Reduced Ang2 expression in aging endothelial cells.

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Hohensinner, P J; Ebenbauer, B; Kaun, C; Maurer, G; Huber, K; Wojta, J

2016-06-03

Aging endothelial cells are characterized by increased cell size, reduced telomere length and increased expression of proinflammatory cytokines. In addition, we describe here that aging reduces the migratory distance of endothelial cells. Furthermore, we observe an increase of the quiescence protein Ang1 and a decrease of the endothelial activation protein Ang2 upon aging. Supplementing Ang2 to aged endothelial cells restored their migratory capacity. We conclude that aging shifts the balance of the Ang1/Ang2 network favouring a quiescent state. Activation of endothelial cells in aging might be necessary to enhance wound healing capacities. Copyright © 2016 Elsevier Inc. All rights reserved.

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

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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

Identification of intracellular proteins and signaling pathways in human endothelial cells regulated by angiotensin-(1-7).

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Meinert, Christian; Gembardt, Florian; Böhme, Ilka; Tetzner, Anja; Wieland, Thomas; Greenberg, Barry; Walther, Thomas

2016-01-01

The study aimed to identify proteins regulated by the cardiovascular protective peptide angiotensin-(1-7) and to determine potential intracellular signaling cascades. Human endothelial cells were stimulated with Ang-(1-7) for 1 h, 3 h, 6 h, and 9 h. Peptide effects on intracellular signaling were assessed via antibody microarray, containing antibodies against 725 proteins. Bioinformatics software was used to identify affected intracellular signaling pathways. Microarray data was verified exemplarily by Western blot, Real-Time RT-PCR, and immunohistochemical studies. The microarray identified 110 regulated proteins after 1 h, 119 after 3 h, 31 after 6 h, and 86 after 9 h Ang-(1-7) stimulation. Regulated proteins were associated with high significance to several metabolic pathways like “Molecular Mechanism of Cancer” and “p53 signaling” in a time dependent manner. Exemplarily, Western blots for the E3-type small ubiquitin-like modifier ligase PIAS2 confirmed the microarray data and displayed a decrease by more than 50% after Ang-(1-7) stimulation at 1 h and 3 h without affecting its mRNA. Immunohistochemical studies with PIAS2 in human endothelial cells showed a decrease in cytoplasmic PIAS2 after Ang-(1-7) treatment. The Ang-(1-7) mediated decrease of PIAS2 was reproduced in other endothelial cell types. The results suggest that angiotensin-(1-7) plays a role in metabolic pathways related to cell death and cell survival in human endothelial cells.

Verocytotoxin-induced apoptosis of human microvascular endothelial cells.

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

2001-04-01

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

Protein S binding to human endothelial cells is required for expression of cofactor activity for activated protein C

NARCIS (Netherlands)

Hackeng, T. M.; Hessing, M.; van 't Veer, C.; Meijer-Huizinga, F.; Meijers, J. C.; de Groot, P. G.; van Mourik, J. A.; Bouma, B. N.

1993-01-01

An important feedback mechanism in blood coagulation is supplied by the protein C/protein S anticoagulant pathway. In this study we demonstrate that the binding of human protein S to cultured human umbilical vein endothelial cells (HUVECs) is required for the expression of cofactor activity of

Characterization of tissue plasminogen activator binding proteins isolated from endothelial cells and other cell types

International Nuclear Information System (INIS)

Beebe, D.P.; Wood, L.L.; Moos, M.

1990-01-01

Human tissue plasminogen activator (t-PA) was shown to bind specifically to human osteosarcoma cells (HOS), and human epidermoid carcinoma cells (A-431 cells). Crosslinking studies with DTSSP demonstrated high molecular weight complexes (130,000) between 125 I-t-PA and cell membrane protein on human umbilical vein endothelial cells (HUVEC), HOS, and A-431 cells. A 48-65,000 molecular weight complex was demonstrated after crosslinking t-PA peptide (res. 7-20) to cells. Ligand blotting of cell lysates which had been passed over a t-PA affinity column revealed binding of t-PA to 54,000 and 95,000 molecular weight proteins. Several t-PA binding proteins were identified in immunopurified cell lysates, including tubulin beta chain, plasminogen activator inhibitor type 1 and single chain urokinase

Bone morphogenic protein 4 produced in endothelial cells by oscillatory shear stress stimulates an inflammatory response

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Sorescu, George P.; Sykes, Michelle; Weiss, Daiana; Platt, Manu O.; Saha, Aniket; Hwang, Jinah; Boyd, Nolan; Boo, Yong C.; Vega, J. David; Taylor, W. Robert;

Signaling hierarchy regulating human endothelial cell development.

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Kelly, Melissa A; Hirschi, Karen K

2009-05-01

Our present knowledge of the regulation of mammalian endothelial cell differentiation has been largely derived from studies of mouse embryonic development. However, unique mechanisms and hierarchy of signals that govern human endothelial cell development are unknown and, thus, explored in these studies. Using human embryonic stem cells as a model system, we were able to reproducibly and robustly generate differentiated endothelial cells via coculture on OP9 marrow stromal cells. We found that, in contrast to studies in the mouse, bFGF and VEGF had no specific effects on the initiation of human vasculogenesis. However, exogenous Ihh promoted endothelial cell differentiation, as evidenced by increased production of cells with cobblestone morphology that coexpress multiple endothelial-specific genes and proteins, form lumens, and exhibit DiI-AcLDL uptake. Inhibition of BMP signaling using Noggin or BMP4, specifically, using neutralizing antibodies suppressed endothelial cell formation; whereas, addition of rhBMP4 to cells treated with the hedgehog inhibitor cyclopamine rescued endothelial cell development. Our studies revealed that Ihh promoted human endothelial cell differentiation from pluripotent hES cells via BMP signaling, providing novel insights applicable to modulating human endothelial cell formation and vascular regeneration for human clinical therapies.

Human endothelial precursor cells express tumor endothelial marker 1/endosialin/CD248.

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Bagley, Rebecca G; Rouleau, Cecile; St Martin, Thia; Boutin, Paula; Weber, William; Ruzek, Melanie; Honma, Nakayuki; Nacht, Mariana; Shankara, Srinivas; Kataoka, Shiro; Ishida, Isao; Roberts, Bruce L; Teicher, Beverly A

2008-08-01

Angiogenesis occurs during normal physiologic processes as well as under pathologic conditions such as tumor growth. Serial analysis of gene expression profiling revealed genes [tumor endothelial markers (TEM)] that are overexpressed in tumor endothelial cells compared with normal adult endothelial cells. Because blood vessel development of malignant tumors under certain conditions may include endothelial precursor cells (EPC) recruited from bone marrow, we investigated TEM expression in EPC. The expression of TEM1 or endosialin (CD248) and other TEM has been discovered in a population of vascular endothelial growth factor receptor 2+/CD31+/CD45-/VE-cadherin+ EPC derived from human CD133+/CD34+ cells. EPC share some properties with fully differentiated endothelial cells from normal tissue, yet reverse transcription-PCR and flow cytometry reveal that EPC express higher levels of endosialin at the molecular and protein levels. The elevated expression of endosialin in EPC versus mature endothelial cells suggests that endosialin is involved in the earlier stages of tumor angiogenesis. Anti-endosialin antibodies inhibited EPC migration and tube formation in vitro. In vivo, immunohistochemistry indicated that human EPC continued to express endosialin protein in a Matrigel plug angiogenesis assay established in nude mice. Anti-endosialin antibodies delivered systemically at 25 mg/kg were also able to inhibit circulating murine EPC in nude mice bearing s.c. SKNAS tumors. EPC and bone marrow-derived cells have been shown previously to incorporate into malignant blood vessels in some instances, yet they remain controversial in the field. The data presented here on endothelial genes that are up-regulated in tumor vasculature and in EPC support the hypothesis that the angiogenesis process in cancer can involve EPC.

Regional differences in endothelial cell cytoskeleton, junctional proteins and phosphorylated tyrosine labeling in the porcine vortex vein system.

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Tan, Priscilla Ern Zhi; Yu, Paula K; Yang, Hongfang; Cringle, Stephen J; Yu, Dao-Yi

2018-07-01

We previously demonstrated endothelial phenotype heterogeneity in the vortex vein system. This study is to further determine whether regional differences are present in the cytoskeleton, junctional proteins and phosphorylated tyrosine labeling within the system. The vortex vein system of twenty porcine eyes was perfused with labels for f-actin, claudin-5, VE-Cadherin, phosphorylated tyrosine and nucleic acid. The endothelial cells of eight different regions (choroidal veins, pre-ampulla, anterior ampulla, mid-ampulla, posterior ampulla, post-ampulla, intra-scleral canal and the extra-ocular vortex vein) were studied using confocal microscopy. There were regional differences in the endothelial cell structures. Cytoskeleton labeling was relatively even in intensity throughout Regions 1 to 6. Overall VE-Cadherin had a non-uniform distribution and thicker width endothelial cell border staining than claudin-5. Progressing downstream there was an increased variation in thickness of VE-cadherin labeling. There was an overlap in phosphorylated tyrosine and VE-Cadherin labeling in the post-ampulla, intra-scleral canal and extra-ocular vortex vein. Intramural cells were observed that were immune-positive for VE-Cadherin and phosphorylated tyrosine. There were significant differences in the number of intramural cells in different regions. Significant regional differences with endothelial cell labeling of cytoskeleton, junction proteins, and phosphorylated tyrosine were found within the vortex vein system. These findings support existing data on endothelial cell phenotype heterogeneity, and may aid in the knowledge of venous pathologies by understanding regions of vulnerability to endothelial damage within the vortex vein system. It could be valuable to further investigate and characterize the VE-cadherin and phosphotyrosine immune-positive intramural cells. Copyright © 2018. Published by Elsevier Ltd.

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

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

2014-04-01

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

Glossogyne Tenuifolia Enhances Posttranslational S-Nitrosylation of Proteins in Vascular Endothelial Cells

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Chao-Ping Wang

2011-06-01

Full Text Available Glossogyne tenuifolia (GT is a traditional Chinese herb that possesses strong antioxidant activity and protects against endothelial cell (EC injury by inhibition of free reactive oxygen species (ROS. The aim of this study was to elucidate the mechanisms by which GT prevents endothelial injury using a proteomics approach. We used a sensitive method to analyze the S- nitrosoproteins utilizing a modified biotin-switch method in order to detect the possible effects of GT on protein posttranslational modification. After treatment of vascular ECs with GT, two proteins HspA9 (IS1, beta-actin (IS2 were observed to have increased posttranslational S-nitrosylation, whereas seven proteins, vimentin (DS2, DS3 and DS5, tropomyosin 3, 4 (DS6 and DS7 and oxidative phosphorylation protein such as ATP synthase, F1 complex (DS1 and 80K-H protein (DS4, were found to have decreased posttranslational S-nitrosylation. Due to S-nitrosylation of HspA9 causing the reduction of intracellular ROS and S-nitrosylation of ATP synthase interfering with ATP production and ROS formation, our study may indicate a novel mechanism in which GT protects EC injury by the inhibition of oxidative reaction.

Sphingosine 1-Phosphate Induces Platelet/Endothelial Cell Adhesion Molecule-1 Tyrosine Phosphorylation in Bovine Aortic Endothelial Cells through a PP2-Inhibitable Mechanism

Directory of Open Access Journals (Sweden)

Yu-Ting Huang

2007-12-01

Full Text Available Sphingosine-1-phosphate (S1P is a low-molecular-weight phospholipid derivative released by activated platelets. S1P transduces signals through a family of G protein-coupled receptors to modulate various physiological behaviors of endothelial cells. Platelet/endothelial cell adhesion molecule-1 (PECAM-1; CD31 is a 130-kDa protein expressed on the surfaces of leukocytes, platelets, and endothelial cells. Upon PECAM-1 activation, its cytoplasmic tyrosine residues become phosphorylated and bind with SH2 domain-containing proteins, thus leading to the downstream functions mediated by PECAM-1. In the present study, we found that S1P induced PECAM-1 tyrosine phosphorylation and SHP-2 association in bovine aortic endothelial cells (BAECs by immunoprecipitation and western blotting. The pretreatment of BAECs with a series of chemical inhibitors to determine the signaling pathway showed that the PECAM-1 phosphorylation was inhibited by PP2, indicating the participation of Src family kinases. These results demonstrated that S1P induced PECAM-1 tyrosine phosphorylation in BAECs through mediation of Src family kinases, and this may regulate the physiological behaviors of endothelial cells.

Syncytin is involved in breast cancer-endothelial cell fusions

DEFF Research Database (Denmark)

Bjerregaard, Bolette; Holck, S.; Christensen, I.J.

2006-01-01

Cancer cells can fuse spontaneously with normal host cells, including endothelial cells, and such fusions may strongly modulate the biological behaviour of tumors. However, the underlying mechanisms are unknown. We now show that human breast cancer cell lines and 63 out of 165 (38%) breast cancer...... specimens express syncytin, an endogenous retroviral envelope protein, previously implicated in fusions between placental trophoblast cells. Additionally, endothelial and cancer cells are shown to express ASCT-2, a receptor for syncytin. Syncytin antisense treatment decreases syncytin expression...... and inhibits fusions between breast cancer cells and endothelial cells. Moreover, a syncytin inhibitory peptide also inhibits fusions between cancer and endothelial cells. These results are the first to show that syncytin is expressed by human cancer cells and is involved in cancer-endothelial cell fusions....

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

DEFF Research Database (Denmark)

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

2016-01-01

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

The molecular mechanism of mediation of adsorbed serum proteins to endothelial cells adhesion and growth on biomaterials.

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Yang, Dayun; Lü, Xiaoying; Hong, Ying; Xi, Tingfei; Zhang, Deyuan

2013-07-01

To explore molecular mechanism of mediation of adsorbed proteins to cell adhesion and growth on biomaterials, this study examined endothelial cell adhesion, morphology and viability on bare and titanium nitride (TiN) coated nickel titanium (NiTi) alloys and chitosan film firstly, and then identified the type and amount of serum proteins adsorbed on the three surfaces by proteomic technology. Subsequently, the mediation role of the identified proteins to cell adhesion and growth was investigated with bioinformatics analyses, and further confirmed by a series of cellular and molecular biological experiments. Results showed that the type and amount of adsorbed serum proteins associated with cell adhesion and growth was obviously higher on the alloys than on the chitosan film, and these proteins mediated endothelial cell adhesion and growth on the alloys via four ways. First, proteins such as adiponectin in the adsorbed protein layer bound with cell surface receptors to generate signal transduction, which activated cell surface integrins through increasing intracellular calcium level. Another way, thrombospondin 1 in the adsorbed protein layer promoted TGF-β signaling pathway activation and enhanced integrins expression. The third, RGD sequence containing proteins such as fibronectin 1, vitronectin and thrombospondin 1 in the adsorbed protein layer bound with activated integrins to activate focal adhesion pathway, increased focal adhesion formation and actin cytoskeleton organization and mediated cell adhesion and spreading. In addition, the activated focal adhesion pathway promoted the expression of cell growth related genes and resulted in cell proliferation. The fourth route, coagulation factor II (F2) and fibronectin 1 in the adsorbed protein layer bound with cell surface F2 receptor and integrin, activated regulation of actin cytoskeleton pathway and regulated actin cytoskeleton organization. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

International Nuclear Information System (INIS)

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

2007-01-01

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

Passive leg movement enhances interstitial VEGF protein, endothelial cell proliferation, and eNOS mRNA content in human skeletal muscle

DEFF Research Database (Denmark)

Hellsten, Ylva; Rufener, Nora; Nielsen, Jens J

2008-01-01

.05) in blood flow without a significant enhancement in oxygen uptake. Muscle interstitial fluid was sampled with microdialysis technique and analyzed for vascular endothelial growth factor (VEGF) protein and for the effect on endothelial cell proliferation. Biopsies obtained from the musculus vastus lateralis...... to cultured endothelial cells revealed that dialysate obtained during leg movement induced a 3.2-fold higher proliferation rate (P level fourfold above resting levels. VEGF mRNA and MMP-2 mRNA levels were...

Mesenchymal Stem/Multipotent Stromal Cells from Human Decidua Basalis Reduce Endothelial Cell Activation.

Science.gov (United States)

Alshabibi, Manal A; Al Huqail, Al Joharah; Khatlani, Tanvir; Abomaray, Fawaz M; Alaskar, Ahmed S; Alawad, Abdullah O; Kalionis, Bill; Abumaree, Mohamed Hassan

2017-09-15

Recently, we reported the isolation and characterization of mesenchymal stem cells from the decidua basalis of human placenta (DBMSCs). These cells express a unique combination of molecules involved in many important cellular functions, which make them good candidates for cell-based therapies. The endothelium is a highly specialized, metabolically active interface between blood and the underlying tissues. Inflammatory factors stimulate the endothelium to undergo a change to a proinflammatory and procoagulant state (ie, endothelial cell activation). An initial response to endothelial cell activation is monocyte adhesion. Activation typically involves increased proliferation and enhanced expression of adhesion and inflammatory markers by endothelial cells. Sustained endothelial cell activation leads to a type of damage to the body associated with inflammatory diseases, such as atherosclerosis. In this study, we examined the ability of DBMSCs to protect endothelial cells from activation through monocyte adhesion, by modulating endothelial proliferation, migration, adhesion, and inflammatory marker expression. Endothelial cells were cocultured with DBMSCs, monocytes, monocyte-pretreated with DBMSCs and DBMSC-pretreated with monocytes were also evaluated. Monocyte adhesion to endothelial cells was examined following treatment with DBMSCs. Expression of endothelial cell adhesion and inflammatory markers was also analyzed. The interaction between DBMSCs and monocytes reduced endothelial cell proliferation and monocyte adhesion to endothelial cells. In contrast, endothelial cell migration increased in response to DBMSCs and monocytes. Endothelial cell expression of adhesion and inflammatory molecules was reduced by DBMSCs and DBMSC-pretreated with monocytes. The mechanism of reduced endothelial proliferation involved enhanced phosphorylation of the tumor suppressor protein p53. Our study shows for the first time that DBMSCs protect endothelial cells from activation by

Adhesive protein interactions with chitosan: consequences for valve endothelial cell growth on tissue-engineering materials.

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Cuy, Janet L; Beckstead, Benjamin L; Brown, Chad D; Hoffman, Allan S; Giachelli, Cecilia M

2003-11-01

Stable endothelialization of a tissue-engineered heart valve is essential for proper valve function, although adhesive characteristics of the native valve endothelial cell (VEC) have rarely been explored. This research evaluated VEC adhesive qualities and attempted to enhance VEC growth on the biopolymer chitosan, a novel tissue-engineering scaffold material with promising biological and chemical properties. Aortic VEC cultures were isolated and found to preferentially adhere to fibronectin, collagen types IV and I over laminin and osteopontin in a dose-dependent manner. Seeding of VEC onto comparison substrates revealed VEC growth and morphology to be preferential in the order: tissue culture polystyrene > gelatin, poly(DL-lactide-co-glycolide), chitosan > poly(hydroxy alkanoate). Adhesive protein precoating of chitosan did not significantly enhance VEC growth, despite equivalent protein adsorption as to polystyrene. Initial cell adhesion to protein-precoated chitosan, however, was higher than for polystyrene. Composite chitosan/collagen type IV films were investigated as an alternative to simple protein precoatings, and were shown to improve VEC growth and morphology over chitosan alone. These findings suggest potential manipulation of chitosan properties to improve amenability to valve tissue-engineering applications. Copyright 2003 Wiley Periodicals, Inc.

Protein kinase Cα phosphorylates a novel argininosuccinate synthase site at serine 328 during calcium-dependent stimulation of endothelial nitric-oxide synthase in vascular endothelial cells.

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Haines, Ricci J; Corbin, Karen D; Pendleton, Laura C; Eichler, Duane C

2012-07-27

Endothelial nitric-oxide synthase (eNOS) utilizes l-arginine as its principal substrate, converting it to l-citrulline and nitric oxide (NO). l-Citrulline is recycled to l-arginine by two enzymes, argininosuccinate synthase (AS) and argininosuccinate lyase, providing the substrate arginine for eNOS and NO production in endothelial cells. Together, these three enzymes, eNOS, AS, and argininosuccinate lyase, make up the citrulline-NO cycle. Although AS catalyzes the rate-limiting step in NO production, little is known about the regulation of AS in endothelial cells beyond the level of transcription. In this study, we showed that AS Ser-328 phosphorylation was coordinately regulated with eNOS Ser-1179 phosphorylation when bovine aortic endothelial cells were stimulated by either a calcium ionophore or thapsigargin to produce NO. Furthermore, using in vitro kinase assay, kinase inhibition studies, as well as protein kinase Cα (PKCα) knockdown experiments, we demonstrate that the calcium-dependent phosphorylation of AS Ser-328 is mediated by PKCα. Collectively, these findings suggest that phosphorylation of AS at Ser-328 is regulated in accordance with the calcium-dependent regulation of eNOS under conditions that promote NO production and are in keeping with the rate-limiting role of AS in the citrulline-NO cycle of vascular endothelial cells.

VEGFR-3 signaling is regulated by a G-protein activator, activator of G-protein signaling 8, in lymphatic endothelial cells.

Science.gov (United States)

Sakima, Miho; Hayashi, Hisaki; Mamun, Abdullah Al; Sato, Motohiko

2018-07-01

Vascular endothelial growth factor C (VEGFC) and its cognate receptor VEGFR-3 play a key role in lymphangiogenesis. We previously reported that an ischemia-inducible Gβγ signal regulator, activator of G-protein signaling 8 (AGS8), regulated the subcellular distribution of vascular endothelial growth factor receptor-2 (VEGFR-2) and influenced VEGFA-induced signaling in vascular endothelial cells. Here, we report that AGS8 regulates VEGFR-3, which is another subtype of the VEGF receptor family, and mediates VEGFC signaling in human dermal lymphatic endothelial cells (HDLECs). VEGFC stimulated the proliferation of HDLECs and tube formation by HDLECs, which were inhibited by knocking down AGS8 by small interfering RNA (siRNA). AGS8 siRNA inhibited VEGFC-mediated phosphorylation of VEGFR-3 and its downstream molecules, including ERK1/2 and AKT. Analysis of fluorescence-activated cell sorting and immunofluorescence staining demonstrated that AGS8 knockdown was associated with a reduction of VEGFR-3 at the cell surface. Endocytosis inhibitors did not rescue the decrease of cell-surface VEGFR-3, suggesting that AGS8 regulated the trafficking of VEGFR-3 to the plasma membrane. An immunoprecipitation assay indicated that VEGFR-3 formed a complex including AGS8 and Gβγ in cells. These data suggest the novel regulation of VEGFC-VEGFR-3 by AGS8 in HDLECs and a potential role for AGS8 in lymphangiogenesis. Copyright © 2018 Elsevier Inc. All rights reserved.

Endothelial microparticle-mediated transfer of MicroRNA-126 promotes vascular endothelial cell repair via SPRED1 and is abrogated in glucose-damaged endothelial microparticles.

Science.gov (United States)

Jansen, Felix; Yang, Xiaoyan; Hoelscher, Marion; Cattelan, Arianna; Schmitz, Theresa; Proebsting, Sebastian; Wenzel, Daniela; Vosen, Sarah; Franklin, Bernardo S; Fleischmann, Bernd K; Nickenig, Georg; Werner, Nikos

2013-10-29

Repair of the endothelium after vascular injury is crucial for preserving endothelial integrity and preventing the development of vascular disease. The underlying mechanisms of endothelial cell repair are largely unknown. We sought to investigate whether endothelial microparticles (EMPs), released from apoptotic endothelial cells (ECs), influence EC repair. Systemic treatment of mice with EMPs after electric denudation of the endothelium accelerated reendothelialization in vivo. In vitro experiments revealed that EMP uptake in ECs promotes EC migration and proliferation, both critical steps in endothelial repair. To dissect the underlying mechanisms, Taqman microRNA array was performed, and microRNA (miR)-126 was identified as the predominantly expressed miR in EMPs. The following experiments demonstrated that miR-126 was transported into recipient human coronary artery endothelial cells by EMPs and functionally regulated the target protein sprouty-related, EVH1 domain-containing protein 1 (SPRED1). Knockdown of miR-126 in EMPs abrogated EMP-mediated effects on human coronary artery endothelial cell migration and proliferation in vitro and reendothelialization in vivo. Interestingly, after simulating diabetic conditions, EMPs derived from glucose-treated ECs contained significantly lower amounts of miR-126 and showed reduced endothelial repair capacity in vitro and in vivo. Finally, expression analysis of miR-126 in circulating microparticles from 176 patients with stable coronary artery disease with and without diabetes mellitus revealed a significantly reduced miR-126 expression in circulating microparticles from diabetic patients. Endothelial microparticles promote vascular endothelial repair by delivering functional miR-126 into recipient cells. In pathological hyperglycemic conditions, EMP-mediated miR-126-induced EC repair is altered.

