phenomena, clearly indicating that these polymers protect human EC against oxidative stress.

  3. Growth factor-and cytokine-stimulated endothelial progenitor cells in post-ischemic cerebral neovascularization

    Institute of Scientific and Technical Information of China (English)

    Philip V.Peplow

    2014-01-01

    Endothelial progenitor cells are resident in the bone marrow blood sinusoids and circulate in the peripheral circulation. They mobilize from the bone marrow after vascular injury and home to the site of injury where they differentiate into endothelial cells. Activation and mobilization of endothelial progenitor cells from the bone marrow is induced via the production and release of endothelial progenitor cell-activating factors and includes speciifc growth factors and cytokines in response to peripheral tissue hypoxia such as after acute ischemic stroke or trauma. Endotheli-al progenitor cells migrate and home to speciifc sites following ischemic stroke via growth factor/cytokine gradients. Some growth factors are less stable under acidic conditions of tissue isch-emia, and synthetic analogues that are stable at low pH may provide a more effective therapeutic approach for inducing endothelial progenitor cell mobilization and promoting cerebral neovas-cularization following ischemic stroke.

  4. Endothelial cell motility, coordination and pattern formation during vasculogenesis.

    Science.gov (United States)

    Czirok, Andras

    2013-01-01

    How vascular networks assemble is a fundamental problem of developmental biology that also has medical importance. To explain the organizational principles behind vascular patterning, we must understand how can tissue level structures be controlled through cell behavior patterns like motility and adhesion that, in turn, are determined by biochemical signal transduction processes? We discuss the various ideas that have been proposed as mechanisms for vascular network assembly: cell motility guided by extracellular matrix alignment (contact guidance), chemotaxis guided by paracrine and autocrine morphogens, and multicellular sprouting guided by cell-cell contacts. All of these processes yield emergent patterns, thus endothelial cells can form an interconnected structure autonomously, without guidance from an external pre-pattern.

  5. Role of NADPH Oxidase-4 in Human Endothelial Progenitor Cells

    Science.gov (United States)

    Hakami, Nora Y.; Ranjan, Amaresh K.; Hardikar, Anandwardhan A.; Dusting, Greg J.; Peshavariya, Hitesh M.

    2017-01-01

    Introduction: Endothelial progenitor cells (EPCs) display a unique ability to promote angiogenesis and restore endothelial function in injured blood vessels. NADPH oxidase 4 (NOX4)-derived hydrogen peroxide (H2O2) serves as a signaling molecule and promotes endothelial cell proliferation and migration as well as protecting against cell death. However, the role of NOX4 in EPC function is not completely understood. Methods: EPCs were isolated from human saphenous vein and mammary artery discarded during bypass surgery. NOX4 gene and protein expression in EPCs were measured by real time-PCR and Western blot analysis respectively. NOX4 gene expression was inhibited using an adenoviral vector expressing human NOX4 shRNA (Ad-NOX4i). H2O2 production was measured by Amplex red assay. EPC migration was evaluated using a transwell migration assay. EPC proliferation and viability were measured using trypan blue counts. Results: Inhibition of NOX4 using Ad-NOX4i reduced Nox4 gene and protein expression as well as H2O2 formation in EPCs. Inhibition of NOX4-derived H2O2 decreased both proliferation and migration of EPCs. Interestingly, pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) decreased NOX4 expression and reduced survival of EPCs. However, the survival of EPCs was further diminished by TNF-α in NOX4-knockdown cells, suggesting that NOX4 has a protective role in EPCs. Conclusion: These findings suggest that NOX4-type NADPH oxidase is important for proliferation and migration functions of EPCs and protects against pro-inflammatory cytokine induced EPC death. These properties of NOX4 may facilitate the efficient function of EPCs which is vital for successful neovascularization.

  6. Levamisole induced apoptosis in cultured vascular endothelial cells

    Science.gov (United States)

    Artwohl, Michaela; Hölzenbein, Thomas; Wagner, Ludwig; Freudenthaler, Angelika; Waldhäusl, Werner; Baumgartner-Parzer, Sabina M

    2000-01-01

    To better understand the anticancer activity of Levamisole (LMS), which serves as an adjuvant in colon cancer therapy in combination with 5-Fluorouracil, this study analyses LMS' ability to induce apoptosis and growth arrest in cultured human micro- and macrovascular endothelial cells (ECs) and fibroblasts. Cells exposed (24 h) to Levamisole (range: 0.5–2 mmol l−1) alone or in combination with antioxidants (10 mmol l−1 glutathione or 5 mmol l−1 N-Acetylcysteine or 0.1 mmol l−1 Tocopherol) were evaluated for apoptosis (3H-thymidine assays, in situ staining), mRNA/protein expression (Northern/Western blot), and proliferation (3H-thymidine incorporation). Levamisole dose-dependently increased apoptosis in ECs to 230% (HUVECs-human umbilical vein ECs), 525% (adult human venous ECs) and 600% (human uterine microvascular ECs) but not in fibroblasts compared to control cells (set as 100%). Levamisole increased in ECs integrin-dependent matrix adhesion, inhibited proliferation (−70%), reduced expression of survival factors such as clusterin (−30%), endothelin-1 (−43%), bcl-2 (−34%), endothelial NO-synthase (−32%) and pRb (Retinoblastoma protein: −89%), and increased that of growth arrest/death signals such as p21 (+73%) and bak (+50%). LMS (2 mmol l−1)-induced apoptosis was inhibited by glutathione (−50%) and N-Acetylcysteine (−36%), which also counteracted reduction by Levamisole of pRb expression, suggesting reactive oxygen species and pRb play a role in these processes. The ability of LMS to selectively induce apoptosis and growth arrest in endothelial cells potentially hints at vascular targeting to contribute to Levamisole's anticancer activity. PMID:11139434

  7. Endothelial RhoGEFs: A systematic analysis of their expression profiles in VEGF-stimulated and tumor endothelial cells.

    Science.gov (United States)

    Hernández-García, Ricardo; Iruela-Arispe, M Luisa; Reyes-Cruz, Guadalupe; Vázquez-Prado, José

    2015-11-01

    Rho guanine nucleotide exchange factors (RhoGEFs) integrate cell signaling inputs into morphological and functional responses. However, little is known about the endothelial repertoire of RhoGEFs and their regulation. Thus, we assessed the expression of 81 RhoGEFs (70 homologous to Dbl and 11 of the DOCK family) in endothelial cells. Further, in the case of DH-RhoGEFs, we also determined their responses to VEGF exposure in vitro and in the context of tumors. A phylogenetic analysis revealed the existence of four groups of DH-RhoGEFs and two of the DOCK family. Among them, we found that the most abundant endothelial RhoGEFs were: Tuba, FGD5, Farp1, ARHGEF17, TRIO, P-Rex1, ARHGEF15, ARHGEF11, ABR, Farp2, ARHGEF40, ALS, DOCK1, DOCK7 and DOCK6. Expression of RASGRF2 and PREX2 increased significantly in response to VEGF, but most other RhoGEFs were unaffected. Interestingly murine endothelial cells isolated from tumors showed that all four phylogenetic subgroups of DH-RhoGEFs were altered when compared to non-tumor endothelial cells. In summary, our results provide a detailed assessment of RhoGEFs expression profiles in the endothelium and set the basis to systematically address their regulation in vascular signaling.

  8. Evaluation of soluble junctional adhesion molecule-A as a biomarker of human brain endothelial barrier breakdown.

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    Axel Haarmann

    Full Text Available BACKGROUND: An inducible release of soluble junctional adhesion molecule-A (sJAM-A under pro-inflammatory conditions was described in cultured non-CNS endothelial cells (EC and increased sJAM-A serum levels were found to indicate inflammation in non-CNS vascular beds. Here we studied the regulation of JAM-A expression in cultured brain EC and evaluated sJAM-A as a serum biomarker of blood-brain barrier (BBB function. METHODOLOGY/PRINCIPAL FINDINGS: As previously reported in non-CNS EC types, pro-inflammatory stimulation of primary or immortalized (hCMEC/D3 human brain microvascular EC (HBMEC induced a redistribution of cell-bound JAM-A on the cell surface away from tight junctions, along with a dissociation from the cytoskeleton. This was paralleled by reduced immunocytochemical staining of occludin and zonula occludens-1 as well as by increased paracellular permeability for dextran 3000. Both a self-developed ELISA test and Western blot analysis detected a constitutive sJAM-A release by HBMEC into culture supernatants, which importantly was unaffected by pro-inflammatory or hypoxia/reoxygenation challenge. Accordingly, serum levels of sJAM-A were unaltered in 14 patients with clinically active multiple sclerosis compared to 45 stable patients and remained unchanged in 13 patients with acute ischemic non-small vessel stroke over time. CONCLUSION: Soluble JAM-A was not suited as a biomarker of BBB breakdown in our hands. The unexpected non-inducibility of sJAM-A release at the human BBB might contribute to a particular resistance of brain EC to inflammatory stimuli, protecting the CNS compartment.

  9. Functional and gene expression analysis of hTERT overexpressed endothelial cells

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    Haruna Takano

    2008-09-01

    Full Text Available Haruna Takano1, Satoshi Murasawa1,2, Takayuki Asahara1,2,31Institute of Biomedical Research and Innovation, Kobe, Japan; 2RIKEN Center for Developmental Biology, Kobe 650-0047, Japan; 3Tokai University of School of Medicine, Tokai, JapanAbstract: Telomerase dysfunction contributes to cellular senescence. Recent advances indicate the importance of senescence in maintaining vascular cell function in vitro. Human telomerase reverse transcriptase (hTERT overexpression is thought to lead to resistance to apoptosis and oxidative stress. However, the mechanism in endothelial lineage cells is unclear. We tried to generate an immortal endothelial cell line from human umbilical vein endothelial cells using a no-virus system and examine the functional mechanisms of hTERT overexpressed endothelial cell senescence in vitro. High levels of hTERT genes and endothelial cell-specific markers were expressed during long-term culture. Also, angiogenic responses were observed in hTERT overexpressed endothelial cell. These cells showed a delay in senescence and appeared more resistant to stressed conditions. PI3K/Akt-related gene levels were enhanced in hTERT overexpressed endothelial cells. An up-regulated PI3K/Akt pathway caused by hTERT overexpression might contribute to anti-apoptosis and survival effects in endothelial lineage cells.Keywords: endothelial, telomerase, senescence, oxidative stress, anti-apoptosis, PI3K/Akt pathway

  10. Vascular endothelial growth factors enhance the permeability of the mouse blood-brain barrier.

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    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.

  11. A novel minimally-invasive method to sample human endothelial cells for molecular profiling.

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    Stephen W Waldo

    Full Text Available The endothelium is a key mediator of vascular homeostasis and cardiovascular health. Molecular research on the human endothelium may provide insight into the mechanisms underlying cardiovascular disease. Prior methodology used to isolate human endothelial cells has suffered from poor yields and contamination with other cell types. We thus sought to develop a minimally invasive technique to obtain endothelial cells derived from human subjects with higher yields and purity.Nine healthy volunteers underwent endothelial cell harvesting from antecubital veins using guidewires. Fluorescence-activated cell sorting (FACS was subsequently used to purify endothelial cells from contaminating cells using endothelial surface markers (CD34/CD105/CD146 with the concomitant absence of leukocyte and platelet specific markers (CD11b/CD45. Endothelial lineage in the purified cell population was confirmed by expression of endothelial specific genes and microRNA using quantitative polymerase chain reaction (PCR.A median of 4,212 (IQR: 2161-6583 endothelial cells were isolated from each subject. Quantitative PCR demonstrated higher expression of von Willebrand Factor (vWF, P<0.001, nitric oxide synthase 3 (NOS3, P<0.001 and vascular cell adhesion molecule 1 (VCAM-1, P<0.003 in the endothelial population compared to similarly isolated leukocytes. Similarly, the level of endothelial specific microRNA-126 was higher in the purified endothelial cells (P<0.001.This state-of-the-art technique isolates human endothelial cells for molecular analysis in higher purity and greater numbers than previously possible. This approach will expedite research on the molecular mechanisms of human cardiovascular disease, elucidating its pathophysiology and potential therapeutic targets.

  12. A Novel Minimally-Invasive Method to Sample Human Endothelial Cells for Molecular Profiling

    Science.gov (United States)

    Waldo, Stephen W.; Brenner, Daniel A.; McCabe, James M.; Dela Cruz, Mark; Long, Brian; Narla, Venkata A.; Park, Joseph; Kulkarni, Ameya; Sinclair, Elizabeth; Chan, Stephen Y.; Schick, Suzaynn F.; Malik, Namita; Ganz, Peter; Hsue, Priscilla Y.

    2015-01-01

    Objective The endothelium is a key mediator of vascular homeostasis and cardiovascular health. Molecular research on the human endothelium may provide insight into the mechanisms underlying cardiovascular disease. Prior methodology used to isolate human endothelial cells has suffered from poor yields and contamination with other cell types. We thus sought to develop a minimally invasive technique to obtain endothelial cells derived from human subjects with higher yields and purity. Methods Nine healthy volunteers underwent endothelial cell harvesting from antecubital veins using guidewires. Fluorescence-activated cell sorting (FACS) was subsequently used to purify endothelial cells from contaminating cells using endothelial surface markers (CD34 / CD105 / CD146) with the concomitant absence of leukocyte and platelet specific markers (CD11b / CD45). Endothelial lineage in the purified cell population was confirmed by expression of endothelial specific genes and microRNA using quantitative polymerase chain reaction (PCR). Results A median of 4,212 (IQR: 2161 – 6583) endothelial cells were isolated from each subject. Quantitative PCR demonstrated higher expression of von Willebrand Factor (vWF, P<0.001), nitric oxide synthase 3 (NOS3, P<0.001) and vascular cell adhesion molecule 1 (VCAM-1, P<0.003) in the endothelial population compared to similarly isolated leukocytes. Similarly, the level of endothelial specific microRNA-126 was higher in the purified endothelial cells (P<0.001). Conclusion This state-of-the-art technique isolates human endothelial cells for molecular analysis in higher purity and greater numbers than previously possible. This approach will expedite research on the molecular mechanisms of human cardiovascular disease, elucidating its pathophysiology and potential therapeutic targets. PMID:25679506

  13. Transplanted microvascular endothelial cells promote oligodendrocyte precursor cell survival in ischemic demyelinating lesions.

    Science.gov (United States)

    Iijima, Keiya; Kurachi, Masashi; Shibasaki, Koji; Naruse, Masae; Puentes, Sandra; Imai, Hideaki; Yoshimoto, Yuhei; Mikuni, Masahiko; Ishizaki, Yasuki

    2015-11-01

    We previously showed that transplantation of brain microvascular endothelial cells (MVECs) greatly stimulated remyelination in the white matter infarct of the internal capsule (IC) induced by endothelin-1 injection and improved the behavioral outcome. In the present study, we examined the effect of MVEC transplantation on the infarct volume using intermittent magnetic resonance image and on the behavior of oligodendrocyte lineage cells histochemically. Our results in vivo show that MVEC transplantation reduced the infarct volume in IC and apoptotic death of oligodendrocyte precursor cells (OPCs). These results indicate that MVECs have a survival effect on OPCs, and this effect might contribute to the recovery of the white matter infarct. The conditioned-medium from cultured MVECs reduced apoptosis of cultured OPCs, while the conditioned medium from cultured fibroblasts did not show such effect. These results suggest a possibility that transplanted MVECs increased the number of OPCs through the release of humoral factors that prevent their apoptotic death. Identification of such humoral factors may lead to the new therapeutic strategy against ischemic demyelinating diseases.

  14. Ginsenoside Rg1 promotes endothelial progenitor cell migration and proliferation

    Institute of Scientific and Technical Information of China (English)

    Ai-wu SHI; Xiao-bin WANG; Feng-xiang LU; Min-min ZHU; Xiang-qing KONG; Ke-jiang CAO

    2009-01-01

    Aim: To investigate the effect of ginsenoside Rgl on the migration, adhesion, proliferation, and VEGF expression of endothe-lial progenitor cells (EPCs).Methods: EPCs were isolated from human peripheral blood and incubated with different concentrations of ginsenoside Rgl (0.1, 0.5, 1.0, and 5.0 μmol/L) and vehicle controls. EPC migration was detected with a modified Boyden chamber assay. EPC adhesion was determined by counting adherent cells on fibronectin-coated culture dishes. EPC proliferation was analyzed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In vitro vasculogenesis was assayed using an in vitro vasculogenesis detection kit. A VEGF-ELISA kit was used to measure the amount of VEGF protein in the cell culture medium.Results: Ginsenoside Rgl promoted EPC adhesionp proliferation, migration and in vitro vasculogenesis in a dose- and time-dependent manner. Cell cycle analysis showed that 5.0 μmol/L of ginsenoside Rgl significantly increased the EPC prolifera-tive phase (S phase) and decreased the resting phase (G0/G1 phase). Ginsenoside Rgl increased vascular endothelial growth factor production.Conclusion: The results indicate that ginsenoside Rgl promotes proliferation, migration, adhesion and in vitro vasculogen-esis.

  15. Study of the Mechanism of Essential Garlic Oil Inhibiting Interleukin-1α-Induced Monocyte Adhesion to Endothelial Cells

    Institute of Scientific and Technical Information of China (English)

    葛璐璐; 张薇; 戴云; 臧燕; 黄纯洁

    2001-01-01

    To observe the effects of essential garlic oil (EGO) on vascular cell adhesive molecule-1 (VCAM-1) expression of endothelial cells and monocyte-endothelial cell adhesion rate induced by interleukin-1α (IL-1α). Methods: Human umbilical vein endothelial cells (HUVEC) were isolated by trypsin digestion method and co-cultured with IL-1α or EGO+IL-1α in the absence or presence of U937 monocyte. Monocyte-endothelial cell adhesion rate was examined with reverted microscope. VCAM-1 expression of endothelial cells was measured by ACAS 570 confocal microscope, and the data were presented as mean fluorescent intensity. Results: EGO significantly inhibited IL-1α-induced endothelial expression of VCAM-1, and thus markedly decreased monocyte-endothelial cell adhesion rate. Conclusion: EGO has the effect on antagonizing adhesion of monocyte and vascular endothelial cell, which might be due to its inhibition on adhesive molecular expression on the surface of endothelial cells.

  16. Cardiotoxic drugs Herceptin and doxorubicin inhibit cardiac microvascular endothelial cell barrier formation resulting in increased drug permeability

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    Emma L. Wilkinson

    2016-10-01

    Full Text Available Cardiotoxicity induced by anti-cancer therapeutics is a severe, and potentially fatal, adverse reaction of the heart in response to certain drugs. Current in vitro approaches to assess cardiotoxicity have focused on analysing cardiomyocytes. More recently it has become apparent that non-cardiomyocyte cells of the heart can potentially contribute to cardiotoxicity. Herceptin and doxorubicin are known to induce cardiotoxicity in the clinic. The effect of these drugs on the endothelial tight junction barrier was tested by analysing tight junction formation and zona occludens-1 (ZO-1 levels, revealing that Herceptin and doxorubicin are able to induce barrier perturbment and decrease barrier function in human cardiac microvascular endothelial cells (HCMECs leading to increased permeability. Herceptin treatment had no effect on the tight junction barrier function in human dermal and human brain microvascular endothelial cells. HCMECs showed detectable levels of HER2 compared with the other endothelial cells suggesting that Herceptin binding to HER2 in these cells may interfere with tight junction formation. Our data suggests that doxorubicin and Herceptin can affect tight junction formation in the cardiac microvasculature leading to increased drug permeability and adverse effects on the cardiac myocytes.

  17. Synergism of matrix stiffness and vascular endothelial growth factor on mesenchymal stem cells for vascular endothelial regeneration.