Infection of endothelial cells by common human viruses.

Science.gov (United States)

Friedman, H M

1989-01-01

Common human viruses were evaluated for their ability to replicate in the endothelial cells of human umbilical vein and bovine thoracic aorta in vitro. Infection occurred with most viruses. The susceptibilities of endothelial cells derived from bovine aorta, pulmonary artery, and vena cava were compared. Among the viruses studied, no differences were noted in the ability to grow in endothelial cells from these three large vessels. One virus, herpes simplex virus type 1, was evaluated for its ability to produce persistent infection of endothelial cells. Infection developed and persisted for up to 3 months. After the first week, productive infection was found in less than 1% of cells. Nevertheless, the infection markedly affected the growth and morphology of the endothelial monolayer. Infection with any of several different viruses was noted to alter endothelial cell functions, including adherence of granulocytes, production of colony-stimulating factor, and synthesis of matrix protein. In addition, herpes simplex virus type 1 induced receptors for the Fc portion of IgG and for complement component C3b. These findings indicate that common human viruses can profoundly affect the biology of the endothelium.

Quantitative Proteomics Analysis of VEGF-Responsive Endothelial Protein S-Nitrosylation Using Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and LC-MS/MS.

Science.gov (United States)

Zhang, Hong-Hai; Lechuga, Thomas J; Chen, Yuezhou; Yang, Yingying; Huang, Lan; Chen, Dong-Bao

2016-05-01

Adduction of a nitric oxide moiety (NO•) to cysteine(s), termed S-nitrosylation (SNO), is a novel mechanism for NO to regulate protein function directly. However, the endothelial SNO-protein network that is affected by endogenous and exogenous NO is obscure. This study was designed to develop a quantitative proteomics approach using stable isotope labeling by amino acids in cell culture for comparing vascular endothelial growth factor (VEGFA)- and NO donor-responsive endothelial nitroso-proteomes. Primary placental endothelial cells were labeled with "light" (L-(12)C6 (14)N4-Arg and L-(12)C6 (14)N2-Lys) or "heavy" (L-(13)C6 (15)N4-Arg and L-(13)C6 (15)N2-Lys) amino acids. The light cells were treated with an NO donor nitrosoglutathione (GSNO, 1 mM) or VEGFA (10 ng/ml) for 30 min, while the heavy cells received vehicle as control. Equal amounts of cellular proteins from the light (GSNO or VEGFA treated) and heavy cells were mixed for labeling SNO-proteins by the biotin switch technique and then trypsin digested. Biotinylated SNO-peptides were purified for identifying SNO-proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Ratios of light to heavy SNO-peptides were calculated for determining the changes of the VEGFA- and GSNO-responsive endothelial nitroso-proteomes. A total of 387 light/heavy pairs of SNO-peptides were identified, corresponding to 213 SNO-proteins that include 125 common and 27 VEGFA- and 61 GSNO-responsive SNO-proteins. The specific SNO-cysteine(s) in each SNO-protein were simultaneously identified. Pathway analysis revealed that SNO-proteins are involved in various endothelial functions, including proliferation, motility, metabolism, and protein synthesis. We collectively conclude that endogenous NO on VEGFA stimulation and exogenous NO from GSNO affect common and different SNO-protein networks, implicating SNO as a critical mechanism for VEGFA stimulation of angiogenesis. © 2016 by the Society for the Study of Reproduction

Radiosensitization of human endothelial cells by IL-24

International Nuclear Information System (INIS)

Meyn, R.E.

2003-01-01

Radiation therapy remains an important cancer treatment modality but despite improvements in dose delivery many patients still fail at their primary tumor site. Therefore, new strategies designed to improve local control are needed. Protocols combining radiation with anti-angiogenic agents might be of particular advantage based on their documented low toxicity. In this regard, we have been conducting preclinical investigations of a novel cytokine, mda7/IL-24. Our collaborators have shown that mda7/IL-24 protein targets the endothelial cells of the tumor microvascular system and has potent anti-angiogenic properties in both in vitro and in vivo assays. Recently, we have demonstrated that recombinant mda7/IL-24 protein radiosensitizes human endothelial cells in vitro. Specifically, 10 ng/ml of recombinant human IL-24 protein for 12 hrs reduced the survival at 2 Gy for human umbilical vein endothelial cells (HUVECs) from 0.33 to 0.12. We are also working on understanding the molecular basis for this radiosensitizing effect. Preliminary data suggest a model whereby mda7/IL-24 engages a specific receptor on the surface of endothelial cells and initiates a signal transduction pathway that modulates the cell's propensity for radiation-induced apoptosis and capacity for repairing radiation-induced DNA double strand breaks. Mechanistic insight gained from these studies may have implications for the actions of other anti-angiogenic agents and may generally explain the regulation of radiosensitivity imparted by growth factors and cytokines

Oxidative Stress Induces Endothelial Cell Senescence via Downregulation of Sirt6

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Rong Liu

2014-01-01

Full Text Available Accumulating evidence has shown that diabetes accelerates aging and endothelial cell senescence is involved in the pathogenesis of diabetic vascular complications, including diabetic retinopathy. Oxidative stress is recognized as a key factor in the induction of endothelial senescence and diabetic retinopathy. However, specific mechanisms involved in oxidative stress-induced endothelial senescence have not been elucidated. We hypothesized that Sirt6, which is a nuclear, chromatin-bound protein critically involved in many pathophysiologic processes such as aging and inflammation, may have a role in oxidative stress-induced vascular cell senescence. Measurement of Sirt6 expression in human endothelial cells revealed that H2O2 treatment significantly reduced Sirt6 protein. The loss of Sirt6 was associated with an induction of a senescence phenotype in endothelial cells, including decreased cell growth, proliferation and angiogenic ability, and increased expression of senescence-associated β-galactosidase activity. Additionally, H2O2 treatment reduced eNOS expression, enhanced p21 expression, and dephosphorylated (activated retinoblastoma (Rb protein. All of these alternations were attenuated by overexpression of Sirt6, while partial knockdown of Sirt6 expression by siRNA mimicked the effect of H2O2. In conclusion, these results suggest that Sirt6 is a critical regulator of endothelial senescence and oxidative stress-induced downregulation of Sirt6 is likely involved in the pathogenesis of diabetic retinopathy.

[The role of endothelial cells and endothelial precursor cells in angiogenesis].

Science.gov (United States)

Poreba, Małgorzata; Usnarska-Zubkiewicz, Lidia; Kuliczkowski, Kazimierz

2006-01-01

Endothelium plays a key role in maintenance of vascular homeostasis in human organism. According to new data endothelial cells and hematopoietic cells have a common precursor in prenatal life--a hemangioblast, which explains the fact of sharing the same determinants on the surface of both type of cells. Circulating endothelial precursors were identified in adults and this suggests that hemangioblasts may be present not only during embriogenesis. In some clinical situations the increased numbers of endothelial cells and endothelial precursors were noted, and especially in patients with neoplastic diseases, which is probably the result of increased angiogenesis. Endothelial precursors are thought to be the promice for therapeutic purposes in future--to increase local angiogenesis.

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

DEFF Research Database (Denmark)

Hellsten, Ylva; Frandsen, Ulrik

1997-01-01

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

The adaptor CRADD/RAIDD controls activation of endothelial cells by proinflammatory stimuli.

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Qiao, Huan; Liu, Yan; Veach, Ruth A; Wylezinski, Lukasz; Hawiger, Jacek

2014-08-08

A hallmark of inflammation, increased vascular permeability, is induced in endothelial cells by multiple agonists through stimulus-coupled assembly of the CARMA3 signalosome, which contains the adaptor protein BCL10. Previously, we reported that BCL10 in immune cells is targeted by the "death" adaptor CRADD/RAIDD (CRADD), which negatively regulates nuclear factor κB (NFκB)-dependent cytokine and chemokine expression in T cells (Lin, Q., Liu, Y., Moore, D. J., Elizer, S. K., Veach, R. A., Hawiger, J., and Ruley, H. E. (2012) J. Immunol. 188, 2493-2497). This novel anti-inflammatory CRADD-BCL10 axis prompted us to analyze CRADD expression and its potential anti-inflammatory action in non-immune cells. We focused our study on microvascular endothelial cells because they play a key role in inflammation. We found that CRADD-deficient murine endothelial cells display heightened BCL10-mediated expression of the pleotropic proinflammatory cytokine IL-6 and chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2) in response to LPS and thrombin. Moreover, these agonists also induce significantly increased permeability in cradd(-/-), as compared with cradd(+/+), primary murine endothelial cells. CRADD-deficient cells displayed more F-actin polymerization with concomitant disruption of adherens junctions. In turn, increasing intracellular CRADD by delivery of a novel recombinant cell-penetrating CRADD protein (CP-CRADD) restored endothelial barrier function and suppressed the induction of IL-6 and MCP-1 evoked by LPS and thrombin. Likewise, CP-CRADD enhanced barrier function in CRADD-sufficient endothelial cells. These results indicate that depletion of endogenous CRADD compromises endothelial barrier function in response to inflammatory signals. Thus, we define a novel function for CRADD in endothelial cells as an inducible suppressor of BCL10, a key mediator of responses to proinflammatory agonists. © 2014 by The American Society for Biochemistry and Molecular Biology

Quantitative Proteomics Analysis of VEGF-Responsive Endothelial Protein S-Nitrosylation Using Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and LC-MS/MS1

Science.gov (United States)

Zhang, Hong-Hai; Lechuga, Thomas J.; Chen, Yuezhou; Yang, Yingying; Huang, Lan; Chen, Dong-Bao

2016-01-01

Adduction of a nitric oxide moiety (NO•) to cysteine(s), termed S-nitrosylation (SNO), is a novel mechanism for NO to regulate protein function directly. However, the endothelial SNO-protein network that is affected by endogenous and exogenous NO is obscure. This study was designed to develop a quantitative proteomics approach using stable isotope labeling by amino acids in cell culture for comparing vascular endothelial growth factor (VEGFA)- and NO donor-responsive endothelial nitroso-proteomes. Primary placental endothelial cells were labeled with “light” (L-12C614N4-Arg and L-12C614N2-Lys) or “heavy” (L-13C615N4-Arg and L-13C615N2-Lys) amino acids. The light cells were treated with an NO donor nitrosoglutathione (GSNO, 1 mM) or VEGFA (10 ng/ml) for 30 min, while the heavy cells received vehicle as control. Equal amounts of cellular proteins from the light (GSNO or VEGFA treated) and heavy cells were mixed for labeling SNO-proteins by the biotin switch technique and then trypsin digested. Biotinylated SNO-peptides were purified for identifying SNO-proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Ratios of light to heavy SNO-peptides were calculated for determining the changes of the VEGFA- and GSNO-responsive endothelial nitroso-proteomes. A total of 387 light/heavy pairs of SNO-peptides were identified, corresponding to 213 SNO-proteins that include 125 common and 27 VEGFA- and 61 GSNO-responsive SNO-proteins. The specific SNO-cysteine(s) in each SNO-protein were simultaneously identified. Pathway analysis revealed that SNO-proteins are involved in various endothelial functions, including proliferation, motility, metabolism, and protein synthesis. We collectively conclude that endogenous NO on VEGFA stimulation and exogenous NO from GSNO affect common and different SNO-protein networks, implicating SNO as a critical mechanism for VEGFA stimulation of angiogenesis. PMID:27075618

CXCL10 can inhibit endothelial cell proliferation independently of CXCR3.

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Gabriele S V Campanella

2010-09-01

Full Text Available CXCL10 (or Interferon-inducible protein of 10 kDa, IP-10 is an interferon-inducible chemokine with potent chemotactic activity on activated effector T cells and other leukocytes expressing its high affinity G protein-coupled receptor CXCR3. CXCL10 is also active on other cell types, including endothelial cells and fibroblasts. The mechanisms through which CXCL10 mediates its effects on non-leukocytes is not fully understood. In this study, we focus on the anti-proliferative effect of CXCL10 on endothelial cells, and demonstrate that CXCL10 can inhibit endothelial cell proliferation in vitro independently of CXCR3. Four main findings support this conclusion. First, primary mouse endothelial cells isolated from CXCR3-deficient mice were inhibited by CXCL10 as efficiently as wildtype endothelial cells. We also note that the proposed alternative splice form CXCR3-B, which is thought to mediate CXCL10's angiostatic activity, does not exist in mice based on published mouse CXCR3 genomic sequences as an in-frame stop codon would terminate the proposed CXCR3-B splice variant in mice. Second, we demonstrate that human umbilical vein endothelial cells and human lung microvascular endothelial cells that were inhibited by CXL10 did not express CXCR3 by FACS analysis. Third, two different neutralizing CXCR3 antibodies did not inhibit the anti-proliferative effect of CXCL10. Finally, fourth, utilizing a panel of CXCL10 mutants, we show that the ability to inhibit endothelial cell proliferation correlates with CXCL10's glycosaminoglycan binding affinity and not with its CXCR3 binding and signaling. Thus, using a very defined system, we show that CXCL10 can inhibit endothelial cell proliferation through a CXCR3-independent mechanism.

Differentiation state determines neural effects on microvascular endothelial cells

International Nuclear Information System (INIS)

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

2012-01-01

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

Effects of SOV-induced phosphatase inhibition and expression of protein tyrosine phosphatases in rat corneal endothelial cells.

Science.gov (United States)

Chen, Wei-Li; Harris, Deshea L; Joyce, Nancy C

2005-11-01

Contact inhibition is an important mechanism for maintaining corneal endothelium in a non-replicative state. Protein tyrosine phosphatases (PTPs) play a role in regulating the integrity of cell-cell contacts, differentiation, and growth. In this study, we aimed to evaluate whether phosphatases are involved in the maintenance of contact-dependent inhibition of proliferation in corneal endothelial cells and to identify candidate PTPs that are expressed in these cells and might be involved in regulation of contact inhibition. Confluent cultures of rat corneal endothelial cells or endothelium in ex vivo corneas were treated with the general phosphatase inhibitor, sodium orthovanadate (SOV). Immunocytochemistry (ICC) evaluated the effect of SOV on cell-cell contacts by staining for ZO-1, and on cell cycle progression by staining for Ki67. Transverse sections of rat cornea and cultured rat corneal endothelial cells were used to test for expression of the candidate PTPs: PTP-mu, PTP-LAR, PTP1B, SHP-1, SHP-2, and PTEN using ICC and either Western blots or RT-PCR. ZO-1 staining demonstrated that SOV induced a time-dependent release of cell-cell contacts in confluent cultures of corneal endothelial cells and in the endothelium of ex vivo corneas. Staining for Ki67 indicated that SOV promoted limited cell cycle progression in the absence of serum. PTP-mu, PTP1B, SHP-1, SHP-2, and PTEN, but not PTP-LAR, were expressed in rat corneal endothelial cells in situ and in culture. The subcellular location of PTP-mu and PTP1B differed in subconfluent and confluent cells, while that of SHP-1, SHP-2, and PTEN was similar, regardless of confluent status. Western blots confirmed the expression of PTP1B, SHP-1, SHP-2, and PTEN. RT-PCR confirmed expression of PTP-mu mRNA. Phosphatases are involved in regulation of junctional integrity and of cell proliferation in corneal endothelial cells. PTP-mu, PTP1B, SHP-1, SHP-2, and PTEN are expressed in rat corneal endothelium and may be involved in

Profiling of kidney vascular endothelial cell plasma membrane proteins by liquid chromatography-tandem mass spectrometry.

Science.gov (United States)

Liu, Zan; Xu, Bo; Nameta, Masaaki; Zhang, Ying; Magdeldin, Sameh; Yoshida, Yutaka; Yamamoto, Keiko; Fujinaka, Hidehiko; Yaoita, Eishin; Tasaki, Masayuki; Nakagawa, Yuki; Saito, Kazuhide; Takahashi, Kota; Yamamoto, Tadashi

2013-06-01

Vascular endothelial cells (VECs) play crucial roles in physiological and pathologic conditions in tissues and organs. Most of these roles are related to VEC plasma membrane proteins. In the kidney, VECs are closely associated with structures and functions; however, plasma membrane proteins in kidney VECs remain to be fully elucidated. Rat kidneys were perfused with cationic colloidal silica nanoparticles (CCSN) to label the VEC plasma membrane. The CCSN-labeled plasma membrane fraction was collected by gradient ultracentrifugation. The VEC plasma membrane or whole-kidney lysate proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and digested with trypsin in gels for liquid chromatography-tandem mass spectrometry. Enrichment analysis was then performed. The VEC plasma membrane proteins were purified by the CCSN method with high yield (approximately 20 μg from 1 g of rat kidney). By Mascot search, 582 proteins were identified in the VEC plasma membrane fraction, and 1,205 proteins were identified in the kidney lysate. In addition to 16 VEC marker proteins such as integrin beta-1 and intercellular adhesion molecule-2 (ICAM-2), 8 novel proteins such as Deltex 3-like protein and phosphatidylinositol binding clathrin assembly protein (PICALM) were identified. As expected, many key functions of plasma membranes in general and of endothelial cells in particular (i.e., leukocyte adhesion) were significantly overrepresented in the proteome of CCSN-labeled kidney VEC fraction. The CCSN method is a reliable technique for isolation of VEC plasma membrane from the kidney, and proteomic analysis followed by bioinformatics revealed the characteristics of in vivo VECs in the kidney.

Telmisartan activates endothelial nitric oxide synthase via Ser1177 phosphorylation in vascular endothelial cells.

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Masahiro Myojo

Full Text Available Because endothelial nitric oxide synthase (eNOS has anti-inflammatory and anti-arteriosclerotic functions, it has been recognized as one of the key molecules essential for the homeostatic control of blood vessels other than relaxation of vascular tone. Here, we examined whether telmisartan modulates eNOS function through its pleiotropic effect. Administration of telmisartan to mice significantly increased the phosphorylation level of eNOS (Ser1177 in the aortic endothelium, but administration of valsartan had no effect. Similarly, telmisartan treatment of human umbilical vein endothelial cells significantly increased the phosphorylation levels of AMP-activated protein kinase (Thr172 and eNOS and the concentration of intracellular guanosine 3',5'-cyclic monophosphate (cGMP. Furthermore, pretreatment with a p38 mitogen-activated protein kinase (p38 MAPK inhibitor suppressed the increased phosphorylation level of eNOS and intracellular cGMP concentration. These data show that telmisartan increases eNOS activity through Ser1177 phosphorylation in vascular endothelial cells mainly via p38 MAPK signaling.

Proline-rich tyrosine kinase 2 (Pyk2) mediates vascular endothelial-cadherin-based cell-cell adhesion by regulating beta-catenin tyrosine phosphorylation

NARCIS (Netherlands)

van Buul, Jaap D.; Anthony, Eloise C.; Fernandez-Borja, Mar; Burridge, Keith; Hordijk, Peter L.

2005-01-01

Vascular endothelial-cadherin (VE-cadherin) controls endothelial cell-cell adhesion and preserves endothelial integrity. In order to maintain endothelial barrier function, VE-cadherin function is tightly regulated through mechanisms that involve protein phosphorylation and cytoskeletal dynamics.

The apelin receptor influences biomechanical and morphological properties of endothelial cells.

Science.gov (United States)

Strohbach, Anne; Pennewitz, Malte; Glaubitz, Michael; Palankar, Raghavendra; Groß, Stefan; Lorenz, Florian; Materzok, Ilka; Rong, Alena; Busch, Mathias C; Felix, Stephan B; Delcea, Mihaela; Busch, Raila

2018-08-01

The adaption of endothelial cells to local flow conditions is a multifunctional process which leads to distinct alterations in cell shape, the subcellular distribution of structural proteins, and cellular function. G-protein-coupled receptors (GPCRs) have been identified to be fundamentally involved in such processes. Recently, we and others have shown that the expression of the endothelial GPCR apelin receptor (APJ) is regulated by fluid flow and that activation of APJ participates in signaling pathways which are related to processes of mechanotransduction. The present study aims to illuminate these findings by further visualization of APJ function. We show that APJ is located to the cellular junctions and might thus be associated with platelet endothelial cell adhesion molecule-1 (PECAM-1) in human umbilical vein endothelial cells (HUVEC). Furthermore, siRNA-mediated silencing of APJ expression influences the shear-induced adaption of HUVEC in terms of cytoskeletal remodeling, cellular elasticity, cellular motility, attachment, and distribution of adhesion complexes. Taken together, our results demonstrate that APJ is crucial for complemented endothelial adaption to local flow conditions. © 2018 Wiley Periodicals, Inc.

Efficient Generation of Human Embryonic Stem Cell-Derived Corneal Endothelial Cells by Directed Differentiation.

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Kathryn L McCabe

Full Text Available To generate human embryonic stem cell derived corneal endothelial cells (hESC-CECs for transplantation in patients with corneal endothelial dystrophies.Feeder-free hESC-CECs were generated by a directed differentiation protocol. hESC-CECs were characterized by morphology, expression of corneal endothelial markers, and microarray analysis of gene expression.hESC-CECs were nearly identical morphologically to primary human corneal endothelial cells, expressed Zona Occludens 1 (ZO-1 and Na+/K+ATPaseα1 (ATPA1 on the apical surface in monolayer culture, and produced the key proteins of Descemet's membrane, Collagen VIIIα1 and VIIIα2 (COL8A1 and 8A2. Quantitative PCR analysis revealed expression of all corneal endothelial pump transcripts. hESC-CECs were 96% similar to primary human adult CECs by microarray analysis.hESC-CECs are morphologically similar, express corneal endothelial cell markers and express a nearly identical complement of genes compared to human adult corneal endothelial cells. hESC-CECs may be a suitable alternative to donor-derived corneal endothelium.

Nitric oxide synthesis-promoting effects of valsartan in human umbilical vein endothelial cells via the Akt/adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway

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Yingshuai Zhao

2017-05-01

Full Text Available Valsartan (VAL, an antagonist of angiotensin II receptor type 1, has antihypertensive and multiple cardiovascular protective effects. The pleiotropic functions of VAL are related to the increased synthesis and biological activity of intravascular nitric oxide (NO. In this study, the role and mechanisms of VAL in the synthesis of NO were examined in human umbilical vein endothelial cells (HUVECs. Ten µmol/L of VAL was used to treat EA.hy926 cells for 30 minutes, 1, 3, 6, 12, and 24 hours, and three concentrations of VAL (i.e., 10, 1, and 0.1 µmol/L were used to treat EA.hy926 cells for 24 hours. The cells were divided into five groups: control, VAL, VAL + Compound C (adenosine monophosphate-activated protein kinase [AMPK] inhibitor, 1 µmol/L, VAL + LY294002 (Akt [protein kinase B] inhibitor, 10 µmol/L, and VAL + L-nitro-arginine methyl ester (L-NAME, endothelial NO synthase [eNOS] inhibitor, 500 µmol/L groups. The NO content in the VAL-treated HUVEC line (EA.hy926 was detected using the nitrate reductase method, and western blot was used to detect the phosphorylation of Akt, AMPK, and eNOS, as well as the changes in total protein levels. VAL increased NO synthesis in EA.hy926 cells in time- and dose-dependent manners (p < 0.05 and the intracellular phosphorylation levels of Akt, AMPK, and eNOS at the corresponding time points. LY294002, Compound C, and L-NAME could inhibit the VAL-promoted NO synthesis. VAL activated Akt, AMPK, and eNOS, thus promoting NO synthesis and playing a protective role in endothelial cells. These results partially explained the mechanisms underlying the cardiovascular protective effects of VAL.

Endothelial-regenerating cells: an expanding universe.

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Steinmetz, Martin; Nickenig, Georg; Werner, Nikos

2010-03-01

Atherosclerosis is the most common cause for cardiovascular diseases and is based on endothelial dysfunction. A growing body of evidence suggests the contribution of bone marrow-derived endothelial progenitor cells, monocytic cells, and mature endothelial cells to vessel formation and endothelial rejuvenation. To this day, various subsets of these endothelial-regenerating cells have been identified according to cellular origin, phenotype, and properties in vivo and in vitro. However, the definition and biology, especially of endothelial progenitor cells, is complex and under heavy debate. In this review, we focus on current definitions of endothelial progenitor cells, highlight the clinical relevance of endothelial-regenerating cells, and provide new insights into cell-cell interactions involved in endothelial cell rejuvenation.