    Science.gov (United States)

    Wingate, Kathryn; Floren, Michael; Tan, Yan; Tseng, Pi Ou Nancy; Tan, Wei

    2014-09-01

    Mesenchymal stem cells (MSCs) hold tremendous potential for vascular tissue regeneration. Research has demonstrated that individual factors in the cell microenvironment such as matrix elasticity and growth factors regulate MSC differentiation to vascular lineage. However, it is not well understood how matrix elasticity and growth factors combine to direct the MSC fate. This study examines the combined effects of matrix elasticity and vascular endothelial growth factor (VEGF) on both MSC differentiation into endothelial lineage and MSC paracrine signaling. MSCs were seeded in soft nanofibrous matrices with or without VEGF, and in Petri dishes with or without VEGF. Only MSCs seeded in three-dimensional soft matrices with VEGF showed significant increases in the expression of endothelial markers (vWF, eNOS, Flt-1, and Flk-1), while eliminating the expression of smooth muscle marker (SM-α-actin). MSCs cultured in VEGF alone on two-dimensional dishes showed increased expression of both early-stage endothelial and smooth muscle markers, indicating immature vascular differentiation. Furthermore, MSCs cultured in soft matrices with VEGF showed faster upregulation of endothelial markers compared with MSCs cultured in VEGF alone. Paracrine signaling studies found that endothelial cells cultured in the conditioned media from MSCs differentiated in the soft matrix and VEGF condition exhibited increased migration and formation of capillary-like structures. These results demonstrate that VEGF and soft matrix elasticity act synergistically to guide MSC differentiation into mature endothelial phenotype while enhancing paracrine signaling. Therefore, it is critical to control both mechanical and biochemical factors to safely regenerate vascular tissues with MSCs.

  18. In Vivo Vascularization of Endothelial Cells Derived from Bone Marrow Mesenchymal Stem Cells in SCID Mouse Model

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    Allameh Abdolamir

    2016-07-01

    Full Text Available Objective In vivo and in vitro stem cell differentiation into endothelial cells is a promising area of research for tissue engineering and cell therapy. Materials and Methods We induced human mesenchymal stem cells (MSCs to differentiate to endothelial cells that had the ability to form capillaries on an extracellular matrix (ECM gel. Thereafter, the differentiated endothelial cells at early stage were characterized by expression of specific markers such as von Willebrand factor (vWF, vascular endothelial growth factor (VEGF receptor 2, and CD31. In this experimental model, the endothelial cells were transplanted into the groins of severe combined immunodeficiency (SCID mice. After 30 days, we obtained tissue biopsies from the transplantation sites. Biopsies were processed for histopathological and double immunohistochemistry (DIHC staining. Results Endothelial cells at the early stage of differentiation expressed endothelial markers. Hematoxylin and eosin (H&E staining, in addition to DIHC demonstrated homing of the endothelial cells that underwent vascularization in the injected site. Conclusion The data clearly showed that endothelial cells at the early stage of differentiation underwent neovascularization in vivo in SCID mice. Endothelial cells at their early stage of differentiation have been proven to be efficient for treatment of diseases with impaired vasculogenesis.

  19. Arginine deiminase modulates endothelial tip cells via excessive synthesis of reactive oxygen species.

    Science.gov (United States)

    Zhuo, Wei; Song, Xiaomin; Zhou, Hao; Luo, Yongzhang

    2011-10-01

    ADI (arginine deiminase), an enzyme that hydrolyses arginine, has been reported as an anti-angiogenesis agent. However, its molecular mechanism is unclear. We have demonstrated for the first time that ADI modulates the angiogenic activity of endothelial tip cells. By arginine depletion, ADI disturbs actin filament in endothelial tip cells, causing disordered migratory direction and decreased migration ability. Furthermore, ADI induces excessive synthesis of ROS (reactive oxygen species), and activates caspase 8-, but not caspase 9-, dependent apoptosis in endothelial cells. These findings provide a novel mechanism by which ADI inhibits tumour angiogenesis through modulating endothelial tip cells.

  20. Effects of blood products on inflammatory response in endothelial cells in vitro.

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    Martin Urner

    Full Text Available BACKGROUND: Transfusing blood products may induce inflammatory reactions within the vascular compartment potentially leading to a systemic inflammatory response. Experiments were designed to assess the inflammatory potential of different blood products in an endothelial cell-based in vitro model and to compare baseline levels of potentially activating substances in transfusion products. METHODS: The inflammatory response from pre-activated (endotoxin-stimulated and non-activated endothelial cells as well as neutrophil endothelial transmigration in response to packed red blood cells (PRBC, platelet concentrates (PC and fresh frozen plasma (FFP was determined. Baseline inflammatory mediator and lipid concentrations in blood products were evaluated. RESULTS: Following incubation with all blood products, an increased inflammatory mediator release from endothelial cells was observed. Platelet concentrates, and to a lesser extent also FFP, caused the most pronounced response, which was accentuated in already pre-stimulated endothelial cells. Inflammatory response of endothelial cells as well as blood product-induced migration of neutrophils through the endothelium was in good agreement with the lipid content of the according blood product. CONCLUSION: Within the group of different blood transfusion products both PC and FFP have a high inflammatory potential with regard to activation of endothelial cells. Inflammation upon blood product exposure is strongly accentuated when endothelial cells are pre-injured. High lipid contents in the respective blood products goes along with an accentuated inflammatory reaction from endothelial cells.

  1. Glycoconjugates and Related Molecules in Human Vascular Endothelial Cells

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    Norihiko Sasaki

    2013-01-01

    Full Text Available Vascular endothelial cells (ECs form the inner lining of blood vessels. They are critically involved in many physiological functions, including control of vasomotor tone, blood cell trafficking, hemostatic balance, permeability, proliferation, survival, and immunity. It is considered that impairment of EC functions leads to the development of vascular diseases. The carbohydrate antigens carried by glycoconjugates (e.g., glycoproteins, glycosphingolipids, and proteoglycans mainly present on the cell surface serve not only as marker molecules but also as functional molecules. Recent studies have revealed that the carbohydrate composition of the EC surface is critical for these cells to perform their physiological functions. In this paper, we consider the expression and functional roles of endogenous glycoconjugates and related molecules (galectins and glycan-degrading enzymes in human ECs.

  2. Exogenous endothelial cells as accelerators of hematopoietic reconstitution

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    Mizer J

    2012-11-01

    Full Text Available Abstract Despite the successes of recombinant hematopoietic-stimulatory factors at accelerating bone marrow reconstitution and shortening the neutropenic period post-transplantation, significant challenges remain such as cost, inability to reconstitute thrombocytic lineages, and lack of efficacy in conditions such as aplastic anemia. A possible means of accelerating hematopoietic reconstitution would be administration of cells capable of secreting hematopoietic growth factors. Advantages of this approach would include: a ability to regulate secretion of cytokines based on biological need; b long term, localized production of growth factors, alleviating need for systemic administration of factors that possess unintended adverse effects; and c potential to actively repair the hematopoietic stem cell niche. Here we overview the field of hematopoietic growth factors, discuss previous experiences with mesenchymal stem cells (MSC in accelerating hematopoiesis, and conclude by putting forth the rationale of utilizing exogenous endothelial cells as a novel cellular therapy for acceleration of hematopoietic recovery.

  3. Glucagon-like peptide-1 activates endothelial nitric oxide synthase in human umbilical vein endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Li DING; Jin ZHANG

    2012-01-01

    To investigate the effects of glucagon-like peptide-1 (GLP-1) on endothelial NO synthase (eNOS) in human umbilical vein endothelial cells (HUVECs),and elucidate whether GLP-1 receptor (GLP-1R) and GLP-1(9-36) are involved in these effects.Methods:HUVECs were used.The activity of eNOS was measured with NOS assay kit.Phosphorylated and total eNOS proteins were detected using Western blot analysis.The level of eNOS mRNA was quantified with real-time RT-PCR.Results:Incubation of HUVECs with GLP-1 (50-5000 pmol/L) for 30 min significantly increased the activity of eNOS.Incubation of HUVECs with GLP-1 (500-5000 pmol/L) for 5 or 10 min increased eNOS phosphorylated at ser-1177.Incubation with GLP-1 (5000 pmol/L) for 48 h elevated the level of eNOS protein,did not affect the level of eNOS mRNA.GLP-1R agonists exenatide and GLP-1(9-36) at the concentration of 5000 pmol/L increased the activity,phosphorylation and protein level of eNOS.GLP-1R antagonist exendin(9-39) or DPP-4 inhibitor sitagliptin,which abolished GLP-1(9-36) formation,at the concentration of 5000 pmol/L partially blocked the effects of GLP-1 on eNOS.Conclusion:GLP-1 upregulated the activity and protein expression of eNOS in HUVECs through the GLP-1R-dependent and GLP-1(9-36)-related pathways.GLP-1 may prevent or delay the formation of atherosclerosis in diabetes mellitus by improving the function of eNOS.

  4. Cell biology of diabetic nephropathy: Roles of endothelial cells, tubulointerstitial cells and podocytes.

    Science.gov (United States)

    Maezawa, Yoshiro; Takemoto, Minoru; Yokote, Koutaro

    2015-01-01

    Diabetic nephropathy is the major cause of end-stage renal failure throughout the world in both developed and developing countries. Diabetes affects all cell types of the kidney, including endothelial cells, tubulointerstitial cells, podocytes and mesangial cells. During the past decade, the importance of podocyte injury in the formation and progression of diabetic nephropathy has been established and emphasized. However, recent findings provide additional perspectives on pathogenesis of diabetic nephropathy. Glomerular endothelial damage is already present in the normoalbuminuric stage of the disease when podocyte injury starts. Genetic targeting of mice that cause endothelial injury leads to accelerated diabetic nephropathy. Tubulointerstitial damage, previously considered to be a secondary effect of glomerular protein leakage, was shown to have a primary significance in the progression of diabetic nephropathy. Emerging evidence suggests that the glomerular filtration barrier and tubulointerstitial compartment is a composite, dynamic entity where any injury of one cell type spreads to other cell types, and leads to the dysfunction of the whole apparatus. Accumulation of novel knowledge would provide a better understanding of the pathogenesis of diabetic nephropathy, and might lead to a development of a new therapeutic strategy for the disease.

  5. Effect of Cytokines Secreted by Human Adipose Stromal Cells on Endothelial Cells

    Institute of Scientific and Technical Information of China (English)

    LI Bingong; ZENG Qiutang; WANG Hongxiang; MAO Xiaobo

    2006-01-01

    To isolate and culture adipose stromal cells (ASCs), and study the effect of cytokines secreted by ASCs on endothelial cells, human adipose tissue was digested with collagenase type Ⅰ solution and ASCs were derived by culture. The cells surface phenotype was examined by flow cytometry. ELISA was used to detect the secretion of VEGF, HGF, SDF-1 α and RT-PCR was employed to detect the expression of their mRNA. Then the ASC medium was utilized to culture human umbilical vein endothelial cells ECV304. Cells were counted by hemacytometer to determine the proliferation and Annexin V/PI was employed for the examination of the apoptosis rate of ECV304. ASCs were derived by culture and expressed CD34, CD105 while they did not express CD31 or CD45. ASCs secreted cytokines such as VEGF, HGF and SDF-1 α so the ASC medium could stimulate proliferation and counteract apoptosis of endothelial cells (P<0.05). Bcl-2 mRNA was also found to be up-regulated in the endothelial cells. It is concluded that ASCs can secrete cytokines and has significant effect on the proliferation of endothelial cells and apoptosis.