A hot water extract of Curcuma longa inhibits adhesion molecule protein expression and monocyte adhesion to TNF-α-stimulated human endothelial cells.

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Kawasaki, Kengo; Muroyama, Koutarou; Yamamoto, Norio; Murosaki, Shinji

2015-01-01

The recruitment of arterial leukocytes to endothelial cells is an important step in the progression of various inflammatory diseases. Therefore, its modulation is thought to be a prospective target for the prevention or treatment of such diseases. Adhesion molecules on endothelial cells are induced by proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), and contribute to the recruitment of leukocytes. In the present study, we investigated the effect of hot water extract of Curcuma longa (WEC) on the protein expression of adhesion molecules, monocyte adhesion induced by TNF-α in human umbilical vascular endothelial cells (HUVECs). Treatment of HUVECs with WEC significantly suppressed both TNF-α-induced protein expression of adhesion molecules and monocyte adhesion. WEC also suppressed phosphorylation and degradation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) induced by TNF-α in HUVECs, suggesting that WEC inhibits the NF-κB signaling pathway.

2-Deoxy-D-glucose treatment of endothelial cells induces autophagy by reactive oxygen species-mediated activation of the AMP-activated protein kinase.

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Qilong Wang

2011-02-01

Full Text Available Autophagy is a cellular self-digestion process activated in response to stresses such as energy deprivation and oxidative stress. However, the mechanisms by which energy deprivation and oxidative stress trigger autophagy remain undefined. Here, we report that activation of AMP-activated protein kinase (AMPK by mitochondria-derived reactive oxygen species (ROS is required for autophagy in cultured endothelial cells. AMPK activity, ROS levels, and the markers of autophagy were monitored in confluent bovine aortic endothelial cells (BAEC treated with the glycolysis blocker 2-deoxy-D-glucose (2-DG. Treatment of BAEC with 2-DG (5 mM for 24 hours or with low concentrations of H(2O(2 (100 µM induced autophagy, including increased conversion of microtubule-associated protein light chain 3 (LC3-I to LC3-II, accumulation of GFP-tagged LC3 positive intracellular vacuoles, and increased fusion of autophagosomes with lysosomes. 2-DG-treatment also induced AMPK phosphorylation, which was blocked by either co-administration of two potent anti-oxidants (Tempol and N-Acetyl-L-cysteine or overexpression of superoxide dismutase 1 or catalase in BAEC. Further, 2-DG-induced autophagy in BAEC was blocked by overexpressing catalase or siRNA-mediated knockdown of AMPK. Finally, pretreatment of BAEC with 2-DG increased endothelial cell viability after exposure to hypoxic stress. Thus, AMPK is required for ROS-triggered autophagy in endothelial cells, which increases endothelial cell survival in response to cell stress.

Role of protein kinase C in regulation of Na+- and K +-dependent ATPase activity and pump function in corneal endothelial cells.

Science.gov (United States)

Hatou, Shin; Yamada, Masakazu; Mochizuki, Hiroshi; Nishida, Teruo

2009-05-01

Na+- and K+-dependent ATPase (Na,K-ATPase) plays an important role in the pump function of the corneal endothelium. We investigated the possible role of protein kinase C (PKC) in regulation of Na,K-ATPase activity and pump function in corneal endothelial cells. Confluent monolayers of mouse corneal endothelial cells were exposed to phorbol 12,13-dibutyrate (PDBu) to induce activation of PKC. ATPase activity of the cells was evaluated by using ammonium molybdate in spectrophotometric measurement of phosphate released from ATP, with Na,K-ATPase activity being defined as the portion of total ATPase activity sensitive to ouabain. Pump function of the cells was measured with a Ussing chamber, with the pump function attributable to Na,K-ATPase activity being defined as the portion of the total short-circuit current sensitive to ouabain. PDBu (10(-7) M) increased the Na,K-ATPase activity and pump function of the cultured cells. These effects of PDBu were potentiated by the cyclooxygenase inhibitor indomethacin and the cytochrome P(450) inhibitor resorufin and were blocked by okadaic acid, an inhibitor of protein phosphatases 1 and 2A. Our results suggest that PKC bidirectionally regulates Na,K-ATPase activity in mouse corneal endothelial cells: it inhibits Na,K-ATPase activity in a cyclooxygenase- and cytochrome P(450)-dependent manner, whereas it stimulates such activity by activating protein phosphatases 1 or 2A.

Endothelial cell senescence with aging in healthy humans: prevention by habitual exercise and relation to vascular endothelial function.

Science.gov (United States)

Rossman, Matthew J; Kaplon, Rachelle E; Hill, Sierra D; McNamara, Molly N; Santos-Parker, Jessica R; Pierce, Gary L; Seals, Douglas R; Donato, Anthony J

2017-11-01

Cellular senescence is emerging as a key mechanism of age-related vascular endothelial dysfunction, but evidence in healthy humans is lacking. Moreover, the influence of lifestyle factors such as habitual exercise on endothelial cell (EC) senescence is unknown. We tested the hypothesis that EC senescence increases with sedentary, but not physically active, aging and is associated with vascular endothelial dysfunction. Protein expression (quantitative immunofluorescence) of p53, a transcription factor related to increased cellular senescence, and the cyclin-dependent kinase inhibitors p21 and p16 were 116%, 119%, and 128% greater (all P age-related differences were not present (all P > 0.05) in venous ECs from older exercising adults (57 ± 1 yr, n = 13). Furthermore, venous EC protein levels of p53 ( r  = -0.49, P = 0.003), p21 ( r  = -0.38, P = 0.03), and p16 ( r  = -0.58, P = 0.002) were inversely associated with vascular endothelial function (brachial artery flow-mediated dilation). Similarly, protein expression of p53 and p21 was 26% and 23% higher (both P healthy older sedentary (63 ± 1 yr, n = 18) versus young sedentary (25 ± 1 yr, n = 9) adults; age-related changes in arterial EC p53 and p21 expression were not observed ( P > 0.05) in older habitually exercising adults (59 ± 1 yr, n = 14). These data indicate that EC senescence is associated with sedentary aging and is linked to endothelial dysfunction. Moreover, these data suggest that prevention of EC senescence may be one mechanism by which aerobic exercise protects against endothelial dysfunction with age. NEW & NOTEWORTHY Our study provides novel evidence in humans of increased endothelial cell senescence with sedentary aging, which is associated with impaired vascular endothelial function. Furthermore, our data suggest an absence of age-related increases in endothelial cell senescence in older exercising adults, which is linked with preserved vascular endothelial function

The Expression Profiles of Lysophospholipid Receptors (LPLRs in Different Endothelial Cells

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Yu-Wei Lee

2006-03-01

Full Text Available Sphingosine-1-phosphate (S1P and lysophosphatidic acid (LPA are two bioactive lysophospholipids (LPLs, stored primarily in platelets and released during platelet activation. Both LPLs are capable of regulating endothelial cell functions. The physiological functions of S1P and LPA are mediated by interacting with eight different G-protein coupled receptors: S1P1 through 5 and LPA1 through 3, which activate three different heterotrimeric GTP proteins－including Gi、Gq and G(12/13. The expression of LPL receptors in endothelial cells would affect the responses of S1P and LPA to these cells. There is no previous report discussing the expression profiles of LPL receptors in different endothelial cells from various species. In this study, we aim to investigate the expression profiles of S1P and LPA receptors in different endothelial cells isolated from human, rat, mouse and bovine origin. We used RT-PCR to determine LPLs receptors expression profiles in different endothelial cells. Our results indicated that endothelial cells from various species express different LPL receptors. Endothelial cells isolated from the same source of different species also had different LPLs receptors expression profiles. Therefore, different endothelial cells should respond to LPLs in different manners.

In vitro evaluation of the interactions between human corneal endothelial cells and extracellular matrix proteins

International Nuclear Information System (INIS)

Choi, Jin San; Kim, Eun Young; Kim, Min Jeong; Giegengack, Matthew; Khan, Faraaz A; Soker, Shay; Khang, Gilson

2013-01-01

The corneal endothelium is the innermost cell layer of the cornea and rests on Descemet's membrane consisting of various extracellular matrix (ECM) proteins which can directly affect the cellular behaviors such as cell adhesion, proliferation, polarity, morphogenesis and function. The objective of this study was to investigate the interactions between the ECM environment and human corneal endothelial cells (HCECs), with the ultimate goal to improve cell proliferation and function in vitro. To evaluate the interaction of HCECs with ECM proteins, cells were seeded on ECM-coated tissue culture dishes, including collagen type I (COL I), collagen type IV (COL IV), fibronectin (FN), FNC coating mix (FNC) and laminin (LM). Cell adhesion and proliferation of HCECs on each substratum and expression of CEC markers were studied. The results showed that HCECs plated on the COL I, COL IV, FN and FNC-coated plates had enhanced cell adhesion initially; the number for COL I, COL IV, FN and FNC was significantly higher than the control (P < 0.05). In addition, cells grown on ECM protein-coated dishes showed more compact cellular morphology and CEC marker expression compared to cells seeded on uncoated dishes. Collectively, our results suggest that an adequate ECM protein combination can provide a long-term culture environment for HCECs for corneal endothelium transplantation. (paper)

PECAM-1 polymorphism affects monocyte adhesion to endothelial cells.

Science.gov (United States)

Goodman, Reyna S; Kirton, Christopher M; Oostingh, Gertie J; Schön, Michael P; Clark, Michael R; Bradley, J Andrew; Taylor, Craig J

2008-02-15

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) plays an important role in leukocyte-endothelial cell adhesion and transmigration. Single nucleotide polymorphisms of PECAM-1 encoding amino acid substitutions at positions 98 leucine/valine (L/V), 536 serine/asparagine (S/N), and 643 arginine/glycine (R/G) occur in strong genetic linkage resulting in two common haplotypes (LSR and VNG). These PECAM-1 polymorphisms are associated with graft-versus-host disease after hematopoietic stem cell transplantation and with cardiovascular disease, but whether they influence PECAM-1 function is unknown. We examined the effect of homozygous and heterozygous expression of the PECAM-1 LSR and VNG genotypes on the adhesive interactions of peripheral blood monocytes and activated endothelial cell monolayers under shear stress in a flow-based cell adhesion assay. There was no difference in monocyte adhesion between the two homozygous genotypes of PECAM-1 but when monocytes expressed both alleles in heterozygous form, firm adhesion of monocytes to endothelial cells was markedly increased. PECAM-1 polymorphism expressed in homozygous or heterozygous form by endothelial cells did not influence monocyte adhesion. This is, to our knowledge, the first demonstration that PECAM-1 genotype can alter the level of monocyte binding to endothelial cells and a demonstration that heterozygous expression of a polymorphic protein may lead to altered function.

Suppression of matrix protein synthesis in endothelial cells by herpes simplex virus is not dependent on viral protein synthesis

International Nuclear Information System (INIS)

Kefalides, N.A.

1986-01-01

The synthesis of matrix proteins by human endothelial cells (EC) in vitro was studied before and at various times after infection with Herpes Simplex virus Type 1 (HSV-1) or 2 (HSV-2). Monolayers of EC were either mock-infected or infected with virus for 1 hr at a multiplicity infection (MOI) of 5 to 20 at 37 0 C. Control and infected cultures were pulse-labeled for 1 or 2 hrs with either [ 14 C]proline or [ 35 S]methionine. Synthesis of labeled matrix proteins was determined by SDS-gel electrophoresis. Suppression of synthesis of fibronectin, Type IV collagen and thrombospondin began as early as 2 hrs and became almost complete by 10 hrs post-infection. The degree of suppression varied with the protein and the virus dose. Suppression of Type IV collagen occurred first followed by that of fibronectin and then thrombospondin. Infection of EC with UV irradiated HSV-1 or HSV-2 resulted in suppression of host-cell protein synthesis as well as viral protein synthesis. Infection with intact virus in the presence of actinomycin-D resulted in suppression of both host-cell and viral protein synthesis. The data indicate that infection of EC with HSV leads to suppression of matrix protein synthesis which does not depend on viral protein synthesis

Resveratrol induces mitochondrial biogenesis in endothelial cells.

Science.gov (United States)

Csiszar, Anna; Labinskyy, Nazar; Pinto, John T; Ballabh, Praveen; Zhang, Hanrui; Losonczy, Gyorgy; Pearson, Kevin; de Cabo, Rafael; Pacher, Pal; Zhang, Cuihua; Ungvari, Zoltan

2009-07-01

Pathways that regulate mitochondrial biogenesis are potential therapeutic targets for the amelioration of endothelial dysfunction and vascular disease. Resveratrol was shown to impact mitochondrial function in skeletal muscle and the liver, but its role in mitochondrial biogenesis in endothelial cells remains poorly defined. The present study determined whether resveratrol induces mitochondrial biogenesis in cultured human coronary arterial endothelial cells (CAECs). In CAECs resveratrol increased mitochondrial mass and mitochondrial DNA content, upregulated protein expression of electron transport chain constituents, and induced mitochondrial biogenesis factors (proliferator-activated receptor-coactivator-1alpha, nuclear respiratory factor-1, mitochondrial transcription factor A). Sirtuin 1 (SIRT1) was induced, and endothelial nitric oxide (NO) synthase (eNOS) was upregulated in a SIRT1-dependent manner. Knockdown of SIRT1 (small interfering RNA) or inhibition of NO synthesis prevented resveratrol-induced mitochondrial biogenesis. In aortas of type 2 diabetic (db/db) mice impaired mitochondrial biogenesis was normalized by chronic resveratrol treatment, showing the in vivo relevance of our findings. Resveratrol increases mitochondrial content in endothelial cells via activating SIRT1. We propose that SIRT1, via a pathway that involves the upregulation of eNOS, induces mitochondrial biogenesis. Resveratrol induced mitochondrial biogenesis in the aortas of type 2 diabetic mice, suggesting the potential for new treatment approaches targeting endothelial mitochondria in metabolic diseases.

Protein C inhibits endocytosis of thrombin-thrombomodulin complexes in A549 lung cancer cells and human umbilical vein endothelial cells

International Nuclear Information System (INIS)

Maruyama, I.; Majerus, P.W.

1987-01-01

We investigated the effect of protein C on the endocytosis of thrombin-thrombomodulin complexes. We previously showed that exposure of umbilical vein endothelial cells to thrombin stimulated the internalization and degradation of thrombin. A similar internalization was stimulated by a monoclonal antithrombomodulin antibody. We have repeated these studies in the presence of protein C and found that endocytosis of 125 I-thrombin-thrombomodulin complexes, but not 125 I-antithrombomodulin-thrombomodulin complexes, is inhibited. Activated protein C did not inhibit endocytosis of thrombin-thrombomodulin complexes. Protein C inhibited both internalization and degradation of 125 I-thrombin and diisopropylphosphoryl (DIP) 125 I-thrombin in human lung cancer cells (A549). These effects were observed at protein C concentrations found in human plasma. Protein S had no effect on the inhibition of endocytosis of thrombin-thrombomodulin complexes by protein C. We propose that protein C may regulate the rate of endocytosis of thrombin-thrombomodulin complexes in vivo and thereby control the capacity for endothelium to activate protein C

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

Science.gov (United States)

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

2017-07-01

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

Protective effect of atorvastatin on radiation-induced endothelial cell injury

Energy Technology Data Exchange (ETDEWEB)

Xinze, Ran; Huaien, Zheng; Fengchao, Wang; Xi, Ran; Aiping, Wang; Jing, Han; Yanqi, Zhang; Jun, Chen [Institute of Combined Injury, State Key Laboratory of Trauma, Burns and Combined Injury, College of Preventive Medicine, Third Military Medical University, Chongqing (China)

2009-04-15

Objective: To explore the protective effect of atorvastatin on irradiated endothelium and the thrombomodulin (TM) expression. Methods: Cultured human coronary artery endothelial cells (HCAEC) and human umbilical vein endothelial cells (HUVEC) were treated by atorvastatin at the final concentration of 10 {mu}mol/ml for 10 min, and then irradiated with 2 and 25 Gy. Cell cycles status and TM expression were quantitatively measured by flow cytometry 24 hours after irradiation. Protein C activation in endothelial cells was also assessod. Results: After administration with atorvastatin for 24 h, the TM expression increased by 77%, 59% and 61% in normal control group, 2 Gy group and 25 Gy group, respectively (t=27.395, 26.420, 58.065; P=0.000). The protein C levels decreased by 23% and 34% compared with the normal group post-irradiation to 2 and 25 Gy, but increased by 79% and 76% compared with the irradiated control group after administration with atorvastatin. The rates of cell apoptosis decreased by 6% and 16% in 2 Gy and 25 Gy groups, respectively after administration with atorvastatin for 24 h (t=4.178, 17.863; P=0.000). Conclusions: Atorva statin can protect endothelia cell from irradiation-induced apeptosis by increasing TM expression and protein C activation. (authors)

Protective effect of atorvastatin on radiation-induced endothelial cell injury

International Nuclear Information System (INIS)

Ran Xinze; Zheng Huaien; Wang Fengchao; Ran Xi; Wang Aiping; Han Jing; Zhang Yanqi; Chen Jun

2009-01-01

Objective: To explore the protective effect of atorvastatin on irradiated endothelium and the thrombomodulin (TM) expression. Methods: Cultured human coronary artery endothelial cells (HCAEC) and human umbilical vein endothelial cells (HUVEC) were treated by atorvastatin at the final concentration of 10 μmol/ml for 10 min, and then irradiated with 2 and 25 Gy. Cell cycles status and TM expression were quantitatively measured by flow cytometry 24 hours after irradiation. Protein C activation in endothelial cells was also assessod. Results: After administration with atorvastatin for 24 h, the TM expression increased by 77%, 59% and 61% in normal control group, 2 Gy group and 25 Gy group, respectively (t=27.395, 26.420, 58.065; P=0.000). The protein C levels decreased by 23% and 34% compared with the normal group post-irradiation to 2 and 25 Gy, but increased by 79% and 76% compared with the irradiated control group after administration with atorvastatin. The rates of cell apoptosis decreased by 6% and 16% in 2 Gy and 25 Gy groups, respectively after administration with atorvastatin for 24 h (t=4.178, 17.863; P=0.000). Conclusions: Atorva statin can protect endothelia cell from irradiation-induced apeptosis by increasing TM expression and protein C activation. (authors)

Endothelial mechanotransduction proteins and vascular function are altered by dietary sucrose supplementation in healthy young male subjects

DEFF Research Database (Denmark)

Gliemann, Lasse; Rytter, Nicolai; Lindskrog, Mads

2017-01-01

Endothelial mechanotransduction is important for vascular function but alterations and activation of vascular mechanosensory proteins have not been investigated in humans. In endothelial cell culture, simple sugars effectively impair mechanosensor proteins. To study mechanosensor- and vascular...... by ultrasound doppler. A muscle biopsy was obtained from the thigh muscle before and after acute passive leg movement, to asses the protein amount and phosphorylation status of mechanosensory proteins and NADPH oxidase. The sucrose intervention led to a reduced flow response to passive movement (by 17 ± 2...... %) and to 12 watts of active exercise (by 9 ± 1 %), indicating impaired vascular function. Reduced flow response to passive and active exercise was paralleled by a significant upregulation of Platelet endothelial cell adhesion molecule (PECAM-1), endothelial nitric oxide synthase, NADPH oxidase and the Rho...

Calmodulin-Dependent Protein Kinase mediates Hypergravity-Induced Changes in F-Actin Expression by Endothelial Cells

Science.gov (United States)

Love, Felisha D.; Melhado, Caroline; Bosah, Francis; Harris-Hooker, Sandra A.; Sanford, Gary L.

1997-01-01

A number of basic cellular functions, e.g., electrolyte concentration cell growth rate, glucose utilization, bone formation, response to growth stimulation and exocytosis are modified by microgravity or during spaceflight. Studies with intact animal during spaceflights have found lipid accumulations within the lumen of the vasculature and degeneration of the vascular wall. Capillary alterations with extensive endothelial invaginations were also seen. Hemodynamic studies have shown that there is a redistribution of blood from the lower extremities to the upper part of the body; this will alter vascular permeability, resulting in leakage into surrounding tissues. These studies indicate that changes in gravity will affect a number of physiological systems, including the vasculature. However, few studies have addressed the effect of microgravity on vascular cell function and metabolism. A major problem with ground based studies is that achieving a true microgravity hand, environment for prolonged period is not possible. On the other increasing gravity (i.e., hypergravity) is easily achieved. Several researchers have shown that hypergravity will increase the proliferation of several different cell limes (e.g., chick embryo fibroblasts) while decreasing cell motility and slowing liver regeneration following partial hepatectomy. These studies suggest that hypergravity will alter the behavior of most cells. Several investigators have shown that hypergravity affects the expression of the early response genes (c-fos and c-myc) and the activation of several protein kinases (PK's) in cells (10,11). In this study we investigated whether hypergravity alters the expression of f-actin by aortic endothelial cells, and the possible role of protein kinases (calmodulin(II)-dependent and PKA) as mediators of these effects.

Apelin is a novel angiogenic factor in retinal endothelial cells

International Nuclear Information System (INIS)

Kasai, Atsushi; Shintani, Norihito; Oda, Maki; Kakuda, Michiya; Hashimoto, Hitoshi; Matsuda, Toshio; Hinuma, Shuji; Baba, Akemichi

2004-01-01

There has been much focus recently on the possible functions of apelin, an endogenous ligand for the orphan G-protein-coupled receptor APJ, in cardiovascular and central nervous systems. We report a new function of apelin as a novel angiogenic factor in retinal endothelial cells. The retinal endothelial cell line RF/6A highly expressed both apelin and APJ transcripts, while human umbilical venous endothelial cells (HUVECs) only expressed apelin mRNA. In accordance with these observations, apelin at concentrations of 1 pM-1 μM significantly enhanced migration, proliferation, and capillary-like tube formation of RF/6A cells, but not those of HUVECs, whereas VEGF stimulates those parameters of both cell types. In vivo Matrigel plug assay for angiogenesis, the inclusion of 1 nM apelin in the Matrigel resulted in clear capillary-like formations with an increase of hemoglobin content in the plug. This is the first report showing that apelin is an angiogenic factor in retinal endothelial cells

Cell-type specific DNA-protein interactions at the tissue-type plasminogen activator promoter in human endothelial and HeLa cells in vivo and in vitro

NARCIS (Netherlands)

Arts, J.; Herr, I.; Lansink, M.; Angel, P.; Kooistra, T.

1997-01-01

Tissue-type plasminogen activator (t-PA) gene expression in human endothelial cells and HeLa cells is stimulated by the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) at the level of transcription. To study the mechanism of transcriptional regulation, we have characterized a

Calpastatin is regulated by protein never in mitosis gene A interacting-1 (PIN1) in endothelial cells

Energy Technology Data Exchange (ETDEWEB)

Liu, Tongzheng, E-mail: liu.tongzheng@mayo.edu [Division of Oncology Research, Department of Oncology, Mayo Clinic, Rochester, MN 55905 (United States); Schneider, Ryan A., E-mail: schneiderr@findlay.edu [College of Pharmacy, The University of Findlay, Findlay, OH 45840 (United States); Hoyt, Dale G., E-mail: hoyt.27@osu.edu [The Dorothy M. Davis Heart and Lung Research Institute, and the Division of Pharmacology, College of Pharmacy, The Ohio State University, 500 West Twelfth Avenue, Columbus, OH 43210 (United States)

2011-10-28

Highlights: Black-Right-Pointing-Pointer Depletion of PIN1 increases inhibitory effect of calpastatin against calpain in endothelial cells. Black-Right-Pointing-Pointer PIN1 associates with calpastatin. Black-Right-Pointing-Pointer PIN1, but not mutants, reduces the inhibitory activity of calpastatin in vitro. Black-Right-Pointing-Pointer Depletion of calpastatin shows that it is required for PIN1 depletion to reduce calpain activity. -- Abstract: The peptidyl-proline isomerase, protein never in mitosis gene A interacting-1 (PIN1) binds and isomerizes proteins phosphorylated on serine/threonine before a proline. It was previously found that depletion of PIN1 greatly increased induction of cyclooxygenase-2 and inducible nitric oxide synthase by lowering calpain activity in murine aortic endothelial cells (MAEC). Here we investigated the effect of PIN1 on the endogenous inhibitor of heterodimeric {mu}- and m-calpains, calpastatin. MAEC were transduced with small hairpin (sh) RNA to knock down PIN1 (KD) or an inactive Control shRNA. Cells were also treated with non-targeted double stranded small inhibitory RNA (siRNA) or siRNA designed to deplete calpastatin. Despite reducing calpain activity, PIN1 KD did not significantly affect the expression of {mu}- and m-calpains, or calpastatin, compared to Control shRNA. Instead, depletion of PIN1 increased the inhibitory activity of calpastatin. Calpastatin co-immunoprecipitated with endogenous PIN1 and was pulled down with glutathione-S-transferase (GST)-PIN1 fusion protein. Adding GST-PIN1 to KD cell extracts lacking PIN1 reduced calpastatin inhibitory activity. Substrate binding and catalytic domain mutants of PIN1 failed to do so. These results suggest that protein interaction and the proline isomerase functions of PIN1 are required for it to inhibit calpastatin. Furthermore, depletion of calpastatin raised calpain activity and reduced calpain inhibitory activity to similar levels in KD and Control MAEC, indicating that

Calpastatin is regulated by protein never in mitosis gene A interacting-1 (PIN1) in endothelial cells

International Nuclear Information System (INIS)

Liu, Tongzheng; Schneider, Ryan A.; Hoyt, Dale G.