  6. Effect of Exogenous Ghrelin on Cell Differentiation Antigen 40 Expression in Endothelial Cells

    Institute of Scientific and Technical Information of China (English)

    Min ZHANG; Fang YUAN; Hui CHEN; Xingbiao QIU; Weiyi FANG

    2007-01-01

    Ghrelin is a brain-gut peptide that serves as a natural ligand for growth hormone secretagogue receptor (GHSR). It also exists abundantly in the cardiovascular system. In order to evaluate the possible role of ghrelin in the development of atherosclerosis, the effect of ghrelin on the expression of cell differentiation antigen 40 (CD40) were studied. Human umbilical vein endothelial cell (HUVEC) line-ECV 304 was pretreated with different concentrations of ghrelin, des-acyl ghrelin or [d-Lys]-GHRP-6 (a ghrelin receptor antagonist), and then induced with tumor necrosis factor-α (TNF-α) and interferon γ (IFN-γ). The mRNA levels of CD40 were analyzed by reverse transcription-polymerase chain reaction, and the expressions of CD40 protein in the cells were measured by flow cytometry (FCM) and Western blotting. The results showed that exogenous ghrelin could significantly inhibit TNF-α/IFN-γinduced CD40 expression in HUVEC cells in a concentration-dependent manner. When treated with 1000 ng/ml of ghrelin, the mRNA level of CD40 in the cells was decreased by approximately 77%, but when treated with both 1000 ng/ml of ghrelin and 1000 ng/ml of [d-Lys]-GHRP-6, the mRNA level of CD40 in the cells was decreased by only 42%,suggesting that [d-Lys]-GHRP-6 could counteract the inhibitory effect of ghrelin in these cells. However,CD40 expression was not inhibited by des-acyl ghrelin at 1000 ng/ml. The results in protein expression analysis detected by FCM and Western blotting further confirmed these results. Our results suggested that in the cardiovascular system, ghrelin not only has an anti-inflammatory effect, but also has a significant immunoregulatory effect that may be mediated through the GHSR-1 a receptor.

  7. Subcellular characterization of glucose uptake in coronary endothelial cells.

    Science.gov (United States)

    Gaudreault, N; Scriven, D R L; Laher, I; Moore, E D W

    2008-01-01

    Despite all the evidence linking glucose toxicity to an increased risk of cardiovascular diseases, very little is known about the regulation of glucose uptake in endothelial cells. We have previously reported an asymmetric distribution of the GLUTs (1-5) and SGLT-1 in en face preparations of rat coronary artery endothelia [Gaudreault N., Scriven D.R., Moore E.D., 2004. Characterisation of glucose transporters in the intact coronary artery endothelium in rats: GLUT-2 upregulated by long-term hyperglycaemia. Diabetologia 47(12),2081-2092]. We assessed this time, through immunocytochemistry and wide field fluorescence microscopy coupled to deconvolution, the presence and subcellular distribution of glucose transporters in cultures of human coronary artery endothelial cells (HCAECs). HCAECs express GLUT-1 to 5 and SGLT-1, but their subcellular distribution lacks the luminal/abluminal asymmetry and the proximity to cell-to-cell junctions observed in intact endothelium. To determine the impact of the transporters' distribution on intracellular glucose accumulation, a fluorescent glucose analog (2-NBDG) was used in conjunction with confocal microscopy to monitor uptake in individual cells; the arteries were mounted in an arteriograph chamber with physiological flow rates. The uptake in both preparations was inhibited by cytochalasin-B and d-glucose and stimulated by insulin, but the distribution of the incorporated 2-NBDG mirrored that of the transporters. In HCAEC it was distributed throughout the cell and in the intact arterial endothelium it was restricted to the narrow cytosolic volume adjacent to the cell-to-cell junctions. We suggest that the latter subcellular organization and compartmentalization may facilitate transendothelial transport of glucose in intact coronary artery.

  8. Functional activities of receptors for tumor necrosis factor-alpha on human vascular endothelial cells.

    NARCIS (Netherlands)

    Paleolog, E.M.; Delasalle, S.A.; Buurman, W.A.; Feldmann, M.

    1994-01-01

    Tumor necrosis factor-alpha (TNF-alpha) plays a critical role in the control of endothelial cell function and hence in regulating traffic of circulating cells into tissues in vivo. Stimulation of endothelial cells in vitro by TNF-alpha increases the surface expression of leukocyte adhesion molecules

  9. Directionally solidified biopolymer scaffolds: Mechanical properties and endothelial cell responses

    Science.gov (United States)

    Meghri, Nicholas W.; Donius, Amalie E.; Riblett, Benjamin W.; Martin, Elizabeth J.; Clyne, Alisa Morss; Wegst, Ulrike G. K.

    2010-07-01

    Vascularization is a primary challenge in tissue engineering. To achieve it in a tissue scaffold, an environment with the appropriate structural, mechanical, and biochemical cues must be provided enabling endothelial cells to direct blood vessel growth. While biochemical stimuli such as growth factors can be added through the scaffold material, the culture medium, or both, a well-designed tissue engineering scaffold is required to provide the necessary local structural and mechanical cues. As chitosan is a well-known carrier for biochemical stimuli, the focus of this study was on structure-property correlations, to evaluate the effects of composition and processing conditions on the three-dimensional architecture and properties of freeze-cast scaffolds; to establish whether freeze-east scaffolds are promising candidates as constructs promoting vascularization; and to conduct initial tissue culture studies with endothelial cells on flat substrates of identical compositions as those of the scaffolds to test whether these are biocompatible and promote cell attachment and proliferation.

  10. Endothelial Progenitor Cells for Diagnosis and Prognosis in Cardiovascular Disease

    Directory of Open Access Journals (Sweden)

    Caterina Oriana Aragona

    2016-01-01

    Full Text Available Objective. To identify, evaluate, and synthesize evidence on the predictive power of circulating endothelial progenitor cells (EPCs in cardiovascular disease, through a systematic review of quantitative studies. Data Sources. MEDLINE was searched using keywords related to “endothelial progenitor cells” and “endothelium” and, for the different categories, respectively, “smoking”; “blood pressure”; “diabetes mellitus” or “insulin resistance”; “dyslipidemia”; “aging” or “elderly”; “angina pectoris” or “myocardial infarction”; “stroke” or “cerebrovascular disease”; “homocysteine”; “C-reactive protein”; “vitamin D”. Study Selection. Database hits were evaluated against explicit inclusion criteria. From 927 database hits, 43 quantitative studies were included. Data Syntheses. EPC count has been suggested for cardiovascular risk estimation in the clinical practice, since it is currently accepted that EPCs can work as proangiogenic support cells, maintaining their importance as regenerative/reparative potential, and also as prognostic markers. Conclusions. EPCs showed an important role in identifying cardiovascular risk conditions, and to suggest their evaluation as predictor of outcomes appears to be reasonable in different defined clinical settings. Due to their capability of proliferation, circulation, and the development of functional progeny, great interest has been directed to therapeutic use of progenitor cells in atherosclerotic diseases. This trial is registered with registration number: Prospero CRD42015023717.

  11. Cationic Nanocylinders Promote Angiogenic Activities of Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Jung Bok Lee

    2016-01-01

    Full Text Available Polymers have been used extensively taking forms as scaffolds, patterned surface and nanoparticle for regenerative medicine applications. Angiogenesis is an essential process for successful tissue regeneration, and endothelial cell–cell interaction plays a pivotal role in regulating their tight junction formation, a hallmark of angiogenesis. Though continuous progress has been made, strategies to promote angiogenesis still rely on small molecule delivery or nuanced scaffold fabrication. As such, the recent paradigm shift from top-down to bottom-up approaches in tissue engineering necessitates development of polymer-based modular engineering tools to control angiogenesis. Here, we developed cationic nanocylinders (NCs as inducers of cell–cell interaction and investigated their effect on angiogenic activities of human umbilical vein endothelial cells (HUVECs in vitro. Electrospun poly (l-lactic acid (PLLA fibers were aminolyzed to generate positively charged NCs. The aninolyzation time was changed to produce two different aspect ratios of NCs. When HUVECs were treated with NCs, the electrostatic interaction of cationic NCs with negatively charged plasma membranes promoted migration, permeability and tubulogenesis of HUVECs compared to no treatment. This effect was more profound when the higher aspect ratio NC was used. The results indicate these NCs can be used as a new tool for the bottom-up approach to promote angiogenesis.

  12. The targeting expression of the vascular endothelial growth factor gene in endothelial cells regulated by HRE.ppET-1

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The success of gene therapy depends largely on the efficacy of gene delivery vector systems that can deliver genes to target organs or cells selectively and efficiently with minimal toxicity. Here, we show that by using the HRE.ppET-1 regulatory element, we were able to restrict expression of the transgene of vascular endothelial growth factor (VEGF) to endothelial cells exclusively in hypoxic conditions. Eukaryotic expression vectors such as pEGFP-HRE.ppET-1, pcDNA3.1-VEGF+Pa, pcDNA3.1-ppET-1+ EGF+Pa, and pcDNA3.1-HRE.ppET-1+VEGF+Pa were constructed by using a series of nuclear molecule handling methods like PCR, enzyme digestion. The recombinant vectors were transfected into HUVEC cells and HL7702 cells by the lipofectin method. GFP expression was observed with a fluorescence microscope to validate the specificity of expression in endothelial cells under the regulation of HRE.ppET-1 element. Cobalt chloride (final concentration 100 μmol/L) was added to the medium to mimic hypoxia in vitro. After transfection of vectors, the expression of VEGF mRNA was detected by RT-PCR, and the expression of VEGF was detected by Western blotting and ELISA methods under normoxia and hypoxia, respectively. The cell proliferation rate was detected by the MTT test. The ex- pression of GFP revealed that the exterior gene was transcripted effectively in endothelial cells regu- lated by the HRE.ppET-1 element, while the expression of GFP was very weak in nonendothelial cells. The results of RT-PCR, Western blotting and ELISA showed that VEGF gene expression in the pcDNA3.1-HRE.ppET-1+VEGF+Pa group and in the pcDNA3.1-ppET-1+VEGF+Pa group was higher in hypoxia than it was in normoxia (P<0.05). The MTT test showed that the proliferation rate of HUVEC transfected with HPVA under hypoxia exceeded that of the control group. We conclude that the HRE.ppET-1 element was expressed specifically in endothelial cells, and can increase the expression of VEGF in hypoxia and stimulate proliferation

  13. Extracellular DNA affects NO content in human endothelial cells.

    Science.gov (United States)

    Efremova, L V; Alekseeva, A Yu; Konkova, M S; Kostyuk, S V; Ershova, E S; Smirnova, T D; Konorova, I L; Veiko, N N

    2010-08-01

    Fragments of extracellular DNA are permanently released into the blood flow due to cell apoptosis and possible de novo DNA synthesis. To find out whether extracellular DNA can affect the synthesis of nitric oxide (NO), one of key vascular tone regulators, we studied in vitro effects of three artificial DNA probes with different sequences and 10 samples of extracellular DNA (obtained from healthy people and patients with hypertension and atherosclerosis) on NO synthesis in endothelial cell culture (HUVEC). For detection of NO in live cells and culture medium, we used a NO-specific agent CuFL penetrating into the cells and forming a fluorescent product FL-NO upon interaction with NO. Human genome DNA fragments affected the content of NO in endothelial cells; this effect depended on both the base sequence and concentration of DNA fragments. Addition of artificial DNA and extracellular DNA from healthy people into the cell culture in a low concentration (5 ng/ml) increased the detected NO concentration by 4-fold at most. Cytosine-guanine (CG)-rich fragment of the transcribed sequence of ribosomal repeat was the most powerful NO-inductor. The effect of DNA fragments on NO synthesis was comparable with that of low doses of oxidizing agents, H(2)O(2) and 17β-estradiol. Extracellular DNA samples obtained from patients with hypertension and atherosclerosis decreased NO content in cells and medium by 1.3-28 times compared to the control; the effect correlated with the content of CG-rich sequences.

  14. Curcumin Attenuates Rapamycin-induced Cell Injury of Vascular Endothelial Cells.

    Science.gov (United States)

    Guo, Ning; Chen, Fangyuan; Zhou, Juan; Fang, Yuan; Li, Hongbing; Luo, Yongbai; Zhang, Yong

    2015-10-01

    Although drug-eluting stents (DES) effectively improve the clinical efficacy of percutaneous coronary intervention, a high risk of late stent thrombosis and in-stent restenosis also exists after DES implantation. Anti-smooth muscle proliferation drugs, such as rapamycin, coating stents, not only inhibit the growth of vascular smooth muscle cells but also inhibit vascular endothelial cells and delay the reendothelialization. Therefore, the development of an ideal agent that protects vascular endothelial cells from rapamycin-eluting stents is of great importance for the next generation of DES. In this study, we demonstrated that rapamycin significantly inhibited the growth of rat aortic endothelial cells in both dose- and time-dependent manner in vitro. Cell apoptosis was increased and migration was decreased by rapamycin treatments in rat aortic endothelial cells in vitro. Surprisingly, treatment with curcumin, an active ingredient of turmeric, significantly reversed these detrimental effects of rapamycin. Moreover, curcumin increased the expression of vascular nitric oxide synthases (eNOS), which was decreased by rapamycin. Furthermore, caveolin-1, the inhibitor of eNOS, was decreased by curcumin. Knockdown of eNOS by small interfering RNA significantly abrogated the protective effects of curcumin. Taken together, our results suggest that curcumin antagonizes the detrimental effect of rapamycin on aortic endothelial cells in vitro through upregulating eNOS. Therefore, curcumin is a promising combined agent for the rescue of DES-induced reendothelialization delay.

  15. Thalidomide effect in endothelial cell of acute radiation proctitis

    Institute of Scientific and Technical Information of China (English)

    Ki-Tae Kim; Hiun-Suk Chae; Jin-Soo Kim; Hyung-Keun Kim; Young-Seok Cho; Whang Choi; Kyu-Yong Choi; Sang-Young Rho; Suk-Jin Kang

    2008-01-01

    AIM: To determine whether thalidomide prevents microvascular injury in acute radiation proctitis in white rats. METHODS: Fourteen female Wistar rats were used:six in the radiation group,six in the thalidomide group,and two in normal controls.The radiation and thalidomide groups were irradiated at the pelvic area using a single 30 Gy exposure.The thalidomide (150 mg/kg) was injected into the peritoneum for 7 d from the day of irradiation.All animals were sacrificed and the rectums were removed on day 8 after irradiation.The microvessels of resected specimens were immunohistochemically stained with thrombomodulin (TM),yon Willebrand Factor (vWF),and vascular endothelial growth factor (VEGF).RESULTS: The microscopic scores did not differ significantly between the radiation and thalidomide groups,but both were higher than in the control group.Expression of TM was significantly lower in the endothelial cells (EC) of the radiation group than in the control and thalidomide groups (P < 0.001).The number of capillaries expressing vWF in the EC was higher in the radiation group (15.3 ± 6.8) than in the control group (3.7 ± 1.7),and the number of capillaries expressing vWF was attenuated by thalidomide (10.8 ± 3.5,P < 0.001).The intensity of VEGF expression in capillaries was greater in the radiation group than in the control group and was also attenuated by thalidomide (P = 0.003).CONCLUSION: The mechanisms of acute radiationinduced proctitis in the rats are related to endothelial cell injury of microvessel,which may be attenuated with thalidomide.

  16. Optical studies of oxidative stress in pulmonary artery endothelial cells

    Science.gov (United States)

    Ghanian, Zahra; Sepehr, Reyhaneh; Eis, Annie; Kondouri, Ganesh; Ranji, Mahsa

    2015-03-01

    Reactive oxygen species (ROS) play an essential role in facilitating signal transduction processes within the cell and modulating the injuries. However, the generation of ROS is tightly controlled both spatially and temporally within the cell, making the study of ROS dynamics particularly difficult. This study present a novel protocol to quantify the dynamic of the mitochondrial superoxide as a precursor of reactive oxygen species. To regulate the mitochondrial superoxide level, metabolic perturbation was induced by administration of potassium cyanide (KCN). The presented method was able to monitor and measure the superoxide production rate over time. Our results demonstrated that the metabolic inhibitor, potassium cyanide (KCN) induced a significant increase in the rate of superoxide production in mitochondria of fetal pulmonary artery endothelial cells (FPAEC). Presented method sets the stage to study different ROS mediated injuries in vitro.

  17. Endothelial cell chimerism by fluorescence in situ hybridization in gender mismatched renal allograft biopsies

    Institute of Scientific and Technical Information of China (English)

    BAI Hong-wei; SHI Bing-yi; QIAN Ye-yong; NA Yan-qun; ZENG Xuan; ZHONG Ding-rong; LU Min; ZOU Wan-zhong; WU Shi-fei

    2007-01-01

    Background The blood vessels of a transplanted organ are the interface between donor and recipient. The endothelium in the blood vessels is thought to be the major target for graft rejection. Endothelial cells of a transplanted organ can be of recipient origin after transplantation. In this study, we tested whether endothelial chimerism correlated with the graft rejection and cold ischemia.Methods We studied the biopsy samples from 34 renal transplants of female recipients who received the kidney from a male donor for the presence of endothelial cells of recipient origin. We examined the tissue sections of renal biopsy samples by fluorescence in situ hybridization (FISH) for the presence of endothelial cells containing two X chromosomes using a biotinylated Y chromosome probe and digoxigenin labelled X chromosome probe, and then analyzed the relationship between the endothelial cell chimerism and the rejection and cold ischemia.Results Endothelial chimerism was common and irrespective of rejections (P>0.05). The cold ischemic time of chimerism group was longer than no chimerism group ((14.83±4.03) hours vs (11.27±3.87) hours, P<0.05).Conclusions There is no correlation between the percentage of recipient endothelial cells in vascular endothelial cells and the type of graft rejection. The endothelium damaged by ischemic injury might be repaired by the endothelial cells from the recipient.

  18. Methylmercury Causes Blood-Brain Barrier Damage in Rats via Upregulation of Vascular Endothelial Growth Factor Expression

    Science.gov (United States)

    Takahashi, Tetsuya; Fujimura, Masatake; Koyama, Misaki; Kanazawa, Masato; Usuki, Fusako; Nishizawa, Masatoyo; Shimohata, Takayoshi

    2017-01-01

    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. PMID:28118383

  19. Tumor endothelial marker 5 expression in endothelial cells during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Vallon, Mario, E-mail: m.vallon@arcor.de [Nuklearmedizinische Klinik und Poliklinik, Technische Universitaet Muenchen, Ismaninger Strasse 22, 81675 Munich (Germany); Rohde, Franziska; Janssen, Klaus-Peter [Chirurgische Klinik und Poliklinik, Technische Universitaet Muenchen, Munich (Germany); Essler, Markus [Nuklearmedizinische Klinik und Poliklinik, Technische Universitaet Muenchen, Ismaninger Strasse 22, 81675 Munich (Germany)

    2010-02-01

    Tumor endothelial marker (TEM) 5 is an adhesion G-protein-coupled receptor upregulated in endothelial cells during tumor and physiologic angiogenesis. So far, the mechanisms leading to upregulation of TEM5 and its function during angiogenesis have not been identified. Here, we report that TEM5 expression in endothelial cells is induced during capillary-like network formation on Matrigel, during capillary morphogenesis in a three-dimensional collagen I matrix, and upon confluence on a two-dimensional matrix. TEM5 expression was not induced by a variety of soluble angiogenic factors, including VEGF and bFGF, in subconfluent endothelial cells. TEM5 upregulation was blocked by toxin B from Clostridium difficile, an inhibitor of the small GTPases Rho, Rac, and Cdc42. The Rho inhibitor C3 transferase from Clostridium botulinum did not affect TEM5 expression, whereas the Rac inhibitor NSC23766 suppressed TEM5 upregulation. An excess of the soluble TEM5 extracellular domain or an inhibitory monoclonal TEM5 antibody blocked contact inhibition of endothelial cell proliferation resulting in multilayered islands within the endothelial monolayer and increased vessel density during capillary formation. Based on our results we conclude that TEM5 expression during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of proliferation in endothelial cells.