2011-01-01

Highlights: ► Depletion of PIN1 increases inhibitory effect of calpastatin against calpain in endothelial cells. ► PIN1 associates with calpastatin. ► PIN1, but not mutants, reduces the inhibitory activity of calpastatin in vitro. ► Depletion of calpastatin shows that it is required for PIN1 depletion to reduce calpain activity. -- Abstract: The peptidyl-proline isomerase, protein never in mitosis gene A interacting-1 (PIN1) binds and isomerizes proteins phosphorylated on serine/threonine before a proline. It was previously found that depletion of PIN1 greatly increased induction of cyclooxygenase-2 and inducible nitric oxide synthase by lowering calpain activity in murine aortic endothelial cells (MAEC). Here we investigated the effect of PIN1 on the endogenous inhibitor of heterodimeric μ- and m-calpains, calpastatin. MAEC were transduced with small hairpin (sh) RNA to knock down PIN1 (KD) or an inactive Control shRNA. Cells were also treated with non-targeted double stranded small inhibitory RNA (siRNA) or siRNA designed to deplete calpastatin. Despite reducing calpain activity, PIN1 KD did not significantly affect the expression of μ- and m-calpains, or calpastatin, compared to Control shRNA. Instead, depletion of PIN1 increased the inhibitory activity of calpastatin. Calpastatin co-immunoprecipitated with endogenous PIN1 and was pulled down with glutathione-S-transferase (GST)–PIN1 fusion protein. Adding GST–PIN1 to KD cell extracts lacking PIN1 reduced calpastatin inhibitory activity. Substrate binding and catalytic domain mutants of PIN1 failed to do so. These results suggest that protein interaction and the proline isomerase functions of PIN1 are required for it to inhibit calpastatin. Furthermore, depletion of calpastatin raised calpain activity and reduced calpain inhibitory activity to similar levels in KD and Control MAEC, indicating that calpastatin is required for PIN1 depletion to lower calpain activity. Thus, PIN1 apparently restrains

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

DEFF Research Database (Denmark)

Burkhart, Annette; Andresen, Thomas Lars; Aigner, Achim

2017-01-01

, as turning BCECs into recombinant protein factories by transfection could result in protein secretion further into the brain. The present study aims to investigate the possibility of transfecting primary rat brain endothelial cells (RBECs) for recombinant protein synthesis and secretion...... of the neuroprotective protein erythropoietin (EPO). We previously showed that 4% of RBECs with BBB properties can be transfected without disrupting the BBB integrity in vitro, but it can be questioned whether this is sufficient to enable protein secretion at therapeutic levels. The present study examined various......-derived neurotropic factor (BDNF). In conclusion, non-viral gene therapy to RBECs leads to protein secretion and signifies a method for therapeutic proteins to target cells inside the CNS otherwise omitted due to the BBB....

Protein hydrolysate from canned sardine and brewing by-products improves TNF-α-induced inflammation in an intestinal-endothelial co-culture cell model.

Science.gov (United States)

Vieira, Elsa F; Van Camp, John; Ferreira, Isabel M P L V O; Grootaert, Charlotte

2017-07-17

The anti-inflammatory activity of sardine protein hydrolysates (SPH) obtained by hydrolysis with proteases from brewing yeast surplus was ascertained. For this purpose, a digested and desalted SPH fraction with molecular weight lower than 10 kDa was investigated using an endothelial cell line (EA.hy926) as such and in a co-culture model with an intestinal cell line (Caco-2). Effects of SPH <10 kDa on nitric oxide (NO) production, reactive oxygen species (ROS) inhibition and secretion of monocyte chemoattractant protein 1 (MCP-1), vascular endothelial growth factor (VEGF), chemokine IL-8 (IL-8) and intercellular adhesion molecule-1 (ICAM-1) were evaluated in TNF-α-treated and untreated cells. Upon TNF-α treatment, levels of NO, MCP-1, VEGF, IL-8, ICAM-1 and endothelial ROS were significantly increased in both mono- and co-culture models. Treatment with SPH <10 kDa (2.0 mg peptides/mL) significantly decreased all the inflammation markers when compared to TNF-α-treated control. This protective effect was more pronounced in the co-culture model, suggesting that SPH <10 kDa Caco-2 cells metabolites produced in the course of intestinal absorption may provide a more relevant protective effect against endothelial dysfunction. Additionally, indirect cross-talk between two cell types was established, suggesting that SPH <10 kDa may also bind to receptors on the Caco-2 cells, thereby triggering a pathway to secrete the pro-inflammatory compounds. Overall, these in vitro screening results, in which intestinal digestion, absorption and endothelial bioactivity are simulated, show the potential of SPH to be used as a functional food with anti-inflammatory properties.

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.

Plastic compressed collagen as a novel carrier for expanded human corneal endothelial cells for transplantation.

Directory of Open Access Journals (Sweden)

Hannah J Levis

Full Text Available Current treatments for reversible blindness caused by corneal endothelial cell failure involve replacing the failed endothelium with donor tissue using a one donor-one recipient strategy. Due to the increasing pressure of a worldwide donor cornea shortage there has been considerable interest in developing alternative strategies to treat endothelial disorders using expanded cell replacement therapy. Protocols have been developed which allow successful expansion of endothelial cells in vitro but this approach requires a supporting material that would allow easy transfer of cells to the recipient. We describe the first use of plastic compressed collagen as a highly effective, novel carrier for human corneal endothelial cells. A human corneal endothelial cell line and primary human corneal endothelial cells retained their characteristic cobblestone morphology and expression of tight junction protein ZO-1 and pump protein Na+/K+ ATPase α1 after culture on collagen constructs for up to 14 days. Additionally, ultrastructural analysis suggested a well-integrated endothelial layer with tightly opposed cells and apical microvilli. Plastic compressed collagen is a superior biomaterial in terms of its speed and ease of production and its ability to be manipulated in a clinically relevant manner without breakage. This method provides expanded endothelial cells with a substrate that could be suitable for transplantation allowing one donor cornea to potentially treat multiple patients.

Release of endothelial cell lipoprotein lipase by plasma lipoproteins and free fatty acids

International Nuclear Information System (INIS)

Saxena, U.; Witte, L.D.; Goldberg, I.J.

1989-01-01

Lipoprotein lipase (LPL) bound to the lumenal surface of vascular endothelial cells is responsible for the hydrolysis of triglycerides in plasma lipoproteins. Studies were performed to investigate whether human plasma lipoproteins and/or free fatty acids would release LPL which was bound to endothelial cells. Purified bovine milk LPL was incubated with cultured porcine aortic endothelial cells resulting in the association of enzyme activity with the cells. When the cells were then incubated with media containing chylomicrons or very low density lipoproteins (VLDL), a concentration-dependent decrease in the cell-associated LPL enzymatic activity was observed. In contrast, incubation with media containing low density lipoproteins or high density lipoproteins produced a much smaller decrease in the cell-associated enzymatic activity. The addition of increasing molar ratios of oleic acid:bovine serum albumin to the media also reduced enzyme activity associated with the endothelial cells. To determine whether the decrease in LPL activity was due to release of the enzyme from the cells or inactivation of the enzyme, studies were performed utilizing radioiodinated bovine LPL. Radiolabeled LPL protein was released from endothelial cells by chylomicrons, VLDL, and by free fatty acids (i.e. oleic acid bound to bovine serum albumin). The release of radiolabeled LPL by VLDL correlated with the generation of free fatty acids from the hydrolysis of VLDL triglyceride by LPL bound to the cells. Inhibition of LPL enzymatic activity by use of a specific monoclonal antibody, reduced the extent of release of 125 I-LPL from the endothelial cells by the added VLDL. These results demonstrated that LPL enzymatic activity and protein were removed from endothelial cells by triglyceride-rich lipoproteins (chylomicrons and VLDL) and oleic acid

Detection and Quantification of Vascular Endothelial Growth Factor Receptor Tyrosine Kinases in Primary Human Endothelial Cells.

Science.gov (United States)

Fearnley, Gareth W; Wheatcroft, Stephen B; Ponnambalam, Sreenivasan

2015-01-01

Proteins differ widely in their pattern of expression depending on organism, tissue, and regulation in response to changing conditions. In the mammalian vasculature, the endothelium responds to vascular endothelial growth factors (VEGFs) via membrane-bound receptor tyrosine kinases (VEGFRs) to modulate many aspects of vascular physiology including vasculogenesis, angiogenesis, and blood pressure. Studies on VEGFR biology are thus dependent on detecting expression levels in different cell types and evaluating how changes in protein levels correlate with changing conditions including circulating VEGF levels. Here, we present a robust immunoblot-based protocol for detecting and quantifying VEGFRs in human endothelial cells. Using internal and external standards, we can rapidly evaluate receptor copy number and assess how this is altered in response to the cellular environment.

The redox mechanism for vascular barrier dysfunction associated with metabolic disorders: Glutathionylation of Rac1 in endothelial cells.

Science.gov (United States)

Han, Jingyan; Weisbrod, Robert M; Shao, Di; Watanabe, Yosuke; Yin, Xiaoyan; Bachschmid, Markus M; Seta, Francesca; Janssen-Heininger, Yvonne M W; Matsui, Reiko; Zang, Mengwei; Hamburg, Naomi M; Cohen, Richard A

2016-10-01

Oxidative stress is implicated in increased vascular permeability associated with metabolic disorders, but the underlying redox mechanism is poorly defined. S-glutathionylation, a stable adduct of glutathione with protein sulfhydryl, is a reversible oxidative modification of protein and is emerging as an important redox signaling paradigm in cardiovascular physiopathology. The present study determines the role of protein S-glutathionylation in metabolic stress-induced endothelial cell permeability. In endothelial cells isolated from patients with type-2 diabetes mellitus, protein S-glutathionylation level was increased. This change was also observed in aortic endothelium in ApoE deficient (ApoE -/- ) mice fed on Western diet. Metabolic stress-induced protein S-glutathionylation in human aortic endothelial cells (HAEC) was positively correlated with elevated endothelial cell permeability, as reflected by disassembly of cell-cell adherens junctions and cortical actin structures. These impairments were reversed by adenoviral overexpression of a specific de-glutathionylation enzyme, glutaredoxin-1 in cultured HAECs. Consistently, transgenic overexpression of human Glrx-1 in ApoE -/- mice fed the Western diet attenuated endothelial protein S-glutathionylation, actin cytoskeletal disorganization, and vascular permeability in the aorta. Mechanistically, glutathionylation and inactivation of Rac1, a small RhoGPase, were associated with endothelial hyperpermeability caused by metabolic stress. Glutathionylation of Rac1 on cysteine 81 and 157 located adjacent to guanine nucleotide binding site was required for the metabolic stress to inhibit Rac1 activity and promote endothelial hyperpermeability. Glutathionylation and inactivation of Rac1 in endothelial cells represent a novel redox mechanism of vascular barrier dysfunction associated with metabolic disorders. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Hepatitis C virus core protein induces dysfunction of liver sinusoidal endothelial cell by down-regulation of silent information regulator 1.

Science.gov (United States)

Sun, Li-Jie; Yu, Jian-Wu; Shi, Yu-Guang; Zhang, Xiao-Yu; Shu, Meng-Ni; Chen, Mo-Yang

2018-05-01

Hepatic fibrosis is a frequent feature of chronic hepatitis C virus (HCV) infection. Some evidence has suggested the potential role of silent information regulator 1 (SIRT1) in organ fibrosis. The aim of this study was to investigate the effect of HCV core protein on expression of SIRT1 of liver sinusoidal endothelial cell (LSEC) and function of LSEC. LSECs were co-cultured with HepG2 cells or HepG2 cells expressing HCV core protein and LSECs cultured alone were used as controls. After co-culture, the activity and expression levels of mRNA and protein of SIRT1 in LSEC were detected by a SIRT1 fluorometric assay kit, real time-PCR (RT-PCR), Western blot, respectively. The levels of adiponectin receptor 2 (AdipoR2), endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) were measured by Western blot. Cluster of differentiation 31 (CD31), CD14, and von Willebrand factor (vWf) of LSECs was performed by flow cytometry. The level of reactive oxygen species (ROS) was assayed. Malondialdehyde (MDA), superoxide dismutase (SOD), adiponectin, nitric oxide (NO), and endothelin-1 (ET-1) levels in the co-culture supernatant were measured. The co-culture supernatant was then used to cultivate LX-2 cells. The levels of α-smooth muscle actin (ASMA) and transforming growth factor-β1 (TGF-β1) protein in LX-2 cells were measured by Western blot. Compared with LSEC co-cultured with HepG2 cells group, in LSEC co-cultured with HepG2-core cells group, the activity and expression level of mRNA and protein of SIRT1 reduced; the level of adiponectin reduced and the expression level of AdipoR2 protein decreased; ROS levels increased; the expression level of eNOS, VEGF protein decreased; and the expression level of CD14 decreased; the expression level of vWf and CD31 increased; NO and SOD levels decreased; whereas ET-1 and MDA levels increased; the levels of ASMA and TGF-β1 protein in LX-2 cells increased. SIRT1 activator improved the above-mentioned changes

Vascular endothelial growth factor A-stimulated signaling from endosomes in primary endothelial cells.

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Fearnley, Gareth W; Smith, Gina A; Odell, Adam F; Latham, Antony M; Wheatcroft, Stephen B; Harrison, Michael A; Tomlinson, Darren C; Ponnambalam, Sreenivasan

2014-01-01

The vascular endothelial growth factor A (VEGF-A) is a multifunctional cytokine that stimulates blood vessel sprouting, vascular repair, and regeneration. VEGF-A binds to VEGF receptor tyrosine kinases (VEGFRs) and stimulates intracellular signaling leading to changes in vascular physiology. An important aspect of this phenomenon is the spatiotemporal coordination of VEGFR trafficking and intracellular signaling to ensure that VEGFR residence in different organelles is linked to downstream cellular outputs. Here, we describe a series of assays to evaluate the effects of VEGF-A-stimulated intracellular signaling from intracellular compartments such as the endosome-lysosome system. These assays include the initial isolation and characterization of primary human endothelial cells, performing reverse genetics for analyzing protein function; methods used to study receptor trafficking, signaling, and proteolysis; and assays used to measure changes in cell migration, proliferation, and tubulogenesis. Each of these assays has been exemplified with studies performed in our laboratories. In conclusion, we describe necessary techniques for studying the role of VEGF-A in endothelial cell function. © 2014 Elsevier Inc. All rights reserved.

CCM proteins control endothelial β1 integrin dependent response to shear stress

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Zuzana Macek Jilkova

2014-11-01

Full Text Available Hemodynamic shear stress from blood flow on the endothelium critically regulates vascular function in many physiological and pathological situations. Endothelial cells adapt to shear stress by remodeling their cytoskeletal components and subsequently by changing their shape and orientation. We demonstrate that β1 integrin activation is critically controlled during the mechanoresponse of endothelial cells to shear stress. Indeed, we show that overexpression of the CCM complex, an inhibitor of β1 integrin activation, blocks endothelial actin rearrangement and cell reorientation in response to shear stress similarly to β1 integrin silencing. Conversely, depletion of CCM2 protein leads to an elongated “shear-stress-like” phenotype even in the absence of flow. Taken together, our findings reveal the existence of a balance between positive extracellular and negative intracellular signals, i.e. shear stress and CCM complex, for the control of β1 integrin activation and subsequent adaptation of vascular endothelial cells to mechanostimulation by fluid shear stress.

A small population of liver endothelial cells undergoes endothelial-to-mesenchymal transition in response to chronic liver injury.

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Ribera, Jordi; Pauta, Montse; Melgar-Lesmes, Pedro; Córdoba, Bernat; Bosch, Anna; Calvo, Maria; Rodrigo-Torres, Daniel; Sancho-Bru, Pau; Mira, Aurea; Jiménez, Wladimiro; Morales-Ruiz, Manuel

2017-11-01

Rising evidence points to endothelial-to-mesenchymal transition (EndMT) as a significant source of the mesenchymal cell population in fibrotic diseases. In this context, we hypothesized that liver endothelial cells undergo EndMT during fibrosis progression. Cirrhosis in mice was induced by CCl 4 A transgenic mouse expressing a red fluorescent protein reporter under the control of Tie2 promoter (Tie2-tdTomato) was used to trace the acquisition of EndMT. Sinusoidal vascular connectivity was evaluated by intravital microscopy and high-resolution three-dimensional confocal microscopy. A modest but significant fraction of liver endothelial cells from both cirrhotic patients and CCl 4 -treated Tie2-tdTomato mice acquired an EndMT phenotype characterized by the coexpression of CD31 and α-smooth muscle actin, compared with noncirrhotic livers. Bone morphogenetic protein-7 (BMP-7) inhibited the acquisition of EndMT induced by transforming growth factor-β1 (TGF-β1) treatment in cultured primary mouse liver endothelial cells from control mice. EndMT was also reduced significantly in vivo in cirrhotic Tie2-tdTomato mice treated intraperitoneally with BMP-7 compared with untreated mice (1.9 ± 0.2 vs. 3.8 ± 0.3%, respectively; P livers correlated with a significant decrease in liver fibrosis ( P livers in both animal models and patients. BMP-7 treatment decreases the occurrence of the EndMT phenotype and has a positive impact on the severity of disease by reducing fibrosis and sinusoidal vascular disorganization. NEW & NOTEWORTHY A subpopulation of liver endothelial cells from cirrhotic patients and mice with liver fibrosis undergoes endothelial-to-mesenchymal transition. Liver endothelial cells from healthy mice could transition into a mesenchymal phenotype in culture in response to TGF-β1 treatment. Fibrotic livers treated chronically with BMP-7 showed lower EndMT acquisition, reduced fibrosis, and improved vascular organization. Copyright © 2017 the American

The effect of ionizing radiation on the filamentous actin of vascular endothelial cell

International Nuclear Information System (INIS)

Yao Xiaowu; Chen Shisheng; Yang Lihe; Lin Juelong; Yang Haiwei

2006-01-01

Objective: To observe the ionizing radiation effect on filamentous actin of vascular endothelial cell and explore its mechanism. Methods: The vascular endothelial cells were irradiated with 0, 2, 4, 6, 8, 10 and 12 Gy 60 Co γ-rays. The cytoskeleton was observed with CLSM at 6 hs after the irradiation and the cytoskeleton protein F-actin detected with flow cytometry after 12 and 24 hs. Results: The damage to cytoskeletons increased with the radiation dose. The cytoskeleton protein F-actin was significantly decreased at 12 hs after the irradiation, and then recovered after 24 hs. Conclusion: Ionizing radiation caused vascular endothelial cell injury by damaging the cytoskeleton and depolymerizating the F-actin. (authors)

A Cell Culture Platform to Maintain Long-term Phenotype of Primary Human Hepatocytes and Endothelial Cells.

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Ware, Brenton R; Durham, Mitchell J; Monckton, Chase P; Khetani, Salman R

2018-03-01

Modeling interactions between primary human hepatocytes (PHHs) and primary human liver sinusoidal endothelial cells (LSECs) in vitro can help elucidate human-specific mechanisms underlying liver physiology/disease and drug responses; however, existing hepatocyte/endothelial coculture models are suboptimal because of their use of rodent cells, cancerous cell lines, and/or nonliver endothelial cells. Hence, we sought to develop a platform that could maintain the long-term phenotype of PHHs and primary human LSECs. Primary human LSECs or human umbilical vein endothelial cells as the nonliver control were cocultivated with micropatterned PHH colonies (to control homotypic interactions) followed by an assessment of PHH morphology and functions (albumin and urea secretion, and cytochrome P-450 2A6 and 3A4 enzyme activities) over 3 weeks. Endothelial phenotype was assessed via gene expression patterns and scanning electron microscopy to visualize fenestrations. Hepatic responses in PHH/endothelial cocultures were benchmarked against responses in previously developed PHH/3T3-J2 fibroblast cocultures. Finally, PHH/fibroblast/endothelial cell tricultures were created and characterized as described previously. LSECs, but not human umbilical vein endothelial cells, induced PHH albumin secretion for ∼11 days; however, neither endothelial cell type could maintain PHH morphology and functions to the same magnitude/longevity as the fibroblasts. In contrast, both PHHs and endothelial cells displayed stable phenotype for 3 weeks in PHH/fibroblast/endothelial cell tricultures; furthermore, layered tricultures in which PHHs and endothelial cells were separated by a protein gel to mimic the space of Disse displayed similar functional levels as the coplanar tricultures. PHH/fibroblast/endothelial tricultures constitute a robust platform to elucidate reciprocal interactions between PHHs and endothelial cells in physiology, disease, and after drug exposure.

Protein Kinases Possibly Mediate Hypergravity-Induced Changes in F-Actin Expression by Endothelial Cells

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Love, Felisha D.; Melhado, Caroline D.; Bosah, Francis N.; Harris-Hooker, Sandra A.; Sanford, Gary L.

1998-01-01

Basic cellular functions such as electrolyte concentration, cell growth rate, glucose utilization, bone formation, response to growth stimulation, and exocytosis are modified in microgravity. These studies indicate that microgravity affects a number of physiological systems and included in this are cell signaling mechanisms. Rijken and coworkers performed growth factor studies that showed PKC signaling and actin microfilament organization appears to be sensitive to microgravity, suggesting that the inhibition of signal transduction by microgravity may be related to alterations in actin microfilament organization. However, similar studies have not been done for vascular cells. Vascular endothelial cells play critical roles in providing nutrients to organ and tissues and in wound repair. The major deterrent to ground-based microgravity studies is that it is impossible to achieved true microgravity for longer than a few minutes on earth. Hence, it has not been possible to conduct prolonged microgravity studies except for two models that simulate certain aspects of microgravity. However, hypergravity is quite easily achieved. Several researchers have shown that hypergravity will increase the proliferation of several different cell lines while decreasing cell motility and slowing liver regeneration following partial hepatectomy, These studies indicate the hypergravity also alters the behavior of most cells. Several investigators have shown that hypergravity affects the activation of several protein kinases (PKs) in cells. In this study, we investigated whether hypergravity alters the expression of f-actin by bovine aortic endothelial cells (BAECs) and the role of PK's (calmodulin 11 dependent, PKA and PKC) as mediators of these effects.

Human alpha-enolase from endothelial cells as a target antigen of anti-endothelial cell antibody in Behçet's disease.

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Lee, Kwang Hoon; Chung, Hae-Shin; Kim, Hyoung Sup; Oh, Sang-Ho; Ha, Moon-Kyung; Baik, Ja-Hyun; Lee, Sungnack; Bang, Dongsik

2003-07-01

To identify and recombine a protein of the human dermal microvascular endothelial cell (HDMEC) that specifically reacts with anti-endothelial cell antibody (AECA) in the serum of patients with Behçet's disease (BD), and to evaluate the usefulness of this protein in BD. The proteomics technique, with 2-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry, was used to identify and recombine HDMEC antigen. Western blotting and enzyme-linked immunosorbent assay (ELISA) of recombinant protein isolated by gene cloning were performed on serum from healthy controls, patients with BD, and patients with other rheumatic diseases (rheumatoid arthritis, systemic lupus erythematosus, and Wegener's granulomatosis). Eighteen of 40 BD patients had serum IgM antibody to HDMEC antigen. The purified protein that reacted with AECA in BD patient sera was found to be alpha-enolase by 2-dimensional gel electrophoresis followed by immunoblotting and MALDI-TOF mass spectrometry. Recombinant alpha-enolase protein was isolated and refined by gene cloning. On Western blots, AECA-positive IgM from the sera of patients with active BD reacted strongly with recombinant human alpha-enolase. BD patient sera positive for anti-alpha-enolase did not react with human gamma-enolase. On dot-blotting, reactivity to human alpha-enolase was detected only in the IgM-positive group. Fifteen of the 18 AECA-positive sera that were positive for the HDMEC antigen showed reactivity to recombinant alpha-enolase IgM antibody by ELISA. The alpha-enolase protein is the target protein of serum AECA in BD patients. This is the first report of the presence of IgM antibodies to alpha-enolase in endothelial cells from the serum of BD patients. Although further studies relating this protein to the pathogenesis of BD will be necessary, alpha-enolase and its antibody may prove useful in the development of new diagnostic and treatment modalities in BD.