  20. Response of the sensorimotor cortex of cerebral palsy rats receiving transplantation of vascular endothelial growth factor 165-transfected neural stem cells

    Institute of Scientific and Technical Information of China (English)

    Jielu Tan; Xiangrong Zheng; Shanshan Zhang; Yujia Yang; Xia Wang; Xiaohe Yu; Le Zhong

    2014-01-01

    Neural stem cells are characterized by the ability to differentiate and stably express exogenous ge-nes. Vascular endothelial growth factor plays a role in protecting local blood vessels and neurons of newborn rats with hypoxic-ischemic encephalopathy. Transplantation of vascular endothelial growth factor-transfected neural stem cells may be neuroprotective in rats with cerebral palsy. In this study, 7-day-old Sprague-Dawley rats were divided into ifve groups: (1) sham operation (control), (2) cerebral palsy model alone or with (3) phosphate-buffered saline, (4) vascular en-dothelial growth factor 165 + neural stem cells, or (5) neural stem cells alone. hTe cerebral palsy model was established by ligating the letf common carotid artery followed by exposure to hypox-ia. Phosphate-buffered saline, vascular endothelial growth factor + neural stem cells, and neural stem cells alone were administered into the sensorimotor cortex using the stereotaxic instrument and microsyringe. Atfer transplantation, the radial-arm water maze test and holding test were performed. Immunohistochemistry for vascular endothelial growth factor and histology using hematoxylin-eosin were performed on cerebral cortex. Results revealed that the number of vas-cular endothelial growth factor-positive cells in cerebral palsy rats transplanted with vascular endothelial growth factor-transfected neural stem cells was increased, the time for ifnding water and the ifnding repetitions were reduced, the holding time was prolonged, and the degree of cell degeneration or necrosis was reduced. hTese ifndings indicate that the transplantation of vascu-lar endothelial growth factor-transfected neural stem cells alleviates brain damage and cognitive deifcits, and is neuroprotective in neonatal rats with hypoxia ischemic-mediated cerebral palsy.

  1. Endothelial-mural cell signaling in vascular development and angiogenesis.

    Science.gov (United States)

    Gaengel, Konstantin; Genové, Guillem; Armulik, Annika; Betsholtz, Christer

    2009-05-01

    Mural cells are essential components of blood vessels and are necessary for normal development, homeostasis, and organ function. Alterations in mural cell density or the stable attachment of mural cells to the endothelium is associated with several human diseases such as diabetic retinopathy, venous malformation, and hereditary stroke. In addition mural cells are implicated in regulating tumor growth and have thus been suggested as potential antiangiogenic targets in tumor therapy. In recent years our knowledge of mural cell function and endothelial-mural cell signaling has increased dramatically, and we now begin to understand the mechanistic basis of the key signaling pathways involved. This is mainly thanks to sophisticated in vivo experiments using a broad repertoire of genetic technologies. In this review, we summarize the five currently best understood signaling pathways implicated in mural cell biology. We discuss PDGFB/PDGFRbeta- dependent pericyte recruitment, as well as the role of angiopoietins and Tie receptors in vascular maturation. In addition, we highlight the effects of sphingosine-1-phosphate signaling on adherens junction assembly and vascular stability, as well as the role of TGF-beta-signaling in mural cell differentiation. We further reflect recent data suggesting an important function for Notch3 signaling in mural cell maturation.

  2. Modulation of the sis Gene Transcript during Endothelial Cell Differentiation in vitro

    Science.gov (United States)

    Jaye, Michael; McConathy, Evelyn; Drohan, William; Tong, Benton; Deuel, Thomas; Maciag, Thomas

    1985-05-01

    Endothelial cells, which line the interior walls of blood vessels, proliferate at the site of blood vessel injury. Knowledge of the factors that control the proliferation of these cells would help elucidate the role of endothelial cells in wound healing, tumor growth, and arteriosclerosis. In vitro, endothelial cells organize into viable, three-dimensional tubular structures in environments that limit cell proliferation. The process of endothelial cell organization was found to result in decreased levels of the sis messenger RNA transcript and increased levels of the messenger RNA transcript for fibronectin. This situation was reversed on transition from the organized structure to a proliferative monolayer. These results suggest a reciprocity for two biological response modifiers involved in the regulation of endothelial cell proliferation and differentiation in vitro.

  3. Hydrogen-Rich Medium Attenuated Lipopolysaccharide-Induced Monocyte-Endothelial Cell Adhesion and Vascular Endothelial Permeability via Rho-Associated Coiled-Coil Protein Kinase.

    Science.gov (United States)

    Xie, Keliang; Wang, Weina; Chen, Hongguang; Han, Huanzhi; Liu, Daquan; Wang, Guolin; Yu, Yonghao

    2015-07-01

    Sepsis is the leading cause of death in critically ill patients. In recent years, molecular hydrogen, as an effective free radical scavenger, has been shown a selective antioxidant and anti-inflammatory effect, and it is beneficial in the treatment of sepsis. Rho-associated coiled-coil protein kinase (ROCK) participates in junction between normal cells, and regulates vascular endothelial permeability. In this study, we used lipopolysaccharide to stimulate vascular endothelial cells and explored the effects of hydrogen-rich medium on the regulation of adhesion of monocytes to endothelial cells and vascular endothelial permeability. We found that hydrogen-rich medium could inhibit adhesion of monocytes to endothelial cells and decrease levels of adhesion molecules, whereas the levels of transepithelial/endothelial electrical resistance values and the expression of vascular endothelial cadherin were increased after hydrogen-rich medium treatment. Moreover, hydrogen-rich medium could lessen the expression of ROCK, as a similar effect of its inhibitor Y-27632. In addition, hydrogen-rich medium could also inhibit adhesion of polymorphonuclear neutrophils to endothelial cells. In conclusion, hydrogen-rich medium could regulate adhesion of monocytes/polymorphonuclear neutrophils to endothelial cells and vascular endothelial permeability, and this effect might be related to the decreased expression of ROCK protein.

  4. The microRNA-dependent cell fate of multipotent stromal cells differentiating to endothelial cells.

    Science.gov (United States)

    Cha, Min-Ji; Choi, Eunhyun; Lee, Seahyoung; Song, Byeong-Wook; Yoon, Cheesoon; Hwang, Ki-Chul

    2016-02-15

    In the endothelial recovery process, bone marrow-derived MSCs are a potential source of cells for both research and therapy, and their capacities to self-renew and to differentiate into all the cell types in the human body make them a promising therapeutic agent for remodeling cellular differentiation and a valuable resource for the treatment of many diseases. Based on the results provided in a miRNA database, we selected miRNAs with unique targets in cell fate-related signaling pathways. The tested miRNAs targeting GSK-3β (miR-26a), platelet-derived growth factor receptor, and CD133 (miR-26a and miR-29b) induced MSC differentiation into functional ECs, whereas miRNAs targeting VEGF receptor (miR-15, miR-144, miR-145, and miR-329) inhibited MSC differentiation into ECs through VEGF stimulation. In addition, the expression levels of these miRNAs were correlated with in vivo physiological endothelial recovery processes. These findings indicate that the miRNA expression profile is distinct for cells in different stages of differentiation from MSCs to ECs and that specific miRNAs can function as regulators of endothelialization.

  5. BIGH3 protein and macrophages in retinal endothelial cell apoptosis.

    Science.gov (United States)

    Mondragon, Albert A; Betts-Obregon, Brandi S; Moritz, Robert J; Parvathaneni, Kalpana; Navarro, Mary M; Kim, Hong Seok; Lee, Chi Fung; LeBaron, Richard G; Asmis, Reto; Tsin, Andrew T

    2015-01-01

    Diabetes is a pandemic disease with a higher occurrence in minority populations. The molecular mechanism to initiate diabetes-associated retinal angiogenesis remains largely unknown. We propose an inflammatory pathway of diabetic retinopathy in which macrophages in the diabetic eye provide TGFβ to retinal endothelial cells (REC) in the retinal microvasculature. In response to TGFβ, REC synthesize and secrete a pro-apoptotic BIGH3 (TGFβ-Induced Gene Human Clone 3) protein, which acts in an autocrine loop to induce REC apoptosis. Rhesus monkey retinal endothelial cells (RhREC) were treated with dMCM (cell media of macrophages treated with high glucose and LDL) and assayed for apoptosis (TUNEL), BIGH3 mRNA (qPCR), and protein (Western blots) expressions. Cells were also treated with ΤGFβ1 and 2 for BIGH3 mRNA and protein expression. Inhibition assays were carried out using antibodies for TGFβ1 and for BIGH3 to block apoptosis and mRNA expression. BIGH3 in cultured RhREC cells were identified by immunohistochemistry (IHC). Distribution of BIGH3 and macrophages in the diabetic mouse retina was examined with IHC. RhRECs treated with dMCM or TGFβ showed a significant increase in apoptosis and BIGH3 protein expression. Recombinant BIGH3 added to RhREC culture medium led to a dose-dependent increase in apoptosis. Antibodies (Ab) directed against BIGH3 and TGFβ, as well as TGFβ receptor blocker resulted in a significant reduction in apoptosis induced by either dMCM, TGFβ or BIGH3. IHC showed that cultured RhREC constitutively expressed BIGH3. Macrophage and BIGH3 protein were co-localized to the inner retina of the diabetic mouse eye. Our results support a novel inflammatory pathway for diabetic retinopathy. This pathway is initiated by TGFβ released from macrophages, which promotes synthesis and release of BIGH3 protein by REC and REC apoptosis.

  6. Endothelial Progenitor Cells in Sprouting Angiogenesis: Proteases Pave the Way.

    Science.gov (United States)

    Laurenzana, A; Fibbi, G; Margheri, F; Biagioni, A; Luciani, C; Del Rosso, M; Chillà, A

    2015-01-01

    Sprouting angiogenesis consists of the expansion and remodelling of existing vessels, where the vascular sprouts connect each other to form new vascular loops. Endothelial Progenitor Cells (EPCs) are a subtype of stem cells, with high proliferative potential, able to differentiate into mature Endothelial Cells (ECs) during the neovascularization process. In addition to this direct structural role EPCs improve neovascularization, also secreting numerous pro-angiogenic factors able to enhance the proliferation, survival and function of mature ECs, and other surrounding progenitor cells. While sprouting angiogenesis by mature ECs involves resident ECs, the vasculogenic contribution of EPCs is a high hurdle race. Bone marrowmobilized EPCs have to detach from the stem cell niche, intravasate into bone marrow vessels, reach the hypoxic area or tumour site, extravasate and incorporate into the new vessel lumen, thus complementing the resident mature ECs in sprouting angiogenesis. The goal of this review is to highlight the role of the main protease systems able to control each of these steps. The pivotal protease systems here described, involved in vascular patterning in sprouting angiogenesis, are the matrix-metalloproteinases (MMPs), the serineproteinases urokinase-type plasminogen activator (uPA) associated with its receptor (uPAR) and receptorassociated plasminogen/plasmin, the neutrophil elastase and the cathepsins. Since angiogenesis plays a critical role not only in physiological but also in pathological processes, such as in tumours, controlling the contribution of EPCs to the angiogenic process, through the regulation of the protease systems involved, could yield new opportunities for the therapeutic prospect of efficient control of pathological angiogenesis.

  7. Vascular endothelial growth factor enhances macrophage clearance of apoptotic cells

    Science.gov (United States)

    Dalal, Samay; Horstmann, Sarah A.; Richens, Tiffany R.; Tanaka, Takeshi; Doe, Jenna M.; Boe, Darren M.; Voelkel, Norbert F.; Taraseviciene-Stewart, Laimute; Janssen, William J.; Lee, Chun G.; Elias, Jack A.; Bratton, Donna; Tuder, Rubin M.; Henson, Peter M.; Vandivier, R. William

    2012-01-01

    Efficient clearance of apoptotic cells from the lung by alveolar macrophages is important for the maintenance of tissue structure and function. Lung tissue from humans with emphysema contains increased numbers of apoptotic cells and decreased levels of vascular endothelial growth factor (VEGF). Mice treated with VEGF receptor inhibitors have increased numbers of apoptotic cells and develop emphysema. We hypothesized that VEGF regulates apoptotic cell clearance by alveolar macrophages (AM) via its interaction with VEGF receptor 1 (VEGF R1). Our data show that the uptake of apoptotic cells by murine AMs and human monocyte-derived macrophages is inhibited by depletion of VEGF and that VEGF activates Rac1. Antibody blockade or pharmacological inhibition of VEGF R1 activity also decreased apoptotic cell uptake ex vivo. Conversely, overexpression of VEGF significantly enhanced apoptotic cell uptake by AMs in vivo. These results indicate that VEGF serves a positive regulatory role via its interaction with VEGF R1 to activate Rac1 and enhance AM apoptotic cell clearance. PMID:22307908

  8. Influence of pro-angiogenic cytokines on proliferative activity and survival of endothelial cells

    Directory of Open Access Journals (Sweden)

    Solyanik G. I.

    2010-04-01

    Full Text Available Aim. Tumor angiogenesis in contrast to physiological one is characterized by high level of malignant cell production of proangiogenic cytokines, which have different influence on functional activity of endothelial cells. The goal of the study – to carry out a comparative analysis of the influence of a vascular endothelial growth factor (VEGF and an epidermal growth factor (EGF on proliferative activity and survival of endothelial cells upon their confluent and exponential growth. Methods. The proliferative activity of endothelial cells was determined by MTT-test and their viability was detected by the trypane blue exclusion test. Results. It was shown that EGF (irrespectively of the level of serum factors in concentrations higher than 10 ng/ml activated the proliferative activity of confluent endotheliocytes in a concentration-dependent manner by 18–36 % (ð < 0.05 as compared to the control, while this cytokine didn’t affect the endothelial cells in the exponential growth phase. VEGF in wide concentration range didn’t display the mitogenic effect on endotheliocytes in both confluent and exponential growth phases. Furthermore, VEGF in concentrations higher than 100 ng/ml inhibited proliferative activity of confluent endothelial cells by 12 % (ð < 0.05. In case of deficiency of nutrients, EGF and VEGF promoted the survival of endothelial cells, considerably decreasing their death. Conclusions. EGF, in contrast to VEGF, stimulates proliferation and survival of the endothelial cells, whereas VEGF has significant influence only on the survival of the cells

  9. Divergent responses of different endothelial cell types to infection with Candida albicans and Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    Kati Seidl

    Full Text Available Endothelial cells are important in the pathogenesis of bloodstream infections caused by Candida albicans and Staphylococcus aureus. Numerous investigations have used human umbilical vein endothelial cells (HUVECs to study microbial-endothelial cell interactions in vitro. However, the use of HUVECs requires a constant supply of umbilical cords, and there are significant donor-to-donor variations in these endothelial cells. The use of an immortalized endothelial cell line would obviate such difficulties. One candidate in this regard is HMEC-1, an immortalized human dermal microvascular endothelial cell line. To determine if HMEC-1 cells are suitable for studying the interactions of C. albicans and S. aureus with endothelial cells in vitro, we compared the interactions of these organisms with HMEC-1 cells and HUVECs. We found that wild-type C. albicans had significantly reduced adherence to and invasion of HMEC-1 cells as compared to HUVECs. Although wild-type S. aureus adhered to and invaded HMEC-1 cells similarly to HUVECs, an agr mutant strain had significantly reduced invasion of HMEC-1 cells, but not HUVECs. Furthermore, HMEC-1 cells were less susceptible to damage induced by C. albicans, but more susceptible to damage caused by S. aureus. In addition, HMEC-1 cells secreted very little IL-8 in response to infection with either organism, whereas infection of HUVECs induced substantial IL-8 secretion. This weak IL-8 response was likely due to the anatomic site from which HMEC-1 cells were obtained because infection of primary human dermal microvascular endothelial cells with C. albicans and S. aureus also induced little increase in IL-8 production above basal levels. Thus, C. albicans and S. aureus interact with HMEC-1 cells in a substantially different manner than with HUVECs, and data obtained with one type of endothelial cell cannot necessarily be extrapolated to other types.

  10. Staphylococcal SSL5 Binding to Human Leukemia Cells Inhibits Cell Adhesion to Endothelial Cells and Platelets

    Directory of Open Access Journals (Sweden)

    Annemiek M. E. Walenkamp

    2010-01-01

    Full Text Available Bacterial proteins provide promising tools for novel anticancer therapies. Staphylococcal superantigen-like 5 (SSL5 was recently described to bind P-selectin glycoprotein ligand-1 (PSGL-1 on leukocytes and to inhibit neutrophil rolling on a P-selectin surface. As leukocytes and tumor cells share many characteristics in migration and dissemination, we explored the potential of SSL5 as an antagonist of malignant cell behavior. Previously, it was demonstrated that rolling of human HL-60 leukemia cells on activated endothelial cells was mediated by P-selectin. In this study, we show that SSL5 targets HL-60 cells. Binding of SSL5 was rapid and without observed toxicity. Competition of SSL5 with the binding of three anti-PSGL-1 antibodies and P-selectin to HL-60 cells identified PSGL-1 as the ligand on HL-60 cells. Presence of sialyl Lewis x epitopes on PSGL-1 was crucial for its interaction with SSL5. Importantly, SSL5 not only inhibited the interaction of HL-60 cells with activated endothelial cells but also with platelets, which both play an important role in growth and metastasis of cancers. These data support the concept that SSL5 could be a lead in the search for novel strategies against hematological malignancies.

  11. Featured Article: Differential regulation of endothelial nitric oxide synthase phosphorylation by protease-activated receptors in adult human endothelial cells.

    Science.gov (United States)

    Tillery, Lakeisha C; Epperson, Tenille A; Eguchi, Satoru; Motley, Evangeline D

    2016-03-01

    Protease-activated receptors have been shown to regulate endothelial nitric oxide synthase through the phosphorylation of specific sites on the enzyme. It has been established that PAR-2 activation phosphorylates eNOS-Ser-1177 and leads to the production of the potent vasodilator nitric oxide, while PAR-1 activation phosphorylates eNOS-Thr-495 and decreases nitric oxide production in human umbilical vein endothelial cells. In this study, we hypothesize a differential coupling of protease-activated receptors to the signaling pathways that regulates endothelial nitric oxide synthase and nitric oxide production in primary adult human coronary artery endothelial cells. Using Western Blot analysis, we showed that thrombin and the PAR-1 activating peptide, TFLLR, lead to the phosphorylation of eNOS-Ser-1177 in human coronary artery endothelial cells, which was blocked by SCH-79797 (SCH), a PAR-1 inhibitor. Using the nitrate/nitrite assay, we also demonstrated that the thrombin- and TFLLR-induced production of nitric oxide was inhibited by SCH and L-NAME, a NOS inhibitor. In addition, we observed that TFLLR, unlike thrombin, significantly phosphorylated eNOS-Thr-495, which may explain the observed delay in nitric oxide production in comparison to that of thrombin. Activation of PAR-2 by SLIGRL, a PAR-2 specific ligand, leads to dual phosphorylation of both catalytic sites but primarily regulated eNOS-Thr-495 phosphorylation with no change in nitric oxide production in human coronary artery endothelial cells. PAR-3, known as the non-signaling receptor, was activated by TFRGAP, a PAR-3 mimicking peptide, and significantly induced the phosphorylation of eNOS-Thr-495 with minimal phosphorylation of eNOS-Ser-1177 with no change in nitric oxide production. In addition, we confirmed that PAR-mediated eNOS-Ser-1177 phosphorylation was Ca(2+)-dependent using the Ca(2+) chelator, BAPTA, while eNOS-Thr-495 phosphorylation was mediated via Rho kinase using the ROCK inhibitor, Y-27632

  12. NF-κB Mediated Transcription in Human Monocytic Cells and Endothelial Cells.

    Science.gov (United States)

    Parry, G C; Mackman, N

    1998-04-01

    Monocytes and endothelial cells become activated at sites of inflammation and contribute to the pathology of many diseases, including septic shock and atherosclerosis. In these cells, induction of genes expressing various inflammatory mediators, such as adhesion molecules, cytokines, and growth factors, is regulated by NF-κB/Rel transcription factors. Recent studies have identified components of the signal transduction pathways leading to the activation of NF-κB/Rel proteins. Inhibition of these signaling pathways provides a novel therapeutic approach to prevent inducible gene expression in both monocytes and endothelial cells. (Trends Cardiovasc Med 1998;8:138-142). © 1998, Elsevier Science Inc.