26S Proteasome regulation of Ankrd1/CARP in adult rat ventricular myocytes and human microvascular endothelial cells

International Nuclear Information System (INIS)

Samaras, Susan E.; Chen, Billy; Koch, Stephen R.; Sawyer, Douglas B.; Lim, Chee Chew; Davidson, Jeffrey M.

2012-01-01

Highlights: ► The 26S proteasome regulates Ankrd1 levels in cardiomyocytes and endothelial cells. ► Ankrd1 protein degrades 60-fold faster in endothelial cells than cardiomyocytes. ► Differential degradation appears related to nuclear vs. sarcolemmal localization. ► Endothelial cell density shows uncoupling of Ankrd1 mRNA and protein levels. -- Abstract: Ankyrin repeat domain 1 protein (Ankrd1), also known as cardiac ankyrin repeat protein (CARP), increases dramatically after tissue injury, and its overexpression improves aspects of wound healing. Reports that Ankrd1/CARP protein stability may affect cardiovascular organization, together with our findings that the protein is crucial to stability of the cardiomyocyte sarcomere and increased in wound healing, led us to compare the contribution of Ankrd1/CARP stability to its abundance. We found that the 26S proteasome is the dominant regulator of Ankrd1/CARP degradation, and that Ankrd1/CARP half-life is significantly longer in cardiomyocytes (h) than endothelial cells (min). In addition, higher endothelial cell density decreased the abundance of the protein without affecting steady state mRNA levels. Taken together, our data and that of others indicate that Ankrd1/CARP is highly regulated at multiple levels of its expression. The striking difference in protein half-life between a muscle and a non-muscle cell type suggests that post-translational proteolysis is correlated with the predominantly structural versus regulatory role of the protein in the two cell types.

Rab GTPases Regulate Endothelial Cell Protein C Receptor-Mediated Endocytosis and Trafficking of Factor VIIa

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Nayak, Ramesh C.; Keshava, Shiva; Esmon, Charles T.; Pendurthi, Usha R.; Rao, L. Vijaya Mohan

2013-01-01

Recent studies have established that factor VIIa (FVIIa) binds to the endothelial cell protein C receptor (EPCR). FVIIa binding to EPCR may promote the endocytosis of this receptor/ligand complex. Rab GTPases are known to play a crucial role in the endocytic and exocytic pathways of receptors or receptor/ligand complexes. The present study was undertaken to investigate the role of Rab GTPases in the intracellular trafficking of EPCR and FVIIa. CHO-EPCR cells and human umbilical vein endothelial cells (HUVEC) were transduced with recombinant adenoviral vectors to express wild-type, constitutively active, or dominant negative mutant of various Rab GTPases. Cells were exposed to FVIIa conjugated with AF488 fluorescent probe (AF488-FVIIa), and intracellular trafficking of FVIIa, EPCR, and Rab proteins was evaluated by immunofluorescence confocal microscopy. In cells expressing wild-type or constitutively active Rab4A, internalized AF488-FVIIa accumulated in early/sorting endosomes and its entry into the recycling endosomal compartment (REC) was inhibited. Expression of constitutively active Rab5A induced large endosomal structures beneath the plasma membrane where EPCR and FVIIa accumulated. Dominant negative Rab5A inhibited the endocytosis of EPCR-FVIIa. Expression of constitutively active Rab11 resulted in retention of accumulated AF488-FVIIa in the REC, whereas expression of a dominant negative form of Rab11 led to accumulation of internalized FVIIa in the cytoplasm and prevented entry of internalized FVIIa into the REC. Expression of dominant negative Rab11 also inhibited the transport of FVIIa across the endothelium. Overall our data show that Rab GTPases regulate the internalization and intracellular trafficking of EPCR-FVIIa. PMID:23555015

Effect of tributyltin on mammalian endothelial cell integrity.

Science.gov (United States)

Botelho, G; Bernardini, C; Zannoni, A; Ventrella, V; Bacci, M L; Forni, M

2015-01-01

Tributyltin (TBT), is a man-made pollutants, known to accumulate along the food chain, acting as an endocrine disruptor in marine organisms, with toxic and adverse effects in many tissues including vascular system. Based on the absence of specific studies of TBT effects on endothelial cells, we aimed to evaluate the toxicity of TBT on primary culture of porcine aortic endothelial cells (pAECs), pig being an excellent model to study human cardiovascular disease. pAECs were exposed for 24h to TBT (100, 250, 500, 750 and 1000nM) showing a dose dependent decrease in cell viability through both apoptosis and necrosis. Moreover the ability of TBT (100 and 500nM) to influence endothelial gene expression was investigated at 1, 7 and 15h of treatment. Gene expression of tight junction molecules, occludin (OCLN) and tight junction protein-1 (ZO-1) was reduced while monocyte adhesion and adhesion molecules ICAM-1 and VCAM-1 (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1) levels increased significantly at 1h. IL-6 and estrogen receptors 1 and 2 (ESR-1 and ESR-2) mRNAs, after a transient decrease, reached the maximum levels after 15h of exposure. Finally, we demonstrated that TBT altered endothelial functionality greatly increasing monocyte adhesion. These findings indicate that TBT deeply alters endothelial profile, disrupting their structure and interfering with their ability to interact with molecules and other cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Cyclosporine Induces Endothelial Cell Release of Complement-Activating Microparticles

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Renner, Brandon; Klawitter, Jelena; Goldberg, Ryan; McCullough, James W.; Ferreira, Viviana P.; Cooper, James E.; Christians, Uwe

2013-01-01

Defective control of the alternative pathway of complement is an important risk factor for several renal diseases, including atypical hemolytic uremic syndrome. Infections, drugs, pregnancy, and hemodynamic insults can trigger episodes of atypical hemolytic uremic syndrome in susceptible patients. Although the mechanisms linking these clinical events with disease flares are unknown, recent work has revealed that each of these clinical conditions causes cells to release microparticles. We hypothesized that microparticles released from injured endothelial cells promote intrarenal complement activation. Calcineurin inhibitors cause vascular and renal injury and can trigger hemolytic uremic syndrome. Here, we show that endothelial cells exposed to cyclosporine in vitro and in vivo release microparticles that activate the alternative pathway of complement. Cyclosporine-induced microparticles caused injury to bystander endothelial cells and are associated with complement-mediated injury of the kidneys and vasculature in cyclosporine-treated mice. Cyclosporine-induced microparticles did not bind factor H, an alternative pathway regulatory protein present in plasma, explaining their complement-activating phenotype. Finally, we found that in renal transplant patients, the number of endothelial microparticles in plasma increases 2 weeks after starting tacrolimus, and treatment with tacrolimus associated with increased C3 deposition on endothelial microparticles in the plasma of some patients. These results suggest that injury-associated release of endothelial microparticles is an important mechanism by which systemic insults trigger intravascular complement activation and complement-dependent renal diseases. PMID:24092930

Protein Profiling of Isolated Leukocytes, Myofibroblasts, Epithelial, Basal, and Endothelial Cells from Normal, Hyperplastic, Cancerous, and Inflammatory Human Prostate Tissues

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Zahraa I. Khamis, Kenneth A. Iczkowski, Ziad J. Sahab, Qing-Xiang Amy Sang

2010-01-01

Full Text Available In situ neoplastic prostate cells are not lethal unless they become invasive and metastatic. For cells to become invasive, the prostate gland must undergo degradation of the basement membrane and disruption of the basal cell layer underneath the luminal epithelia. Although the roles of proteinases in breaking down the basement membrane have been well-studied, little is known about the factors that induce basal cell layer disruption, degeneration, and its eventual disappearance in invasive cancer. It is hypothesized that microenvironmental factors may affect the degradation of the basal cell layer, which if protected may prevent tumor progression and invasion. In this study, we have revealed differential protein expression patterns between epithelial and stromal cells isolated from different prostate pathologies and identified several important epithelial and stromal proteins that may contribute to inflammation and malignant transformation of human benign prostate tissues to cancerous tissues using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and proteomics methods. Cellular retinoic acid-binding protein 2 was downregulated in basal cells of benign prsotate. Caspase-1 and interleukin-18 receptor 1 were highly expressed in leukocytes of prostate cancer. Proto-oncogene Wnt-3 was downregulated in endothelial cells of prostatitis tissue and tyrosine phosphatase non receptor type 1 was only found in normal and benign endothelial cells. Poly ADP-ribose polymerase 14 was downregulated in myofibroblasts of prostatitis tissue. Interestingly, integrin alpha-6 was upregulated in epithelial cells but not detected in myofibroblasts of prostate cancer. Further validation of these proteins may generate new strategies for the prevention of basal cell layer disruption and subsequent cancer invasion.

The tyrosine phosphatase SHP-1 regulates hypoxia inducible factor-1α (HIF-1α protein levels in endothelial cells under hypoxia.

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Stefan K Alig

Full Text Available The tyrosine phosphatase SHP-1 negatively influences endothelial function, such as VEGF signaling and reactive oxygen species (ROS formation, and has been shown to influence angiogenesis during tissue ischemia. In ischemic tissues, hypoxia induced angiogenesis is crucial for restoring oxygen supply. However, the exact mechanism how SHP-1 affects endothelial function during ischemia or hypoxia remains unclear. We performed in vitro endothelial cell culture experiments to characterize the role of SHP-1 during hypoxia.SHP-1 knock-down by specific antisense oligodesoxynucleotides (AS-Odn increased cell growth as well as VEGF synthesis and secretion during 24 hours of hypoxia compared to control AS-Odn. This was prevented by HIF-1α inhibition (echinomycin and apigenin. SHP-1 knock-down as well as overexpression of a catalytically inactive SHP-1 (SHP-1 CS further enhanced HIF-1α protein levels, whereas overexpression of a constitutively active SHP-1 (SHP-1 E74A resulted in decreased HIF-1α levels during hypoxia, compared to wildtype SHP-1. Proteasome inhibition (MG132 returned HIF-1α levels to control or wildtype levels respectively in these cells. SHP-1 silencing did not alter HIF-1α mRNA levels. Finally, under hypoxic conditions SHP-1 knock-down enhanced intracellular endothelial reactive oxygen species (ROS formation, as measured by oxidation of H2-DCF and DHE fluorescence.SHP-1 decreases half-life of HIF-1α under hypoxic conditions resulting in decreased cell growth due to diminished VEGF synthesis and secretion. The regulatory effect of SHP-1 on HIF-1α stability may be mediated by inhibition of endothelial ROS formation stabilizing HIF-1α protein. These findings highlight the importance of SHP-1 in hypoxic signaling and its potential as therapeutic target in ischemic diseases.

Advanced glycation end products impair function of late endothelial progenitor cells through effects on protein kinase Akt and cyclooxygenase-2

International Nuclear Information System (INIS)

Chen Qin; Dong Li; Wang Lian; Kang Lina; Xu Biao

2009-01-01

Endothelial progenitor cells (EPCs) exhibit impaired function in the context of diabetes, and advanced glycation end products (AGEs), which accumulate in diabetes, may contribute to this. In the present study, we investigated the mechanism by which AGEs impair late EPC function. EPCs from human umbilical cord blood were isolated, and incubated with AGE-modified albumin (AGE-albumin) at different concentrations found physiologically in plasma. Apoptosis, migration, and tube formation assays were used to evaluate EPC function including capacity for vasculogenesis, and expression of the receptor for AGEs (RAGE), Akt, endothelial nitric oxide synthase (eNOS), and cycloxygenase-2 (COX-2) were determined. Anti-RAGE antibody was used to block RAGE function. AGE-albumin concentration-dependently enhanced apoptosis and depressed migration and tube formation, but did not affect proliferation, of late EPCs. High AGE-albumin increased RAGE mRNA and protein expression, and decreased Akt and COX-2 protein expression, whilst having no effect on eNOS mRNA or protein in these cells. These effects were inhibited by co-incubation with anti-RAGE antibody. These results suggest that RAGE mediates the AGE-induced impairment of late EPC function, through down-regulation of Akt and COX-2 in these cells.

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

Science.gov (United States)

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

2004-01-01

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

Low-Intensity Pulsed Ultrasound Prevents the Oxidative Stress Induced Endothelial-Mesenchymal Transition in Human Aortic Endothelial Cells

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Jiamin Li

2018-02-01

Full Text Available Background/Aims: Endothelial-mesenchymal transition (EndMT has been shown to take part in the generation and progression of diverse diseases, involving a series of changes leading to a loss of their endothelial characteristics and an acquirement of properties typical of mesenchymal cells. Low-intensity pulsed ultrasound (LIPUS is a new therapeutic option that has been successfully used in fracture healing. However, whether LIPUS can inhibit oxidative stress-induced endothelial cell damages through inhibiting EndMT remained unknown. This study aimed to investigate the protective effects of LIPUS against oxidative stress-induced endothelial cell damages and the underlying mechanisms. Methods: EndMT was induced by H2O2 (100 µm for seven days. Human aortic endothelial cells (HAECs were exposed to H2O2 with or without LIPUS treatment for seven days. The expression of EndMT markers (CD31, VE-cadherin, FSP1 and α-SMA were analyzed. The levels of total and phosphorylated PI3K and AKT proteins were detected by Western Blot analysis. Cell chemotaxis was determined by wound healing and transwell assay. Results: LIPUS relieved EndMT by decreasing ROS accumulation and increasing activation of the PI3K signaling cascade. LIPUS alleviated the migration of EndMT-derived mesenchymal-like cells through reducing extracellular matrix (ECM deposition that is associated with matrix metallopeptidase (MMP proteolytic activity and collagen production. Conclusion: LIPUS produces cytoprotective effects against oxidative injuries to endothelial cells through suppressing the oxidative stress-induced EndMT, activating the PI3K/AKT pathway under oxidative stress, and limiting cell migration and excessive ECM deposition.

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

Mechanotransduction in Endothelial Cells Studied with Fluorescence Imaging

Energy Technology Data Exchange (ETDEWEB)

Chien Shu [Departments of Bioengineering and Medicine and Institute of Engineering in Medicine, University of California, San Diego, La Jolla, California 92093-0427 (United States)

2011-01-01

Mechanotransduction involves the conversion of mechanical stimuli to intracellular signaling to modulate gene and protein expressions and hence cellular functions in endothelial cells, thus playing importance roles in the regulation of homeostasis in health and disease. The aim of this paper is to investigate the dynamics of mechanotransduction in endothelial cells by the use of fluorescent resonance energy transfer (FRET) to study the temporal and spatial activation of Src kinase and focal adhesion kinase, both of which play critical roles in many cellular processes. The results have contributed to the elucidation of the roles of these two important signaling molecules and their interactions in mediating mechanotransduction.

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.

Nanofiber density determines endothelial cell behavior on hydrogel matrix

International Nuclear Information System (INIS)

Berti, Fernanda V.; Rambo, Carlos R.; Dias, Paulo F.; Porto, Luismar M.

2013-01-01

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

Flow-induced endothelial cell alignment requires the RhoGEF Trio as a scaffold protein to polarize active Rac1 distribution.

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Kroon, Jeffrey; Heemskerk, Niels; Kalsbeek, Martin J T; de Waard, Vivian; van Rijssel, Jos; van Buul, Jaap D

2017-07-01

Endothelial cells line the lumen of the vessel wall and are exposed to flow. In linear parts of the vessel, the endothelial cells experience laminar flow, resulting in endothelial cell alignment in the direction of flow, thereby protecting the vessel wall from inflammation and permeability. In order for endothelial cells to align, they undergo rapid remodeling of the actin cytoskeleton by local activation of the small GTPase Rac1. However, it is not clear whether sustained and local activation of Rac1 is required for long-term flow-induced cell alignment. Using a FRET-based DORA Rac1 biosensor, we show that local Rac1 activity remains for 12 h upon long-term flow. Silencing studies show that the RhoGEF Trio is crucial for keeping active Rac1 at the downstream side of the cell and, as a result, for long-term flow-induced cell alignment. Surprisingly, Trio appears to be not involved in flow-induced activation of Rac1. Our data show that flow induces Rac1 activity at the downstream side of the cell in a Trio-dependent manner and that Trio functions as a scaffold protein rather than a functional GEF under long-term flow conditions. © 2017 Kroon et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Ectopic expression of the calcium-binding protein parvalbumin in mouse liver endothelial cells

DEFF Research Database (Denmark)

Castillo, M B; Berchtold, M W; Rülicke, T

1997-01-01

To elucidate the physiological role of the Ca2+ binding protein parvalbumin, we have generated transgenic mice carrying the full-length complementary DNA (cDNA) of rat parvalbumin under the control of the heavy-metal inducible metallothionein IIA promoter. Immunohistochemical and biochemical...... methods have been used to detect the presence of ectopic parvalbumin expression in different tissues. Here we show the expression of parvalbumin in endothelial cells lining the liver sinusoids in situ and after isolation in vitro. The hemodynamic effects of endothelin 1, a peptide hormone mediating potent...... vasoconstriction via calcium signalling, were investigated in the mouse liver perfused in situ. Vasoconstriction, thought to be mediated by the Ito cell, was not affected in the transgenic animals, whereas microvascular exchange, probed with the multiple indicator dilution technique, was markedly decreased...

DC8 and DC13 var genes associated with severe malaria bind avidly to diverse endothelial cells.

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Marion Avril

Full Text Available During blood stage infection, Plasmodium falciparum infected erythrocytes (IE bind to host blood vessels. This virulence determinant enables parasites to evade spleen-dependent killing mechanisms, but paradoxically in some cases may reduce parasite fitness by killing the host. Adhesion of infected erythrocytes is mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1, a family of polymorphic adhesion proteins encoded by var genes. Whereas cerebral binding and severe malaria are associated with parasites expressing DC8 and DC13 var genes, relatively little is known about the non-brain endothelial selection on severe malaria adhesive types. In this study, we selected P. falciparum-IEs on diverse endothelial cell types and demonstrate that DC8 and DC13 var genes were consistently among the major var transcripts selected on non-brain endothelial cells (lung, heart, bone marrow. To investigate the molecular basis for this avid endothelial binding activity, recombinant proteins were expressed from the predominant upregulated DC8 transcript, IT4var19. In-depth binding comparisons revealed that multiple extracellular domains from this protein bound brain and non-brain endothelial cells, and individual domains largely did not discriminate between different endothelial cell types. Additionally, we found that recombinant DC8 and DC13 CIDR1 domains exhibited a widespread endothelial binding activity and could compete for DC8-IE binding to brain endothelial cells, suggesting they may bind the same host receptor. Our findings provide new insights into the interaction of severe malaria adhesive types and host blood vessels and support the hypothesis that parasites causing severe malaria express PfEMP1 variants with a superior ability to adhere to diverse endothelial cell types, and may therefore endow these parasites with a growth and transmission advantage.

Endogenous Vascular Endothelial Growth Factor-A (VEGF-A) Maintains Endothelial Cell Homeostasis by Regulating VEGF Receptor-2 Transcription*

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E, Guangqi; Cao, Ying; Bhattacharya, Santanu; Dutta, Shamit; Wang, Enfeng; Mukhopadhyay, Debabrata

2012-01-01

Vascular endothelial growth factor A (VEGF-A) is one of the most important factors controlling angiogenesis. Although the functions of exogenous VEGF-A have been widely studied, the roles of endogenous VEGF-A remain unclear. Here we focused on the mechanistic functions of endogenous VEGF-A in endothelial cells. We found that it is complexed with VEGF receptor 2 (VEGFR-2) and maintains a basal expression level for VEGFR-2 and its downstream signaling activation. Endogenous VEGF-A also controls expression of key endothelial specific genes including VEGFR-2, Tie-2, and vascular endothelial cadherin. Of importance, endogenous VEGF-A differs from exogenous VEGF-A by regulating VEGFR-2 transcription through mediation of FoxC2 binding to the FOX:ETS motif, and the complex formed by endogenous VEGF-A with VEGFR-2 is localized within the EEA1 (early endosome antigen 1) endosomal compartment. Taken together, our results emphasize the importance of endogenous VEGF-A in endothelial cells by regulating key vascular proteins and maintaining the endothelial homeostasis. PMID:22167188

The Effect of a p38 Mitogen-Activated Protein Kinase Inhibitor on Cellular Senescence of Cultivated Human Corneal Endothelial Cells.

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Hongo, Akane; Okumura, Naoki; Nakahara, Makiko; Kay, EunDuck P; Koizumi, Noriko

2017-07-01

We have begun a clinical trial of a cell-based therapy for corneal endothelial dysfunction in Japan. The purpose of this study was to investigate the usefulness of a p38 MAPK inhibitor for prevention cellular senescence in cultivated human corneal endothelial cells (HCECs). HCECs of 10 donor corneas were divided and cultured with or without SB203580 (a p38 MAPK inhibitor). Cell density and morphology were evaluated by phase-contrast microscopy. Expression of function-related proteins was examined by immunofluorescent staining. Cellular senescence was evaluated by SA-β-gal staining and Western blotting for p16 and p21. Senescence-associated factors were evaluated by membrane blotting array, quantitative PCR, and ELISA. Phase-contrast microscopy showed a significantly higher cell density for HCECs cultured with SB203580 than without SB203580 (2623 ± 657 cells/mm2 and 1752 ± 628 cells/mm2, respectively). The HCECs cultured with SB203580 maintained a hexagonal morphology and expressed ZO-1, N-cadherin, and Na+/K+-ATPase in the plasma membrane, whereas the control HCECs showed an altered staining pattern for these marker proteins. HCECs cultured without SB203580 showed high positive SA-β-gal staining, a low nuclear/cytoplasm ratio, and expression of p16 and p21. IL-6, IL-8, CCL2, and CXCL1 were observed at high levels in low cell density HCECs cultured without SB203580. Activation of p38 MAPK signaling due to culture stress might be a causative factor that induces cellular senescence; therefore, the use of p38 MAPK inhibitor to counteract senescence may achieve sufficient numbers of HCECs for tissue engineering therapy for corneal endothelial dysfunction.

ITE inhibits growth of human pulmonary artery endothelial cells.

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Pang, Ling-Pin; Li, Yan; Zou, Qing-Yun; Zhou, Chi; Lei, Wei; Zheng, Jing; Huang, Shi-An

2017-10-01

Pulmonary arterial hypertension (PAH), a deadly disorder is associated with excessive growth of human pulmonary artery endothelial (HPAECs) and smooth muscle (HPASMCs) cells. Current therapies primarily aim at promoting vasodilation, which only ameliorates clinical symptoms without a cure. 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) is an endogenous aryl hydrocarbon receptor (AhR) ligand, and mediates many cellular function including cell growth. However, the roles of ITE in human lung endothelial cells remain elusive. Herein, we tested a hypothesis that ITE inhibits growth of human pulmonary artery endothelial cells via AhR. Immunohistochemistry was performed to localize AhR expression in human lung tissues. The crystal violet method and MTT assay were used to determine ITE's effects on growth of HPAECs. The AhR activation in HPAECs was confirmed using Western blotting and RT-qPCR. The role of AhR in ITE-affected proliferation of HPAECs was assessed using siRNA knockdown method followed by the crystal violet method. Immunohistochemistry revealed that AhR was present in human lung tissues, primarily in endothelial and smooth muscle cells of pulmonary veins and arteries, as well as in bronchial and alveolar sac epithelia. We also found that ITE dose- and time-dependently inhibited proliferation of HPAECs with a maximum inhibition of 83% at 20 µM after 6 days of treatment. ITE rapidly decreased AhR protein levels, while it increased mRNA levels of cytochrome P450 (CYP), family 1, member A1 (CYP1A1) and B1 (CYP1B1), indicating activation of the AhR/CYP1A1 and AhR/CYP1B1 pathways in HPAECs. The AhR siRNA significantly suppressed AhR protein expression, whereas it did not significantly alter ITE-inhibited growth of HPAECs. ITE suppresses growth of HPAECs independent of AhR, suggesting that ITE may play an important role in preventing excessive growth of lung endothelial cells.

Particulate matter air pollution disrupts endothelial cell barrier via calpain-mediated tight junction protein degradation

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Wang Ting

2012-08-01

Full Text Available Abstract Background Exposure to particulate matter (PM is a significant risk factor for increased cardiopulmonary morbidity and mortality. The mechanism of PM-mediated pathophysiology remains unknown. However, PM is proinflammatory to the endothelium and increases vascular permeability in vitro and in vivo via ROS generation. Objectives We explored the role of tight junction proteins as targets for PM-induced loss of lung endothelial cell (EC barrier integrity and enhanced cardiopulmonary dysfunction. Methods Changes in human lung EC monolayer permeability were assessed by Transendothelial Electrical Resistance (TER in response to PM challenge (collected from Ft. McHenry Tunnel, Baltimore, MD, particle size >0.1 μm. Biochemical assessment of ROS generation and Ca2+ mobilization were also measured. Results PM exposure induced tight junction protein Zona occludens-1 (ZO-1 relocation from the cell periphery, which was accompanied by significant reductions in ZO-1 protein levels but not in adherens junction proteins (VE-cadherin and β-catenin. N-acetyl-cysteine (NAC, 5 mM reduced PM-induced ROS generation in ECs, which further prevented TER decreases and atteneuated ZO-1 degradation. PM also mediated intracellular calcium mobilization via the transient receptor potential cation channel M2 (TRPM2, in a ROS-dependent manner with subsequent activation of the Ca2+-dependent protease calpain. PM-activated calpain is responsible for ZO-1 degradation and EC barrier disruption. Overexpression of ZO-1 attenuated PM-induced endothelial barrier disruption and vascular hyperpermeability in vivo and in vitro. Conclusions These results demonstrate that PM induces marked increases in vascular permeability via ROS-mediated calcium leakage via activated TRPM2, and via ZO-1 degradation by activated calpain. These findings support a novel mechanism for PM-induced lung damage and adverse cardiovascular outcomes.

Coordinated Molecular Cross-Talk between Staphylococcus aureus, Endothelial Cells and Platelets in Bloodstream Infection

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Carolina D. Garciarena

2015-12-01

Full Text Available Staphylococcus aureus is an opportunistic pathogen often carried asymptomatically on the human body. Upon entry to the otherwise sterile environment of the cardiovascular system, S. aureus can lead to serious complications resulting in organ failure and death. The success of S. aureus as a pathogen in the bloodstream is due to its ability to express a wide array of cell wall proteins on its surface that recognise host receptors, extracellular matrix proteins and plasma proteins. Endothelial cells and platelets are important cells in the cardiovascular system and are a major target of bloodstream infection. Endothelial cells form the inner lining of a blood vessel and provide an antithrombotic barrier between the vessel wall and blood. Platelets on the other hand travel throughout the cardiovascular system and respond by aggregating around the site of injury and initiating clot formation. Activation of either of these cells leads to functional dysregulation in the cardiovascular system. In this review, we will illustrate how S. aureus establish intimate interactions with both endothelial cells and platelets leading to cardiovascular dysregulation.

Suppression of complement regulatory protein C1 inhibitor in vascular endothelial activation by inhibiting vascular cell adhesion molecule-1 action

International Nuclear Information System (INIS)

Zhang, Haimou; Qin, Gangjian; Liang, Gang; Li, Jinan; Chiu, Isaac; Barrington, Robert A.; Liu, Dongxu

2007-01-01

Increased expression of adhesion molecules by activated endothelium is a critical feature of vascular inflammation associated with the several diseases such as endotoxin shock and sepsis/septic shock. Our data demonstrated complement regulatory protein C1 inhibitor (C1INH) prevents endothelial cell injury. We hypothesized that C1INH has the ability of an anti-endothelial activation associated with suppression of expression of adhesion molecule(s). C1INH blocked leukocyte adhesion to endothelial cell monolayer in both static assay and flow conditions. In inflammatory condition, C1INH reduced vascular cell adhesion molecule (VCAM-1) expression associated with its cytoplasmic mRNA destabilization and nuclear transcription level. Studies exploring the underlying mechanism of C1INH-mediated suppression in VCAM-1 expression were related to reduction of NF-κB activation and nuclear translocation in an IκBα-dependent manner. The inhibitory effects were associated with reduction of inhibitor IκB kinase activity and stabilization of the NF-κB inhibitor IκB. These findings indicate a novel role for C1INH in inhibition of vascular endothelial activation. These observations could provide the basis for new therapeutic application of C1INH to target inflammatory processes in different pathologic situations

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

International Nuclear Information System (INIS)

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

2007-01-01

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

Astrocytes Can Adopt Endothelial Cell Fates in a p53-Dependent Manner.

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Brumm, Andrew J; Nunez, Stefanie; Doroudchi, Mehdi M; Kawaguchi, Riki; Duan, Jinhzu; Pellegrini, Matteo; Lam, Larry; Carmichael, S Thomas; Deb, Arjun; Hinman, Jason D

2017-08-01

Astrocytes respond to a variety of CNS injuries by cellular enlargement, process outgrowth, and upregulation of extracellular matrix proteins that function to prevent expansion of the injured region. This astrocytic response, though critical to the acute injury response, results in the formation of a glial scar that inhibits neural repair. Scar-forming cells (fibroblasts) in the heart can undergo mesenchymal-endothelial transition into endothelial cell fates following cardiac injury in a process dependent on p53 that can be modulated to augment cardiac repair. Here, we sought to determine whether astrocytes, as the primary scar-forming cell of the CNS, are able to undergo a similar cellular phenotypic transition and adopt endothelial cell fates. Serum deprivation of differentiated astrocytes resulted in a change in cellular morphology and upregulation of endothelial cell marker genes. In a tube formation assay, serum-deprived astrocytes showed a substantial increase in vessel-like morphology that was comparable to human umbilical vein endothelial cells and dependent on p53. RNA sequencing of serum-deprived astrocytes demonstrated an expression profile that mimicked an endothelial rather than astrocyte transcriptome and identified p53 and angiogenic pathways as specifically upregulated. Inhibition of p53 with genetic or pharmacologic strategies inhibited astrocyte-endothelial transition. Astrocyte-endothelial cell transition could also be modulated by miR-194, a microRNA downstream of p53 that affects expression of genes regulating angiogenesis. Together, these studies demonstrate that differentiated astrocytes retain a stimulus-dependent mechanism for cellular transition into an endothelial phenotype that may modulate formation of the glial scar and promote injury-induced angiogenesis.

Akt/FOXO3a signaling modulates the endothelial stress response through regulation of heat shock protein 70 expression.

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Kim, Hyo-Soo; Skurk, Carsten; Maatz, Henrike; Shiojima, Ichiro; Ivashchenko, Yuri; Yoon, Suk-Won; Park, Young-Bae; Walsh, Kenneth

2005-06-01

To identify new antiapoptotic targets of the PI3K-Akt signaling pathway in endothelial cells, adenovirus-mediated Akt1 gene transfer and oligonucleotide microarrays were used to examine Akt-regulated transcripts. DNA microarray analysis revealed that HSP70 expression underwent the greatest fold activation of 12,532 transcripts examined in human umbilical vein endothelial cells (HUVEC) transduced with constitutively active Akt1. Akt1 gene transfer increased HSP70 transcript expression by 24.8-fold as determined by quantitative PCR and promoted a dose-dependent up-regulation of HSP70 protein as determined by Western immunoblot analysis. Gene transfer of FOXO3a, a downstream target of Akt in endothelial cells, significantly suppressed both basal and stress-induced HSP70 protein expression. FOXO3a induced caspase-9-dependent apoptosis in HUVEC, and cotransduction with Ad-HSP70 rescued endothelial cells from FOXO3a-induced apoptosis under basal and stress conditions. Our results identify HSP70 as a new antiapoptotic target of Akt-FOXO3a signaling in endothelial cells that controls viability through modulation of the stress-induced intrinsic cell death pathway.

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.

Chemical functionalization of bioceramics to enhance endothelial cells adhesion for tissue engineering.

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Borcard, Françoise; Staedler, Davide; Comas, Horacio; Juillerat, Franziska Krauss; Sturzenegger, Philip N; Heuberger, Roman; Gonzenbach, Urs T; Juillerat-Jeanneret, Lucienne; Gerber-Lemaire, Sandrine

2012-09-27

To control the selective adhesion of human endothelial cells and human serum proteins to bioceramics of different compositions, a multifunctional ligand containing a cyclic arginine-glycine-aspartate (RGD) peptide, a tetraethylene glycol spacer, and a gallate moiety was designed, synthesized, and characterized. The binding of this ligand to alumina-based, hydroxyapatite-based, and calcium phosphate-based bioceramics was demonstrated. The conjugation of this ligand to the bioceramics induced a decrease in the nonselective and integrin-selective binding of human serum proteins, whereas the binding and adhesion of human endothelial cells was enhanced, dependent on the particular bioceramics.

Protein kinase A-alpha directly phosphorylates FoxO1 in vascular endothelial cells to regulate expression of vascular cellular adhesion molecule-1 mRNA.

Science.gov (United States)

Lee, Ji-Won; Chen, Hui; Pullikotil, Philomena; Quon, Michael J

2011-02-25

FoxO1, a forkhead box O class transcription factor, is abundant in insulin-responsive tissues. Akt, downstream from phosphatidylinositol 3-kinase in insulin signaling, phosphorylates FoxO1 at Thr(24), Ser(256), and Ser(319), negatively regulating its function. We previously reported that dehydroepiandrosterone-stimulated phosphorylation of FoxO1 in endothelial cells requires cAMP-dependent protein kinase α (PKA-α). Therefore, we hypothesized that FoxO1 is a novel direct substrate for PKA-α. Using an immune complex kinase assay with [γ-(32)P]ATP, purified PKA-α directly phosphorylated wild-type FoxO1 but not FoxO1-AAA (mutant with alanine substitutions at known Akt phosphorylation sites). Phosphorylation of wild-type FoxO1 (but not FoxO1-AAA) was detectable using phospho-specific antibodies. Similar results were obtained using purified GST-FoxO1 protein as the substrate. Thus, FoxO1 is a direct substrate for PKA-α in vitro. In bovine aortic endothelial cells, interaction between endogenous PKA-α and endogenous FoxO1 was detected by co-immunoprecipitation. In human aortic endothelial cells (HAEC), pretreatment with H89 (PKA inhibitor) or siRNA knockdown of PKA-α decreased forskolin- or prostaglandin E(2)-stimulated phosphorylation of FoxO1. In HAEC transfected with a FoxO-promoter luciferase reporter, co-expression of the catalytic domain of PKA-α, catalytically inactive mutant PKA-α, or siRNA against PKA-α caused corresponding increases or decreases in transactivation of the FoxO promoter. Expression of vascular cellular adhesion molecule-1 mRNA, up-regulated by FoxO1 in endothelial cells, was enhanced by siRNA knockdown of PKA-α or treatment of HAEC with the PKA inhibitor H89. Adhesion of monocytes to endothelial cells was enhanced by H89 treatment or overexpression of FoxO1-AAA, similar to effects of TNF-α treatment. We conclude that FoxO1 is a novel physiological substrate for PKA-α in vascular endothelial cells.

Endothelial cell oxidative stress and signal transduction

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ROCIO FONCEA

2000-01-01

Full Text Available Endothelial dysfunction (ED is an early event in atherosclerotic disease, preceding clinical manifestations and complications. Increased reactive oxygen species (ROS have been implicated as important mechanisms that contribute to ED, and ROS’s may function as intracellular messengers that modulate signaling pathways. Several intracellular signal events stimulated by ROS have been defined, including the identification of two members of the mitogen activated protein kinase family (ERK1/2 and big MAP kinase, BMK1, tyrosine kinases (Src and Syk and different isoenzymes of PKC as redox-sensitive kinases. ROS regulation of signal transduction components include the modification in the activity of transcriptional factors such as NFkB and others that result in changes in gene expression and modifications in cellular responses. In order to understand the intracellular mechanisms induced by ROS in endothelial cells (EC, we are studying the response of human umbilical cord vein endothelial cells to increased ROS generation by different pro-atherogenic stimuli. Our results show that Homocysteine (Hcy and oxidized LDL (oxLDL enhance the activity and expression of oxidative stress markers, such as NFkB and heme oxygenase 1. These results suggest that these pro-atherogenic stimuli increase oxidative stress in EC, and thus explain the loss of endothelial function associated with the atherogenic process

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

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

2015-12-01

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

Matrin 3 as a key regulator of endothelial cell survival

International Nuclear Information System (INIS)

Przygodzka, Patrycja; Boncela, Joanna; Cierniewski, Czeslaw S.

2011-01-01

Matrin 3 is an integral component of nuclear matrix architecture that has been implicated in interacting with other nuclear proteins and thus modulating the activity of proximal promoters. In this study, we evaluated the contribution of this protein to proliferation of endothelial cells. To selectively modulate matrin 3 expression, we used siRNA oligonucleotides and transfection of cells with a pEGFP-N1-Mtr3. Our data indicate that downregulation of matrin 3 is responsible for reduced proliferation and leads to necrosis of endothelial cells. This conclusion is supported by observations that reducing matrin 3 expression results in (a) producing signs of necrosis detected by PI staining, LDH release, and scatter parameters in flow cytometry, (b) affecting cell cycle progression. It does not cause (c) membrane asymmetry of cells as indicated by lack of Annexin V binding as well as (d) activation of caspase 3 and cleavage of PARP. We conclude that matrin 3 plays a significant role in controlling cell growth and proliferation, probably via formation of complexes with nuclear proteins that modulate pro- and antiapoptotic signaling pathways. Thus, degradation of matrin 3 may be a switching event that induces a shift from apoptotic to necrotic death of cells.

Endothelial MMP14 is required for endothelial-dependent growth support of human airway basal cells

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Ding, Bi-Sen; Gomi, Kazunori; Rafii, Shahin; Crystal, Ronald G.; Walters, Matthew S.

2015-01-01

ABSTRACT Human airway basal cells are the stem (or progenitor) population of the airway epithelium, and play a central role in anchoring the epithelium to the basement membrane. The anatomic position of basal cells allows for potential paracrine signaling between them and the underlying non-epithelial stromal cells. In support of this, we have previously demonstrated that endothelial cells support growth of basal cells during co-culture through vascular endothelial growth factor A (VEGFA)-mediated signaling. Building on these findings, we found, by RNA sequencing analysis, that basal cells expressed multiple fibroblast growth factor (FGF) ligands (FGF2, FGF5, FGF11 and FGF13) and that only FGF2 and FGF5 were capable of functioning in a paracrine manner to activate classical FGF receptor (FGFR) signaling. Antibody-mediated blocking of FGFR1 during basal-cell–endothelial-cell co-culture significantly reduced the endothelial-cell-dependent basal cell growth. Stimulation of endothelial cells with basal-cell-derived growth factors induced endothelial cell expression of matrix metallopeptidase 14 (MMP14), and short hairpin RNA (shRNA)-mediated knockdown of endothelial cell MMP14 significantly reduced the endothelial-cell-dependent growth of basal cells. Overall, these data characterize a new growth-factor-mediated reciprocal ‘crosstalk’ between human airway basal cells and endothelial cells that regulates proliferation of basal cells. PMID:26116571

In vivo endothelization of tubular vascular grafts through in situ recruitment of endothelial and endothelial progenitor cells by RGD-fused mussel adhesive proteins

International Nuclear Information System (INIS)

Kang, Tae-Yun; Lee, Jung Ho; Kang, Jo-A; Rhie, Jong-Won; Kim, Bum Jin; Cha, Hyung Joon; Hong, Jung Min; Kim, Byoung Soo; Cho, Dong-Woo

2015-01-01

The use of tissue mimics in vivo, including patterned vascular networks, is expected to facilitate the regeneration of functional tissues and organs with large volumes. Maintaining patency of channels in contact with blood is an important issue in the development of a functional vascular network. Endothelium is the only known completely non-thrombogenic material; however, results from treatments to induce endothelialization are inconclusive. The present study was designed to evaluate the clinical applicability of in situ recruitment of endothelial cells/endothelial progenitor cells (EC/EPC) and pre-endothelization using a recombinant mussel adhesive protein fused with arginine–glycine–aspartic acid peptide (MAP-RGD) coating in a model of vascular graft implantation. Microporous polycaprolactone (PCL) scaffolds were fabricated with salt leaching methods and their surfaces were modified with collagen and MAP-RGD. We then evaluated their anti-thrombogenicity with an in vitro hemocompatibility assessment and a 4-week implantation in the rabbit carotid artery. We observed that MAP-RGD coating reduced the possibility of early in vivo graft failure and enhanced re-endothelization by in situ recruitment of EC/EPC (patency rate: 2/3), while endothelization prior to implantation aggravated the formation of thrombosis and/or IH (patency rate: 0/3). The results demonstrated that in situ recruitment of EC/EPC by MAP-RGD could be a promising strategy for vascular applications. In addition, it rules out several issues associated with pre-endothelization, such as cell source, purity, functional modulation and contamination. Further evaluation of long term performance and angiogenesis from the luminal surface may lead to the clinical use of MAP-RGD for tubular vascular grafts and regeneration of large-volume tissues with functional vascular networks. (paper)

Effect of serum proteins on polystyrene nanoparticle uptake and intracellular trafficking in endothelial cells

International Nuclear Information System (INIS)

Guarnieri, Daniela; Guaccio, Angela; Fusco, Sabato; Netti, Paolo A.

2011-01-01

The physico-chemical properties of nanoparticles (NPs), such as small dimensions, surface charge and surface functionalization, control their capability to interact with cells and, in particular, with sub-cellular components. This interaction can be also influenced by the adsorption of molecules present in biological fluids, like blood, on NP surface. Here, we analysed the effect of serum proteins on 49 and 100 nm red fluorescent polystyrene NP uptake in porcine aortic endothelial (PAE) cells, as a model for vascular transport. To this aim, NP uptake kinetic, endocytic pathway and intracellular trafficking were studied by monitoring NPs inside cells through confocal microscopy and multiple particle tracking (MPT). We demonstrated that NPs are rapidly internalized by cells in serum-free (SF) medium, according to a saturation kinetic. Conversely, in 10% foetal bovine serum-enriched (SE) medium, NP uptake rate results drastically reduced. Moreover, NP internalization depends on an active endocytic mechanism that does not involve clathrin- and caveolae-mediated vesicular transport, in both SE and SF media. Furthermore, MPT data indicate that NP intracellular trafficking is unaffected by protein presence. Indeed, approximately 50–60% of internalized NPs is characterized by a sub-diffusive behaviour, whereas the remaining fraction shows an active motion. These findings demonstrate that the unspecific protein adsorption on NP surface can affect cellular uptake in terms of internalization kinetics, but it is not effective in controlling active and cellular-mediated uptake mechanisms of NPs and their intracellular routes.

N-Isopropylacrylamide-co-glycidylmethacrylate as a Thermoresponsive Substrate for Corneal Endothelial Cell Sheet Engineering

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Bernadette K. Madathil

2014-01-01

Full Text Available Endothelial keratoplasty is a recent shift in the surgical treatment of corneal endothelial dystrophies, where the dysfunctional endothelium is replaced whilst retaining the unaffected corneal layers. To overcome the limitation of donor corneal shortage, alternative use of tissue engineered constructs is being researched. Tissue constructs with intact extracellular matrix are generated using stimuli responsive polymers. In this study we evaluated the feasibility of using the thermoresponsive poly(N-isopropylacrylamide-co-glycidylmethacrylate polymer as a culture surface to harvest viable corneal endothelial cell sheets. Incubation below the lower critical solution temperature of the polymer allowed the detachment of the intact endothelial cell sheet. Phase contrast and scanning electron microscopy revealed the intact architecture, cobble stone morphology, and cell-to-cell contact in the retrieved cell sheet. Strong extracellular matrix deposition was also observed. The RT-PCR analysis confirmed functionally active endothelial cells in the cell sheet as evidenced by the positive expression of aquaporin 1, collagen IV, Na+-K+ ATPase, and FLK-1. Na+-K+ ATPase protein expression was also visualized by immunofluorescence staining. These results suggest that the in-house developed thermoresponsive culture dish is a suitable substrate for the generation of intact corneal endothelial cell sheet towards transplantation for endothelial keratoplasty.

Restoration of autophagy in endothelial cells from patients with diabetes mellitus improves nitric oxide signaling.

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Fetterman, Jessica L; Holbrook, Monica; Flint, Nir; Feng, Bihua; Bretón-Romero, Rosa; Linder, Erika A; Berk, Brittany D; Duess, Mai-Ann; Farb, Melissa G; Gokce, Noyan; Shirihai, Orian S; Hamburg, Naomi M; Vita, Joseph A

2016-04-01

Endothelial dysfunction contributes to cardiovascular disease in diabetes mellitus. Autophagy is a multistep mechanism for the removal of damaged proteins and organelles from the cell. Under diabetic conditions, inadequate autophagy promotes cellular dysfunction and insulin resistance in non-vascular tissue. We hypothesized that impaired autophagy contributes to endothelial dysfunction in diabetes mellitus. We measured autophagy markers and endothelial nitric oxide synthase (eNOS) activation in freshly isolated endothelial cells from diabetic subjects (n = 45) and non-diabetic controls (n = 41). p62 levels were higher in cells from diabetics (34.2 ± 3.6 vs. 20.0 ± 1.6, P = 0.001), indicating reduced autophagic flux. Bafilomycin inhibited insulin-induced activation of eNOS (64.7 ± 22% to -47.8 ± 8%, P = 0.04) in cells from controls, confirming that intact autophagy is necessary for eNOS signaling. In endothelial cells from diabetics, activation of autophagy with spermidine restored eNOS activation, suggesting that impaired autophagy contributes to endothelial dysfunction (P = 0.01). Indicators of autophagy initiation including the number of LC3-bound puncta and beclin 1 expression were similar in diabetics and controls, whereas an autophagy terminal phase indicator, the lysosomal protein Lamp2a, was higher in diabetics. In endothelial cells under diabetic conditions, the beneficial effect of spermidine on eNOS activation was blocked by autophagy inhibitors bafilomycin or 3-methyladenine. Blocking the terminal stage of autophagy with bafilomycin increased p62 (P = 0.01) in cells from diabetics to a lesser extent than in cells from controls (P = 0.04), suggesting ongoing, but inadequate autophagic clearance. Inadequate autophagy contributes to endothelial dysfunction in patients with diabetes and may be a target for therapy of diabetic vascular disease. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

The coffee diterpene kahweol inhibits tumor necrosis factor-α-induced expression of cell adhesion molecules in human endothelial cells

International Nuclear Information System (INIS)

Kim, Hyung Gyun; Kim, Ji Young; Hwang, Yong Pil; Lee, Kyung Jin; Lee, Kwang Youl; Kim, Dong Hee; Kim, Dong Hyun; Jeong, Hye Gwang

2006-01-01

Endothelial cells produce adhesion molecules after being stimulated with various inflammatory cytokines. These adhesion molecules play an important role in the development of atherogenesis. Recent studies have highlighted the chemoprotective and anti-inflammatory effects of kahweol, a coffee-specific diterpene. This study examined the effects of kahweol on the cytokine-induced monocyte/human endothelial cell interaction, which is a crucial early event in atherogenesis. Kahweol inhibited the adhesion of TNFα-induced monocytes to endothelial cells and suppressed the TNFα-induced protein and mRNA expression of the cell adhesion molecules, VCAM-1 and ICAM-1. Furthermore, kahweol inhibited the TNFα-induced JAK2-PI3K/Akt-NF-κB activation pathway in these cells. Overall, kahweol has anti-inflammatory and anti-atherosclerotic activities, which occurs partly by down-regulating the pathway that affects the expression and interaction of the cell adhesion molecules on endothelial cells

Targeting NCK-Mediated Endothelial Cell Front-Rear Polarity Inhibits Neovascularization.