  13. Double suicide genes selectively kill human umbilical vein endothelial cells

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    Liu Lunxu

    2011-02-01

    Full Text Available Abstract Background To construct a recombinant adenovirus containing CDglyTK double suicide genes and evaluate the killing effect of the double suicide genes driven by kinase domain insert containing receptor (KDR promoter on human umbilical vein endothelial cells. Methods Human KDR promoter, Escherichia coli (E. coli cytosine deaminase (CD gene and the herpes simplex virus-thymidine kinase (TK gene were cloned using polymerase chain reaction (PCR. Plasmid pKDR-CDglyTK was constructed with the KDR promoter and CDglyTK genes. A recombinant adenoviral plasmid AdKDR-CDglyTK was then constructed and transfected into 293 packaging cells to grow and harvest adenoviruses. KDR-expressing human umbilical vein endothelial cells (ECV304 and KDR-negative liver cancer cell line (HepG2 were infected with the recombinant adenoviruses at different multiplicity of infection (MOI. The infection rate was measured by green fluorescent protein (GFP expression. The infected cells were cultured in culture media containing different concentrations of prodrugs ganciclovir (GCV and/or 5-fluorocytosine (5-FC. The killing effects were measured using two different methods, i.e. annexin V-FITC staining and terminal transferase-mediated dUTP nick end-labeling (TUNEL staining. Results Recombinant adenoviruses AdKDR-CDglyTK were successfully constructed and they infected ECV304 and HepG2 cells efficiently. The infection rate was dependent on MOI of recombinant adenoviruses. ECV304 cells infected with AdKDR-CDglyTK were highly sensitive to GCV and 5-FC. The cell survival rate was dependent on both the concentration of the prodrugs and the MOI of recombinant adenoviruses. In contrast, there were no killing effects in the HepG2 cells. The combination of two prodrugs was much more effective in killing ECV304 cells than GCV or 5-FC alone. The growth of transgenic ECV304 cells was suppressed in the presence of prodrugs. Conclusion AdKDR-CDglyTK/double prodrog system may be a useful

  14. The impact of microgravity and hypergravity on endothelial cells.

    Science.gov (United States)

    Maier, Jeanette A M; Cialdai, Francesca; Monici, Monica; Morbidelli, Lucia

    2015-01-01

    The endothelial cells (ECs), which line the inner surface of vessels, play a fundamental role in maintaining vascular integrity and tissue homeostasis, since they regulate local blood flow and other physiological processes. ECs are highly sensitive to mechanical stress, including hypergravity and microgravity. Indeed, they undergo morphological and functional changes in response to alterations of gravity. In particular microgravity leads to changes in the production and expression of vasoactive and inflammatory mediators and adhesion molecules, which mainly result from changes in the remodelling of the cytoskeleton and the distribution of caveolae. These molecular modifications finely control cell survival, proliferation, apoptosis, migration, and angiogenesis. This review summarizes the state of the art on how microgravity and hypergravity affect cultured ECs functions and discusses some controversial issues reported in the literature.

  15. The Impact of Microgravity and Hypergravity on Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Jeanette A. M. Maier

    2015-01-01

    Full Text Available The endothelial cells (ECs, which line the inner surface of vessels, play a fundamental role in maintaining vascular integrity and tissue homeostasis, since they regulate local blood flow and other physiological processes. ECs are highly sensitive to mechanical stress, including hypergravity and microgravity. Indeed, they undergo morphological and functional changes in response to alterations of gravity. In particular microgravity leads to changes in the production and expression of vasoactive and inflammatory mediators and adhesion molecules, which mainly result from changes in the remodelling of the cytoskeleton and the distribution of caveolae. These molecular modifications finely control cell survival, proliferation, apoptosis, migration, and angiogenesis. This review summarizes the state of the art on how microgravity and hypergravity affect cultured ECs functions and discusses some controversial issues reported in the literature.

  16. INSULIN INDUCES NITRIC OXIDE PRODUCTION IN BOVINEAORTIC ENDOTHELIAL CELLS

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective To examine the effects of insulin on cell proliferation, nitric oxide (NO) release and nitric oxide synthase (NOS) gene expression in bovine aortic endothelial cells ( BAEC ) . Methods The mi togenesis was assessed by MTT method; the products of NO in the culture media, by Griess reaction; and the levels of NOS mRNA in BAEC , by RT/PCR tech nique. Results BAEC were not responsive to the growth-promoting effects of insulin. Stimulation with insulin resulted a dose-dependent rise of NO in the culture supernatants 2h later, with a maximum at 12~24h and a decline at 24h. This rise was inhibited by an inhibitor of NOS (L-NAME). NOS mRNA increased slightly in BAEC without statistical significance. Conelu sion The study suggested that the insulin-induced NO release might be caused directly by NOS activation.

  17. Nerve growth factor modulate proliferation of cultured rabbit corneal endothelial cells and epithelial cells.

    Science.gov (United States)

    Li, Xinyu; Li, Zhongguo; Qiu, Liangxiu; Zhao, Changsong; Hu, Zhulin

    2005-01-01

    In order to investigate the effect of nerve growth factor (NGF) on the proliferation of rabbit corneal endothelial cells and epithelial cells, the in vitro cultured rabbit corneal endothelial cells and epithelial cells were treated with different concentrations of NGF. MTT assay was used to examine the clonal growth and proliferation of the cells by determining the absorbency values at 570 nm. The results showed that NGF with three concentrations ranging from 5 U/mL to 500 U/mL enhanced the proliferation of rabbit corneal endothelial cells in a concentration-dependent manner. 50 U/mL and 500 U/mL NGF got more increase of proliferation than that of 5 U/mL NGF did. Meanwhile, 50 U/mL and 500 U/mL NGF could promote the proliferation of the rabbit corneal epithelial cells significantly in a concentration-dependent manner. However, 5 U/mL NGF did not enhance the proliferation of epithelial cells. It was suggested that exogenous NGF can stimulate the proliferation of both rabbit corneal endothelial and epithelial cells, but the extent of modulation is different.

  18. Glycosaminoglycan mimetic improves enrichment and cell functions of human endothelial progenitor cell colonies.

    Science.gov (United States)

    Chevalier, Fabien; Lavergne, Mélanie; Negroni, Elisa; Ferratge, Ségolène; Carpentier, Gilles; Gilbert-Sirieix, Marie; Siñeriz, Fernando; Uzan, Georges; Albanese, Patricia

    2014-05-01

    Human circulating endothelial progenitor cells isolated from peripheral blood generate in culture cells with features of endothelial cells named late-outgrowth endothelial colony-forming cells (ECFC). In adult blood, ECFC display a constant quantitative and qualitative decline during life span. Even after expansion, it is difficult to reach the cell dose required for cell therapy of vascular diseases, thus limiting the clinical use of these cells. Glycosaminoglycans (GAG) are components from the extracellular matrix (ECM) that are able to interact and potentiate heparin binding growth factor (HBGF) activities. According to these relevant biological properties of GAG, we designed a GAG mimetic having the capacity to increase the yield of ECFC production from blood and to improve functionality of their endothelial outgrowth. We demonstrate that the addition of [OTR(4131)] mimetic during the isolation process of ECFC from Cord Blood induces a 3 fold increase in the number of colonies. Moreover, addition of [OTR(4131)] to cell culture media improves adhesion, proliferation, migration and self-renewal of ECFC. We provide evidence showing that GAG mimetics may have great interest for cell therapy applied to vascular regeneration therapy and represent an alternative to exogenous growth factor treatments to optimize potential therapeutic properties of ECFC.

  19. Atherogenic Cytokines Regulate VEGF-A-Induced Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells into Endothelial Cells

    OpenAIRE

    Izuagie Attairu Ikhapoh; Pelham, Christopher J.; Agrawal, Devendra K

    2015-01-01

    Coronary artery stenting or angioplasty procedures frequently result in long-term endothelial dysfunction or loss and complications including arterial thrombosis and myocardial infarction. Stem cell-based therapies have been proposed to support endothelial regeneration. Mesenchymal stem cells (MSCs) differentiate into endothelial cells (ECs) in the presence of VEGF-A in vitro. Application of VEGF-A and MSC-derived ECs at the interventional site is a complex clinical challenge. In this study, ...

  20. Synergistic effect of angiotensin II on vascular endothelial growth factor-A-mediated differentiation of bone marrow-derived mesenchymal stem cells into endothelial cells

    OpenAIRE

    Ikhapoh, Izuagie Attairu; Pelham, Christopher J; Agrawal, Devendra K.

    2015-01-01

    Introduction Increased levels of angiotensin II (Ang II) and activity of Ang II receptor type 1 (AT1R) elicit detrimental effects in cardiovascular disease. However, the role of Ang II receptor type 2 (AT2R) remains poorly defined. Mesenchymal stem cells (MSCs) replenish and repair endothelial cells in the cardiovascular system. Herein, we investigated a novel role of angiotensin signaling in enhancing vascular endothelial growth factor (VEGF)-A-mediated differentiation of MSCs into endotheli...

  1. Effects of TNF-alpha on Endothelial Cell Collective Migration

    Science.gov (United States)

    Chen, Desu; Wu, Di; Helim Aranda-Espinoza, Jose; Losert, Wolfgang

    2013-03-01

    Tumor necrosis factor (TNF-alpha) is a small cell-signaling protein usually released by monocytes and macrophages during an inflammatory response. Previous work had shown the effects of TNF-alpha on single cell morphology, migration, and biomechanical properties. However, the effect on collective migrations remains unexplored. In this work, we have created scratches on monolayers of human umbilical endothelial cells (HUVECs) treated with 25ng/mL TNF-alpha on glass substrates. The wound healing like processes were imaged with phase contrast microscopy. Quantitative analysis of the collective migration of cells treated with TNF-alpha indicates that these cells maintain their persistent motion and alignment better than untreated cells. In addition, the collective migration was characterized by measuring the amount of non-affine deformations of the wound healing monolayer. We found a lower mean non-affinity and narrower distribution of non-affinities upon TNF-alpha stimulation. These results suggest that TNF-alpha introduces a higher degree of organized cell collective migration.

  2. Bioluminescence imaging of transplanted human