Science.gov (United States)

Dubrac, Alexandre; Genet, Gael; Ola, Roxana; Zhang, Feng; Pibouin-Fragner, Laurence; Han, Jinah; Zhang, Jiasheng; Thomas, Jean-Léon; Chedotal, Alain; Schwartz, Martin A; Eichmann, Anne

2016-01-26

Sprouting angiogenesis is a key process driving blood vessel growth in ischemic tissues and an important drug target in a number of diseases, including wet macular degeneration and wound healing. Endothelial cells forming the sprout must develop front-rear polarity to allow sprout extension. The adaptor proteins Nck1 and 2 are known regulators of cytoskeletal dynamics and polarity, but their function in angiogenesis is poorly understood. Here, we show that the Nck adaptors are required for endothelial cell front-rear polarity and migration downstream of the angiogenic growth factors VEGF-A and Slit2. Mice carrying inducible, endothelial-specific Nck1/2 deletions fail to develop front-rear polarized vessel sprouts and exhibit severe angiogenesis defects in the postnatal retina and during embryonic development. Inactivation of NCK1 and 2 inhibits polarity by preventing Cdc42 and Pak2 activation by VEGF-A and Slit2. Mechanistically, NCK binding to ROBO1 is required for both Slit2- and VEGF-induced front-rear polarity. Selective inhibition of polarized endothelial cell migration by targeting Nck1/2 prevents hypersprouting induced by Notch or Bmp signaling inhibition, and pathological ocular neovascularization and wound healing, as well. These data reveal a novel signal integration mechanism involving NCK1/2, ROBO1/2, and VEGFR2 that controls endothelial cell front-rear polarity during sprouting angiogenesis. © 2015 American Heart Association, Inc.

Hyaluronan protection of corneal endothelial cells against extracellular histones after phacoemulsification.

Science.gov (United States)

Kawano, Hiroki; Sakamoto, Taiji; Ito, Takashi; Miyata, Kazunori; Hashiguchi, Teruto; Maruyama, Ikuro

2014-11-01

To determine the effect of histones on corneal endothelial cells generated during cataract surgery. Kagoshima University Hospital, Kagoshima, Japan. Experimental study. Standard phacoemulsification was performed on enucleated pig eyes. Histones in the anterior segment of the eye were determined by immunohistochemistry. Cultured human corneal endothelial cells were exposed to histones for 18 hours, and cell viability was determined by 2-(2-methoxy-4-nitrophenyl)-3-(4-nitro-phenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt assay. The concentration of interleukin-6 (IL-6) in the culture medium of human corneal endothelial cells was measured using enzyme-linked immunosorbent assay. The effects of signal inhibitors U0126, SB203580, and SP600125 were evaluated. The protective effect of hyaluronan against histones was evaluated in human corneal endothelial cells with and without hyaluronan. Cellular debris containing histones was observed in the anterior chamber of pig eyes after phacoemulsification. Exposure of human corneal endothelial cells to 50 μg/mL of histones or more led to cytotoxic effects. The IL-6 concentration was significantly increased dose dependently after exposure of human corneal endothelial cells to histones (Phistone-induced IL-6 production was significantly decreased by extracellular signal-regulated kinases 1/2 and p-38 mitogen-activated protein kinase inhibitors (Phistones caused formation of histone aggregates, decreased the cytotoxic effects of the histones, and blocked the increase in IL-6 (PHistones were released extracellularly during phacoemulsification and exposure of human corneal endothelial cells to histones increased the IL-6 secretion. The intraoperative use of hyaluronan may decrease the cytotoxic effects of histones released during cataract surgery. No author has a financial or proprietary interest in any material or method mentioned. Copyright © 2014 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.

The matricellular protein TSP1 promotes human and mouse endothelial cell senescence through CD47 and Nox1.

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Meijles, Daniel N; Sahoo, Sanghamitra; Al Ghouleh, Imad; Amaral, Jefferson H; Bienes-Martinez, Raquel; Knupp, Heather E; Attaran, Shireen; Sembrat, John C; Nouraie, Seyed M; Rojas, Mauricio M; Novelli, Enrico M; Gladwin, Mark T; Isenberg, Jeffrey S; Cifuentes-Pagano, Eugenia; Pagano, Patrick J

2017-10-17

Senescent cells withdraw from the cell cycle and do not proliferate. The prevalence of senescent compared to normally functioning parenchymal cells increases with age, impairing tissue and organ homeostasis. A contentious principle governing this process has been the redox theory of aging. We linked matricellular protein thrombospondin 1 (TSP1) and its receptor CD47 to the activation of NADPH oxidase 1 (Nox1), but not of the other closely related Nox isoforms, and associated oxidative stress, and to senescence in human cells and aged tissue. In human endothelial cells, TSP1 promoted senescence and attenuated cell cycle progression and proliferation. At the molecular level, TSP1 increased Nox1-dependent generation of reactive oxygen species (ROS), leading to the increased abundance of the transcription factor p53. p53 mediated a DNA damage response that led to senescence through Rb and p21 cip , both of which inhibit cell cycle progression. Nox1 inhibition blocked the ability of TSP1 to increase p53 nuclear localization and p21 cip abundance and its ability to promote senescence. Mice lacking TSP1 showed decreases in ROS production, p21 cip expression, p53 activity, and aging-induced senescence. Conversely, lung tissue from aging humans displayed increases in the abundance of vascular TSP1, Nox1, p53, and p21 cip Finally, genetic ablation or pharmacological blockade of Nox1 in human endothelial cells attenuated TSP1-mediated ROS generation, restored cell cycle progression, and protected against senescence. Together, our results provide insights into the functional interplay between TSP1 and Nox1 in the regulation of endothelial senescence and suggest potential targets for controlling the aging process at the molecular level. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Regulation of Ecto-5´-Nucleotidase by Docosahexaenoic Acid in Human Endothelial Cells

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Vu Thi Thom

2013-08-01

Full Text Available Background/Aims: Modulation of extracellular adenine nucleotide and adenosine concentrations is one potential mechanism by which docosahexaenoic acid (DHA may exert beneficial effects in critically ill patients. This study assessed DHA effects on extracellular adenine purines. Methods: Experiments used human pulmonary endothelial cells (HPMEC and umbilical vein endothelial cells (HUVEC treated with DHA (48 h. mRNA level (real-time PCR, expression (western blot, flow cytometry and activities (hydrolysis of etheno(ε-purines and fluorescence HPLC of CD73 (ecto-5´-nucleotidase and CD39 (ecto-NTPDase-1 were quantified. Results: DHA elevated total CD73 membrane protein expression concentration-dependently but CD73 mRNA level did not change. Increased expression was paralleled by increased enzyme activity. Effects observed on membrane level were reversed in intact cells, in which ε-AMP hydrolysis decreased after DHA. In intact endothelial cells ATP release was enhanced and CD39 activity blunted following DHA treatment. Hence, extracellular ATP and ADP concentrations increased and this inhibited ε-AMP hydrolysis. Conclusion: In human endothelial cells DHA caused 1 up-regulation of CD73 protein content and increased AMP hydrolysis at the cell membrane level, 2 increased cellular ATP release, and 3 decreased extracellular ATP/ADP hydrolysis. Thus, reorganization of the extracellular adenine-nucleotide-adenosine axis in response to DHA resulted in an increased extracellular ATP/adenosine ratio.

Expression of Toll-Like Receptor 4 in Glomerular Endothelial Cells under Diabetic Conditions

International Nuclear Information System (INIS)

Takata, Shunsuke; Sawa, Yoshihiko; Uchiyama, Takanobu; Ishikawa, Hiroyuki

2013-01-01

Diabetic conditions promote glomerulosclerosis by mesangial cells but the mechanisms are not fully elucidated. The present study evaluated the expression of toll-like receptor 4 in glomerular endothelial cells in the streptozotocin (STZ)-induced type 1 diabetic mouse (ICR-STZ) and the type 2 diabetic KK/TaJcl mouse which were fed a high fat diet feed (KK/Ta-HF). In the ICR-STZ and KK/Ta-HF almost glomeruli were immunostained with anti-TLR4 but there was no glomerulus immunostained by ani-TLR4 in the control ICR and KK/Ta. Laser-scanning confocal microscopy showed that the TLR4-positive region did not coincide with the podoplanin-positive region but coincide with the PECAM-1- and VE-cadherin-positive regions in the glomeruli of the ICR-STZ and KK/Ta-HF. The in situ hybridization showed that almost signals for TLR4 mRNA were present in the glomerulus of the ICR-STZ and KK/Ta-HF to a stronger extent than in the control ICR and KK/Ta. These suggest that glomerular endothelial cells usually express the TLR4 gene and hyperglycemia in the diabetic condition induces the TLR4 protein expression in the glomerular capillary endothelial cells. Cytokine productions through the TLR signaling pathway in glomerular endothelial cells may allow mesangial cells to produce extracellular matrix proteins in the diabetic milieu

Lenalidomide, an anti-tumor drug, regulates retinal endothelial cell function: Implication for treating ocular neovascular disorder

International Nuclear Information System (INIS)

Dong, Ling-Feng; Yao, Jin; Wang, Xiao-Qun; Shan, Kun; Yang, Hong; Yan, Biao; Jiang, Qin

2015-01-01

Ocular angiogenesis is an important pathologic character of several ocular diseases, such as retinopathy of prematurity, diabetic retinopathy and age-related macular degeneration (AMD). Inhibition of ocular angiogenesis has great therapeutic value for treating these dieses. Here we show that lenalidomide, an anti-tumor drug, has great anti-angiogenic potential in ocular diseases. Lenalidomide inhibits retinal endothelial cell viability in normal and pathological condition, and inhibits VEGF-induced endothelial cell migration and tube formation in vitro. Moreover, lenalidomide inhibits ocular angiogenesis in vivo through the reduction of angiogenesis- and inflammation-related protein expression. Collectively, lenalidomide is a promising drug for treating ocular angiogenesis through its anti-proliferative and anti-inflammatory property. - Highlights: • Lenalidomide inhibits retinal endothelial cell viability in vitro. • Lenalidomide inhibits retinal endothelial cell migration and tube formation. • Lenalidomide inhibits pathological ocular angiogenesis in vivo. • Lenalidomide inhibits angiogenesis- and inflammation-related protein expression.

Lenalidomide, an anti-tumor drug, regulates retinal endothelial cell function: Implication for treating ocular neovascular disorder

Energy Technology Data Exchange (ETDEWEB)

Dong, Ling-Feng; Yao, Jin; Wang, Xiao-Qun; Shan, Kun; Yang, Hong [Eye Hospital, Nanjing Medical University, Nanjing (China); The Fourth School of Clinical Medicine, Nanjing Medical University, Nanjing (China); Yan, Biao, E-mail: yanbiao1982@hotmail.com [Eye Hospital, Nanjing Medical University, Nanjing (China); The Fourth School of Clinical Medicine, Nanjing Medical University, Nanjing (China); Jiang, Qin, E-mail: jiangqin710@126.com [Eye Hospital, Nanjing Medical University, Nanjing (China); The Fourth School of Clinical Medicine, Nanjing Medical University, Nanjing (China)

2015-10-02

Ocular angiogenesis is an important pathologic character of several ocular diseases, such as retinopathy of prematurity, diabetic retinopathy and age-related macular degeneration (AMD). Inhibition of ocular angiogenesis has great therapeutic value for treating these dieses. Here we show that lenalidomide, an anti-tumor drug, has great anti-angiogenic potential in ocular diseases. Lenalidomide inhibits retinal endothelial cell viability in normal and pathological condition, and inhibits VEGF-induced endothelial cell migration and tube formation in vitro. Moreover, lenalidomide inhibits ocular angiogenesis in vivo through the reduction of angiogenesis- and inflammation-related protein expression. Collectively, lenalidomide is a promising drug for treating ocular angiogenesis through its anti-proliferative and anti-inflammatory property. - Highlights: • Lenalidomide inhibits retinal endothelial cell viability in vitro. • Lenalidomide inhibits retinal endothelial cell migration and tube formation. • Lenalidomide inhibits pathological ocular angiogenesis in vivo. • Lenalidomide inhibits angiogenesis- and inflammation-related protein expression.

Endothelial mechanotransduction proteins and vascular function are altered by dietary sucrose supplementation in healthy young male subjects.

Science.gov (United States)

Gliemann, Lasse; Rytter, Nicolai; Lindskrog, Mads; Slingsby, Martina H Lundberg; Åkerström, Thorbjörn; Sylow, Lykke; Richter, Erik A; Hellsten, Ylva

2017-08-15

Mechanotransduction in endothelial cells is a central mechanism in the regulation of vascular tone and vascular remodelling Mechanotransduction and vascular function may be affected by high sugar levels in plasma because of a resulting increase in oxidative stress and increased levels of advanced glycation end-products (AGE). In healthy young subjects, 2 weeks of daily supplementation with 3 × 75 g of sucrose was found to reduce blood flow in response to passive lower leg movement and in response to 12 W of knee extensor exercise. This vascular impairment was paralleled by up-regulation of platelet endothelial cell adhesion molecule (PECAM)-1, endothelial nitric oxide synthase, NADPH oxidase and Rho family GTPase Rac1 protein expression, an increased basal phosphorylation status of vascular endothelial growth factor receptor 2 and a reduced phosphorylation status of PECAM-1. There were no measurable changes in AGE levels. The findings of the present study demonstrate that daily high sucrose intake markedly affects mechanotransduction proteins and has a detrimental effect on vascular function. Endothelial mechanotransduction is important for vascular function but alterations and activation of vascular mechanosensory proteins have not been investigated in humans. In endothelial cell culture, simple sugars effectively impair mechanosensor proteins. To study mechanosensor- and vascular function in humans, 12 young healthy male subjects supplemented their diet with 3 × 75 g sucrose day -1 for 14 days in a randomized cross-over design. Before and after the intervention period, the hyperaemic response to passive lower leg movement and active knee extensor exercise was determined by ultrasound doppler. A muscle biopsy was obtained from the thigh muscle before and after acute passive leg movement to allow assessment of protein amounts and the phosphorylation status of mechanosensory proteins and NADPH oxidase. The sucrose intervention led to a reduced flow

Endothelial cell repopulation after stenting determines in-stent neointima formation: effects of bare-metal vs. drug-eluting stents and genetic endothelial cell modification.

Science.gov (United States)

Douglas, Gillian; Van Kampen, Erik; Hale, Ashley B; McNeill, Eileen; Patel, Jyoti; Crabtree, Mark J; Ali, Ziad; Hoerr, Robert A; Alp, Nicholas J; Channon, Keith M

2013-11-01

Understanding endothelial cell repopulation post-stenting and how this modulates in-stent restenosis is critical to improving arterial healing post-stenting. We used a novel murine stent model to investigate endothelial cell repopulation post-stenting, comparing the response of drug-eluting stents with a primary genetic modification to improve endothelial cell function. Endothelial cell repopulation was assessed en face in stented arteries in ApoE(-/-) mice with endothelial-specific LacZ expression. Stent deployment resulted in near-complete denudation of endothelium, but was followed by endothelial cell repopulation, by cells originating from both bone marrow-derived endothelial progenitor cells and from the adjacent vasculature. Paclitaxel-eluting stents reduced neointima formation (0.423 ± 0.065 vs. 0.240 ± 0.040 mm(2), P = 0.038), but decreased endothelial cell repopulation (238 ± 17 vs. 154 ± 22 nuclei/mm(2), P = 0.018), despite complete strut coverage. To test the effects of selectively improving endothelial cell function, we used transgenic mice with endothelial-specific overexpression of GTP-cyclohydrolase 1 (GCH-Tg) as a model of enhanced endothelial cell function and increased NO production. GCH-Tg ApoE(-/-) mice had less neointima formation compared with ApoE(-/-) littermates (0.52 ± 0.08 vs. 0.26 ± 0.09 mm(2), P = 0.039). In contrast to paclitaxel-eluting stents, reduced neointima formation in GCH-Tg mice was accompanied by increased endothelial cell coverage (156 ± 17 vs. 209 ± 23 nuclei/mm(2), P = 0.043). Drug-eluting stents reduce not only neointima formation but also endothelial cell repopulation, independent of strut coverage. In contrast, selective targeting of endothelial cell function is sufficient to improve endothelial cell repopulation and reduce neointima formation. Targeting endothelial cell function is a rational therapeutic strategy to improve vascular healing and decrease neointima formation after stenting.

Characterization of human endothelial cell urokinase-type plasminogen activator receptor protein and messenger RNA

DEFF Research Database (Denmark)

Barnathan, E S; Kuo, A; Karikó, K

1990-01-01

Human umbilical vein endothelial cells in culture (HUVEC) express receptors for urokinase-type plasminogen activators (u-PA). The immunochemical nature of this receptor and its relationship to u-PA receptors expressed by other cell types is unknown. Cross-linking active site-blocked u-PA to HUVEC...... an endothelial cell cDNA library using the polymerase chain reaction (PCR) and oligonucleotide primers corresponding to the DNA sequence of the receptor cloned from transformed human fibroblasts (Roldan et al, EMBO J 9:467, 1990). The size of the cDNA (approximately 1,054 base pairs, bp) and the presence...

Sickle erythrocytes inhibit human endothelial cell DNA synthesis

International Nuclear Information System (INIS)

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

1990-01-01

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

Atherosclerosis-Associated Endothelial Cell Apoptosis by MiR-429-Mediated Down Regulation of Bcl-2

Directory of Open Access Journals (Sweden)

Tao Zhang

2015-10-01

Full Text Available Background/Aims: Endothelial cell injury and subsequent apoptosis play a key role in the development and pathogenesis of atherosclerosis, which is hallmarked by dysregulated lipid homeostasis, aberrant immunity and inflammation, and plaque-instability-associated coronary occlusion. Nevertheless, our understanding of the mechanisms underlying endothelial cell apoptosis is still limited. MicroRNA-429 (miR-29 is a known cancer suppressor that promotes cancer cell apoptosis. However, it is unknown whether miR-429 may be involved in the development of atherosclerosis through similar mechanisms. We addressed these questions in the current study. Methods: We examined the levels of endothelial cell apoptosis in ApoE (-/- mice suppled with high-fat diet (HFD, a mouse model for atherosclerosis (simplified as HFD mice. We analyzed the levels of anti-apoptotic protein Bcl-2 and the levels of miR-429 in the purified CD31+ endothelial cells from mouse aorta. Prediction of the binding between miR-429 and 3'-UTR of Bcl-2 mRNA was performed by bioinformatics analyses and confirmed by a dual luciferase reporter assay. The effects of miR-429 were further analyzed in an in vitro model using oxidized low-density lipoprotein (ox-LDL-treated human aortic endothelial cells (HAECs. Results: HFD mice developed atherosclerosis in 12 weeks, while the control ApoE (-/- mice that had received normal diet (simplified as NOR mice did not. HFD mice had significantly lower percentage of endothelial cells and significantly higher percentage of mesenchymal cells in the aorta than NOR mice. Significantly higher levels of endothelial cell apoptosis were detected in HFD mice, resulting from decreases in Bcl-2 protein, but not mRNA. The decreases in Bcl-2 in endothelial cells were due to increased levels of miR-429, which suppressed the translation of Bcl-2 mRNA via 3'-UTR binding. These in vivo findings were reproduced in vitro on ox-LDL-treated HAECs. Conclusion: Atherosclerosis

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

Defibrotide Stimulates Angiogenesis and Protects Endothelial Cells from Calcineurin Inhibitor-Induced Apoptosis via Upregulation of AKT/Bcl-xL.

Science.gov (United States)

Wang, Xiangmin; Pan, Bin; Hashimoto, Yuko; Ohkawara, Hiroshi; Xu, Kailin; Zeng, Lingyu; Ikezoe, Takayuki

2018-01-01

Sinusoidal obstruction syndrome is a life-threatening complication that can occur after haematopoietic stem cell transplantation. Defibrotide (DF) has been approved for the treatment of individuals with severe sinusoidal obstruction syndrome following haematopoietic stem cell transplantation in the European Union and the United States. However, the precise mechanisms by which DF protects endothelial cells remain to be elucidated. In this study, we found that DF stimulated angiogenesis in vitro and in vivo as assessed by vascular tube formation, scratch-wound repair and Matrigel plug assays. These effects were associated with an activation of pro-survival signalling pathways, including AKT (protein kinase B), ERK (extracellular signal-regulated kinases) and p38. More importantly, DF alleviated calcineurin inhibitor-induced growth inhibition and apoptosis of human umbilical vein endothelial cells and human hepatic sinusoidal endothelial cells in parallel with upregulation of anti-apoptotic protein B-cell lymphoma-extra-large (Bcl-xL), which was mediated by AKT (protein kinase B). Notably, these effects were abrogated when Bcl-xL was depleted by small interfering RNA (ribonucleic acid). In addition, DF counteracted calcineurin inhibitor-induced activation of nuclear factor-κB and Janus kinase 2 (JAK2)/Signal Transducer and Activator of Transcription 3 (STAT3) signalling and production of cytokines in vascular endothelial cell-derived EA.hy926 cells. Taken together, DF has pro-angiogenic, anti-apoptotic and anti-inflammatory effects on endothelial cells. DF is a potentially useful agent to prevent the development of, and treat individuals with, endothelial cell injury-related complications after haematopoietic stem cell transplantation. Schattauer GmbH Stuttgart.

Hormonal regulation of Na+/K+-dependent ATPase activity and pump function in corneal endothelial cells.

Science.gov (United States)

Hatou, Shin

2011-10-01

Na- and K-dependent ATPase (Na,K-ATPase) in the basolateral membrane of corneal endothelial cells plays an important role in the pump function of the corneal endothelium. We investigated the role of dexamethasone in the regulation of Na,K-ATPase activity and pump function in these cells. Mouse corneal endothelial cells were exposed to dexamethasone or insulin. ATPase activity was evaluated by spectrophotometric measurement, and pump function was measured using an Ussing chamber. Western blotting and immunocytochemistry were performed to measure the expression of the Na,K-ATPase α1-subunit. Dexamethasone increased Na,K-ATPase activity and the pump function of endothelial cells. Western blot analysis indicated that dexamethasone increased the expression of the Na,K-ATPase α1-subunit but decreased the ratio of active to inactive Na,K-ATPase α1-subunit. Insulin increased Na,K-ATPase activity and pump function of cultured corneal endothelial cells. These effects were transient and blocked by protein kinase C inhibitors and inhibitors of protein phosphatases 1 (PP1) and 2A (PP2A). Western blot analysis indicated that insulin decreased the amount of inactive Na,K-ATPase α1-subunit, but the expression of total Na,K-ATPase α1-subunit was unchanged. Immunocytochemistry showed that insulin increased cell surface expression of the Na,K-ATPase α1-subunit. Our results suggest that dexamethasone and insulin stimulate Na,K-ATPase activity in mouse corneal endothelial cells. The effect of dexamethasone activation in these cells was mediated by Na,K-ATPase synthesis and an increased enzymatic activity because of dephosphorylation of Na,K-ATPase α1-subunits. The effect of insulin is mediated by the protein kinase C, PP1, and/or PP2A pathways.

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......-endothelial electrical resistance above 200 ohm*cm2, indicating that the BCECs formed a tight polar monolayer with functional tight junctions. This was confirmed by immunostaining for the thigh junction protein ZO-1. Rat BCECs were transfected with a red fluorescence protein Hc-RED for 24 hours. Positive transfection...

XIAP reverses various functional activities of FRNK in endothelial cells

International Nuclear Information System (INIS)

Ahn, Sunyoung; Kim, Hyun Jeong; Chi, Sung-Gil; Park, Heonyong

2012-01-01

Highlights: ► FRNK domain is recruited into focal adhesion (FA), controlling endothelial cell adhesion. ► XIAP binds the FRNK domain of FAK. ► XIAP inhibits recruitment of FRNK into Fas and FRNK-promoted cell adhesion. ► XIAP plays a key role in vascular functions of FRNK or FRNK domain-mediated vascular functions of FAK. -- Abstract: In endothelial cells, focal adhesion kinase (FAK) regulates cell proliferation, migration, adhesion, and shear-stimulated activation of MAPK. We recently found that FAK is recruited into focal adhesion (FA) sites through interactions with XIAP (X-chromosome linked inhibitor of apoptosis protein) and activated by Src kinase in response to shear stress. In this study, we examined which domain(s) of FAK is(are) important for various vascular functions such as FA recruiting, XIAP-binding and shear stress-stimulated ERK activation. Through a series of experiments, we determined that the FRNK domain is recruited into FA sites and promotes endothelial cell adhesion. Interestingly, XIAP knockdown was shown to reduce FA recruitment of FRNK and the cell adhesive effect of FRNK. In addition, we found that XIAP interacts with FRNK, suggesting cross-talk between XIAP and FRNK. We also demonstrated that FRNK inhibits endothelial cell migration and shear-stimulated ERK activation. These inhibitory effects of FRNK were reversed by XIAP knockdown. Taken together, we can conclude that XIAP plays a key role in vascular functions of FRNK or FRNK domain-mediated vascular functions of FAK.

Endothelial progenitor cells in chronic obstructive pulmonary disease and emphysema

Science.gov (United States)

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

2017-01-01

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

Leonurine protects against tumor necrosis factor-α-mediated inflammation in human umbilical vein endothelial cells.

Science.gov (United States)

Liu, Xinhua; Pan, Lilong; Wang, Xianli; Gong, Qihai; Zhu, Yi Zhun

2012-05-01

Leonurine, a bioactive alkaloid compound in Herba leonuri, has various pharmacological activities, including antioxidant and anti-apoptotic capacities. This study was conducted to test the hypothesis that leonurine was able to attenuate tumor necrosis factor (TNF)-α-induced human umbilical vein endothelial cells (HUVEC) activation and the underlying molecular mechanisms. Mitogen-activated protein kinases (MAPK) activation, nuclear factor-κB (NF-κB) activation, and inflammatory mediators expression were detected by Western blot or enzyme-liked immunosorbent assay, intracellular reactive oxygen species (ROS) and NF-κB p65 translocation were measured by immunofluorescence, endothelial cell-monocyte interaction was detected by microscope. Leonurine inhibited U937 cells adhesion to TNF-α-activated HUVEC in a concentration dependent manner. Treatment with leonurine blocked TNF-α-induced mRNA and protein expression of adhesion molecules (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1), cyclooxygenase-2, and monocyte chemoattractant protein-1 in endothelial cells. In addition, leonurine attenuated TNF-α-induced intracellular ROS production in HUVEC. Furthermore, leonurine also suppressed the TNF-α-activated p38 phosphorylation and IκBα degradation. Subsequently, reduced NF-κB p65 phosphorylation, nuclear translocation, and DNA-binding activity were also observed. Our results demonstrated for the first time that the anti-inflammatory properties of leonurine in endothelial cells, at least in part, through suppression of NF-κB activation, which may have a potential therapeutic use for inflammatory vascular diseases. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Interleukin-6 and intercellular cell adhesion molecule-1 expression remains elevated in revived live endothelial cells following spaceflight.

Science.gov (United States)

Muid, S; Froemming, G R A; Ali, A M; Nawawi, H

2013-12-01

The effects of spaceflight on cardiovascular health are not necessarily seen immediately after astronauts have returned but can be delayed. It is important to investigate the long term effects of spaceflight on protein and gene expression of inflammation and endothelial activation as a predictor for the development of atherosclerosis and potential cardiovascular problems. The objectives of this study were to investigate the (a) protein and gene expression of inflammation and endothelial activation, (b) expression of nuclear factor kappa B (NFκB), signal transducer and activator of transcription-3 (STAT-3) and endothelial nitric oxide synthase (eNOS) in human umbilical vein endothelial cells (HUVEC) 3 months post-space flight travel compared to ground controls. HUVEC cultured on microcarriers in fluid processing apparatus were flown to the International Space Station (ISS) by the Soyuz TMA-11 rocket. After landing, the cells were detached from microcarriers and recultured in T-25 cm(2) culture flasks (Revived HUVEC). Soluble protein expression of IL-6, TNF-α, ICAM-1, VCAM-1 and e-selectin were measured by ELISA. Gene expression of these markers and in addition NFκB, STAT-3 and eNOS were measured. Spaceflight induced IL-6 and ICAM-1 remain elevated even after 3 months post spaceflight travel and this is mediated via STAT-3 pathway. The downregulation of eNOS expression in revived HUVEC cells suggests a reduced protection of the cells and the surrounding vessels against future insults that may lead to atherosclerosis. It would be crucial to explore preventive measures, in relation to atherosclerosis and its related complications.

Targeting NCK-Mediated Endothelial Cell Front-Rear Polarity Inhibits Neo-Vascularization

Science.gov (United States)

Dubrac, Alexandre; Genet, Gael; Ola, Roxana; Zhang, Feng; Pibouin-Fragner, Laurence; Han, Jinah; Zhang, Jiasheng; Thomas, Jean-Léon; Chedotal, Alain; Schwartz, Martin A.; Eichmann, Anne

2015-01-01

Background Sprouting angiogenesis is a key process driving blood vessel growth in ischemic tissues and an important drug target in a number of diseases, including wet macular degeneration and wound healing. Endothelial cells forming the sprout must develop front-rear polarity to allow sprout extension. The adaptor proteins Nck1 and 2 are known regulators of cytoskeletal dynamics and polarity, but their function in angiogenesis is poorly understood. Here we show that the Nck adaptors are required for endothelial cell front-rear polarity and migration downstream of the angiogenic growth factors VEGF-A and Slit2. Methods and Results Mice carrying inducible, endothelial-specific Nck1/2 deletions fail to develop front-rear polarized vessel sprouts and exhibit severe angiogenesis defects in the postnatal retina and during embryonic development. Inactivation of NCK1 and 2 inhibits polarity by preventing Cdc42 and Pak2 activation by VEGF-A and Slit2. Mechanistically, NCK binding to ROBO1 is required for both Slit2 and VEGF induced front-rear polarity. Selective inhibition of polarized endothelial cell migration by targeting Nck1/2 prevents hypersprouting induced by Notch or Bmp signaling inhibition, as well as pathological ocular neovascularization and wound healing. Conclusions These data reveal a novel signal integration mechanism involving NCK1/2, ROBO1/2 and VEGFR2 that controls endothelial cell front-rear polarity during sprouting angiogenesis. PMID:26659946

Strategies to reverse endothelial progenitor cell dysfunction in diabetes.

Science.gov (United States)

Petrelli, Alessandra; Di Fenza, Raffaele; Carvello, Michele; Gatti, Francesca; Secchi, Antonio; Fiorina, Paolo

2012-01-01

Bone-marrow-derived cells-mediated postnatal vasculogenesis has been reported as the main responsible for the regulation of vascular homeostasis in adults. Since their discovery, endothelial progenitor cells have been depicted as mediators of postnatal vasculogenesis for their peculiar phenotype (partially staminal and partially endothelial), their ability to differentiate in endothelial cell line and to be incorporated into the vessels wall during ischemia/damage. Diabetes mellitus, a condition characterized by cardiovascular disease, nephropathy, and micro- and macroangiopathy, showed a dysfunction of endothelial progenitor cells. Herein, we review the mechanisms involved in diabetes-related dysfunction of endothelial progenitor cells, highlighting how hyperglycemia affects the different steps of endothelial progenitor cells lifetime (i.e., bone marrow mobilization, trafficking into the bloodstream, differentiation in endothelial cells, and homing in damaged tissues/organs). Finally, we review preclinical and clinical strategies that aim to revert diabetes-induced dysfunction of endothelial progenitor cells as a means of finding new strategies to prevent diabetic complications.

Strategies to Reverse Endothelial Progenitor Cell Dysfunction in Diabetes

Directory of Open Access Journals (Sweden)

Alessandra Petrelli

2012-01-01

Full Text Available Bone-marrow-derived cells-mediated postnatal vasculogenesis has been reported as the main responsible for the regulation of vascular homeostasis in adults. Since their discovery, endothelial progenitor cells have been depicted as mediators of postnatal vasculogenesis for their peculiar phenotype (partially staminal and partially endothelial, their ability to differentiate in endothelial cell line and to be incorporated into the vessels wall during ischemia/damage. Diabetes mellitus, a condition characterized by cardiovascular disease, nephropathy, and micro- and macroangiopathy, showed a dysfunction of endothelial progenitor cells. Herein, we review the mechanisms involved in diabetes-related dysfunction of endothelial progenitor cells, highlighting how hyperglycemia affects the different steps of endothelial progenitor cells lifetime (i.e., bone marrow mobilization, trafficking into the bloodstream, differentiation in endothelial cells, and homing in damaged tissues/organs. Finally, we review preclinical and clinical strategies that aim to revert diabetes-induced dysfunction of endothelial progenitor cells as a means of finding new strategies to prevent diabetic complications.

Endothelial induced EMT in breast epithelial cells with stem cell properties

DEFF Research Database (Denmark)

Sigurdsson, Valgardur; Hilmarsdottir, Bylgja; Sigmundsdottir, Hekla

2011-01-01

endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M) derived from D492, show reduced expression...... of keratins, a switch from E-Cadherin (E-Cad) to N-Cadherin (N-Cad) and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44(high)/CD24(low) ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D......492 was partially blocked by inhibition of HGF signaling. Basal-like breast cancer, a vascular rich cancer with stem cell properties and adverse prognosis has been linked with EMT. We immunostained several basal-like breast cancer samples for endothelial and EMT markers. Cancer cells close...

Up-regulation of endothelial monocyte chemoattractant protein-1 by coplanar PCB77 is caveolin-1-dependent

International Nuclear Information System (INIS)

Majkova, Zuzana; Smart, Eric; Toborek, Michal; Hennig, Bernhard

2009-01-01

Atherosclerosis, the primary cause of heart disease and stroke is initiated in the vascular endothelium, and risk factors for its development include environmental exposure to persistent organic pollutants. Caveolae are membrane microdomains involved in regulation of many signaling pathways, and in particular in endothelial cells. We tested the hypothesis that intact caveolae are required for coplanar PCB77-induced up-regulation of monocyte chemoattractant protein-1 (MCP-1), an endothelium-derived chemokine that attracts monocytes into sub-endothelial space in early stages of the atherosclerosis development. Atherosclerosis-prone LDL-R -/- mice (control) or caveolin-1 -/- /LDL-R -/- mice were treated with PCB77. PCB77 induced aortic mRNA expression and plasma protein levels of MCP-1 in control, but not caveolin-1 -/- /LDL-R -/- mice. To study the mechanism of this effect, primary endothelial cells were used. PCB77 increased MCP-1 levels in endothelial cells in a time- and concentration-dependent manner. This effect was abolished by caveolin-1 silencing using siRNA. Also, MCP-1 up-regulation by PCB77 was prevented by inhibiting p38 and c-Jun N-terminal kinase (JNK), but not ERK1/2, suggesting regulatory functions via p38 and JNK MAPK pathways. Finally, pre-treatment of endothelial cells with the aryl hydrocarbon receptor (AhR) inhibitor α-naphthoflavone (α-NF) partially blocked MCP-1 up-regulation. Thus, our data demonstrate that coplanar PCB77 can induce MCP-1 expression by endothelial cells and that this effect is mediated by AhR, as well as p 38 and JNK MAPK pathways. Intact caveolae are required for these processes both in vivo and in vitro. This further supports a key role for caveolae in vascular inflammation induced by persistent organic pollutants.

Endothelial cells in the eyes of an immunologist.

Science.gov (United States)

Young, M Rita

2012-10-01

Endothelial cell activation in the process of tumor angiogenesis and in various aspects of vascular biology has been extensively studied. However, endothelial cells also function in other capacities, including in immune regulation. Compared to the more traditional immune regulatory populations (Th1, Th2, Treg, etc.), endothelial cells have received far less credit as being immune regulators. Their regulatory capacity is multifaceted. They are critical in both limiting and facilitating the trafficking of various immune cell populations, including T cells and dendritic cells, out of the vasculature and into tissue. They also can be induced to stimulate immune reactivity or to be immune inhibitory. In each of these parameters (trafficking, immune stimulation and immune inhibition), their role can be physiological, whereby they have an active role in maintaining health. Alternatively, their role can be pathological, whereby they contribute to disease. In theory, endothelial cells are in an ideal location to recruit cells that can mediate immune reactivity to tumor tissue. Furthermore, they can activate the immune cells as they transmigrate across the endothelium into the tumor. However, what is seen is the absence of these protective effects of endothelial cells and, instead, the endothelial cells succumb to the defense mechanisms of the tumor, resulting in their acquisition of a tumor-protective role. To understand the immune regulatory potential of endothelial cells in protecting the host versus the tumor, it is useful to better understand the other circumstances in which endothelial cells modulate immune reactivities. Which of the multitude of immune regulatory roles that endothelial cells can take on seems to rely on the type of stimulus that they are encountering. It also depends on the extent to which they can be manipulated by potential dangers to succumb and contribute toward attack on the host. This review will explore the physiological and pathological roles

Hypoxia-induced mitogenic factor enhances angiogenesis by promoting proliferation and migration of endothelial cells

International Nuclear Information System (INIS)

Tong Qiangsong; Zheng Liduan; Li Bo; Wang Danming; Huang Chuanshu; Matuschak, George M.; Li Dechun

2006-01-01

Our previous studies have indicated that hypoxia-induced mitogenic factor (HIMF) has angiogenic properties in an in vivo matrigel plug model and HIMF upregulates expression of vascular endothelial growth factor (VEGF) in mouse lungs and cultured lung epithelial cells. However, whether HIMF exerts angiogenic effects through modulating endothelial cell function remains unknown. In this study, mouse aortic rings cultured with recombinant HIMF protein resulted in enhanced vascular sprouting and increased endothelial cell spreading as confirmed by Dil-Ac-LDL uptake, von Willebrand factor and CD31 staining. In cultured mouse endothelial cell line SVEC 4-10, HIMF dose-dependently enhanced cell proliferation, in vitro migration and tubulogenesis, which was not attenuated by SU1498, a VEGFR2/Flk-1 receptor tyrosine kinase inhibitor. Moreover, HIMF stimulation resulted in phosphorylation of Akt, p38 and ERK1/2 kinases in SVEC 4-10 cells. Treatment of mouse aortic rings and SVEC 4-10 cells with LY294002, but not SB203580, PD098059 or U0126, abolished HIMF-induced vascular sprouting and angiogenic responses. In addition, transfection of a dominant-negative mutant of phosphatidylinositol 3-kinase (PI-3K), Δp85, blocked HIMF-induced phosphorylation of Akt, endothelial activation and tubulogenesis. These results indicate that HIMF enhances angiogenesis by promoting proliferation and migration of endothelial cells via activation of the PI-3K/Akt pathways

Putative outer membrane proteins of Leptospira interrogans stimulate human umbilical vein endothelial cells (HUVECS) and express during infection.

Science.gov (United States)

Gómez, Ricardo M; Vieira, Monica L; Schattner, Mirta; Malaver, Elisa; Watanabe, Monica M; Barbosa, Angela S; Abreu, Patricia A E; de Morais, Zenaide M; Cifuente, Javier O; Atzingen, Marina V; Oliveira, Tatiane R; Vasconcellos, Silvio A; Nascimento, Ana L T O

2008-01-01

Cell adhesion molecules (CAMs) are surface receptors present in eukaryotic cells that mediate cell-cell or cell-extracellular matrix interactions. Vascular endothelium stimulation in vitro that lead to the upregulation of CAMs was reported for the pathogenic spirochaetes, including rLIC10365 of Leptospira interrogans. In this study, we report the cloning of LIC10507, LIC10508, LIC10509 genes of L. interrogans using Escherichia coli as a host system. The rational for selecting these sequences is due to their location in L. interrogans serovar Copenhageni genome that has a potential involvement in pathogenesis. The genes encode for predicted lipoproteins with no assigned functions. The purified recombinant proteins were capable to promote the upregulation of intercellular adhesion molecule 1 (ICAM-1) and E-selectin on monolayers of human umbilical vein endothelial cells (HUVECS). In addition, the coding sequences are expressed in the renal tubules of animal during bacterial experimental infection. The proteins are probably located at the outer membrane of the bacteria since they are detected in detergent-phase of L. interrogans Triton X-114 extract. Altogether our data suggest a possible involvement of these proteins during bacterial infection and provide new insights into the role of this region in the pathogenesis of Leptospira.

Indirect induction of endothelial cell injury by PU- or PTFE-mediated activation of monocytes.

Science.gov (United States)

Liu, Xin; Xue, Yang; Sun, Jiao

2010-01-01

Polyurethanes (PUs) and polytetrafluoroethylene (PTFE) are widely used for making cardiovascular devices, but thrombus formation on the surfaces of these devices is inevitable. Since endothelial injury can lead to thrombosis, most of the studies on PUs or PTFE focused on their damage to endothelial cells. However, few studies have attempted to clarify whether the use of foreign objects as biomaterials can cause endothelial injury by activating the innate immune system. In this study, we aimed to investigate the roles of PU- or PTFE-stimulated immune cells in endothelial-cell injury. First, monocytes (THP-1 cells) were stimulated with PU or PTFE for 24 h and, subsequently, human umbilical vein endothelial cells (HUVECs) were treated with the supernatants of the stimulated cells for 24 h. We measured the generation of intracellular reactive oxygen species (ROS) from THP-1 cells treated with PU and PTFE for 24 h, meanwhile hydrogen dioxide (H(2)O(2)), tumor necrosis factor (TNF)-α and interleukin (IL)-1β in the supernatants were also detected. Then, we assessed the apoptosis rate of the HUVECs and determined the expression of NO, inducible nitric oxide synthase (iNOS), and apoptosis-related proteins (p53, Bax, Bcl-2) in the HUVECs. The results showed that large amounts of ROS and low levels of pro-inflammatory cytokines (TNF-α and IL-1β) were produced by the stimulated THP-1 cells. After culturing with the supernatants of the PU- or PTFE-stimulated THP-1 cells, the apoptosis rate, NO production and expression of iNOS, p53 and Bax in the HUVECs were up-regulated, while Bcl-2 expression was down-regulated. In conclusion, the release of ROS by PU- or PTFE-treated THP-1 cells may induce iNOS expression and cause apoptosis in HUVECs via the p53, Bax and Bcl-2 proteins. These data provide the interesting finding that endothelial injury in the process of biomaterial-induced thrombosis can be initiated through the release of soluble mediators by monocytes.

Magnetizable stent-grafts enable endothelial cell capture

Energy Technology Data Exchange (ETDEWEB)

Tefft, Brandon J. [Department of Cardiovascular Diseases, Mayo Clinic, Rochester, MN (United States); Uthamaraj, Susheil [Division of Engineering, Mayo Clinic, Rochester, MN (United States); Harburn, J. Jonathan [School of Medicine, Pharmacy and Health, Durham University, Stockton-on-Tees (United Kingdom); Hlinomaz, Ota [Department of Cardioangiology, St. Anne' s University Hospital, Brno (Czech Republic); Lerman, Amir [Department of Cardiovascular Diseases, Mayo Clinic, Rochester, MN (United States); Dragomir-Daescu, Dan [Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN (United States); Sandhu, Gurpreet S., E-mail: sandhu.gurpreet@mayo.edu [Department of Cardiovascular Diseases, Mayo Clinic, Rochester, MN (United States)

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. - Highlights: • Magnetic stent-grafts were made from 2205 steel stents and polyurethane nanofibers. • Stent-grafts remained patent and formed a thin and uniform neointima when implanted. • Stent-grafts captured endothelial cells labeled with magnetic nanoparticles. �

Flow-induced endothelial cell alignment requires the RhoGEF Trio as a scaffold protein to polarize active Rac1 distribution

NARCIS (Netherlands)

Kroon, Jeffrey; Heemskerk, Niels; Kalsbeek, Martin J. T.; de Waard, Vivian; van Rijssel, Jos; van Buul, Jaap D.

2017-01-01

Endothelial cells line the lumen of the vessel wall and are exposed to flow. In linear parts of the vessel, the endothelial cells experience laminar flow, resulting in endothelial cell alignment in the direction of flow, thereby protecting the vessel wall from inflammation and permeability. In order

Histones Induce the Procoagulant Phenotype of Endothelial Cells through Tissue Factor Up-Regulation and Thrombomodulin Down-Regulation.

Science.gov (United States)

Kim, Ji Eun; Yoo, Hyun Ju; Gu, Ja Yoon; Kim, Hyun Kyung

2016-01-01

The high circulating levels of histones found in various thrombotic diseases may compromise the anticoagulant barrier of endothelial cells. We determined how histones affect endothelial procoagulant tissue factor (TF) and anticoagulant thrombomodulin (TM). Surface antigens, soluble forms, and mRNA levels of TF and TM were measured by flow cytometry, ELISA, and real-time RT-PCR, respectively. TF and TM activity were measured using procoagulant activity, thrombin generation, or chromogenic assays. Involvement of the toll-like receptor (TLR) was assessed using the neutralizing antibodies. Histones dose-dependently induced surface antigens, activity and mRNA levels of endothelial TF. Histone-treated endothelial cells significantly shortened the lag time and enhanced the endogenous thrombin potential of normal plasma, which was normalized by a TF neutralizing antibody. Histones induced phosphatidylserine and protein-disulfide isomerase expression in endothelial cells. Histones also reduced the surface antigen, activity, and mRNA levels of endothelial TM. Polysialic acid and heparin reversed the histone-induced TF up-regulation and TM down-regulation. Activated protein C did not affect the TF up-regulation, but interrupted TM down-regulation. TLR2, and TLR4 inhibitors partially blocked the TF up-regulation. Histones induced the endothelial procoagulant phenotype through TF up-regulation and TM down-regulation. The effects of histones were partly mediated by TLR2, TLR4. Strategies to inhibit the harmful effects of histones in endothelial cells may be required in order to prevent a thrombotic environment.

Protein tyrosine phosphatase, PTP1B, expression and activity in rat corneal endothelial cells

Science.gov (United States)

Harris, Deshea L.

2007-01-01

Purpose The current studies were conducted to determine whether the protein tyrosine phosphatase, PTP1B, plays a role in regulating epidermal growth factor receptor (EGFR) Tyr992 phosphorylation and cell cycle entry in rat corneal endothelial cells. Methods Corneas were obtained from male Sprague-Dawley rats. PTP1B mRNA and protein expression were compared in confluent and subconfluent cells by RT-PCR and western blots. Immunocytochemistry was used to determine the subcellular localization of both PTP1B and EGFR following epidermal growth factor (EGF) stimulation. Western blots were used to analyze the time-dependent effect of EGF on phosphorylation of EGFR Tyr992 plus or minus CinnGEL 2Me, an inhibitor of PTP1B activity. The effect of PTP1B inhibition on cell cycle entry was determined by calculating the percent of Ki67-positive cells following EGF treatment. Results PTP1B mRNA expression was similar in confluent and subconfluent cells, but PTP1B protein was expressed at 3 fold higher levels in subconfluent cells. Positive staining for PTP1B was localized in vesicular structures below the plasma membrane. EGFR staining was located at cell-cell borders in untreated endothelium, but was mainly cytoplasmic by 15 min after EGF treatment. In control cultures, phosphorylation of EGFR Tyr992 peaked by 5 min following EGF stimulation and rapidly decreased to basal levels by 30 min. In cultures pretreated with CinnGEL 2Me, Tyr992 phosphorylation peaked 2 min following EGF addition and was consistently sustained at a higher level than controls until 60 min after treatment. By 18 h following EGF treatment, cultures pretreated with CinnGEL 2Me exhibited a 1.7 fold increase in the number of Ki67-positive cells compared with control cultures. Conclusions Comparison of PTP1B mRNA and protein levels indicates that PTP1B expression is regulated mainly at the protein level and is higher in subconfluent cells. PTP1B was located in vesicles below the plasma membrane. The fact that

RhoA GTPase regulates radiation-induced alterations in endothelial cell adhesion and migration

International Nuclear Information System (INIS)

Rousseau, Matthieu; Gaugler, Marie-Hélène; Rodallec, Audrey; Bonnaud, Stéphanie; Paris, François; Corre, Isabelle

2011-01-01

Highlights: ► We explore the role of RhoA in endothelial cell response to ionizing radiation. ► RhoA is rapidly activated by single high-dose of radiation. ► Radiation leads to RhoA/ROCK-dependent actin cytoskeleton remodeling. ► Radiation-induced apoptosis does not require the RhoA/ROCK pathway. ► Radiation-induced alteration of endothelial adhesion and migration requires RhoA/ROCK. -- Abstract: Endothelial cells of the microvasculature are major target of ionizing radiation, responsible of the radiation-induced vascular early dysfunctions. Molecular signaling pathways involved in endothelial responses to ionizing radiation, despite being increasingly investigated, still need precise characterization. Small GTPase RhoA and its effector ROCK are crucial signaling molecules involved in many endothelial cellular functions. Recent studies identified implication of RhoA/ROCK in radiation-induced increase in endothelial permeability but other endothelial functions altered by radiation might also require RhoA proteins. Human microvascular endothelial cells HMEC-1, either treated with Y-27632 (inhibitor of ROCK) or invalidated for RhoA by RNA interference were exposed to 15 Gy. We showed a rapid radiation-induced activation of RhoA, leading to a deep reorganisation of actin cytoskeleton with rapid formation of stress fibers. Endothelial early apoptosis induced by ionizing radiation was not affected by Y-27632 pre-treatment or RhoA depletion. Endothelial adhesion to fibronectin and formation of focal adhesions increased in response to radiation in a RhoA/ROCK-dependent manner. Consistent with its pro-adhesive role, ionizing radiation also decreased endothelial cells migration and RhoA was required for this inhibition. These results highlight the role of RhoA GTPase in ionizing radiation-induced deregulation of essential endothelial functions linked to actin cytoskeleton.

Tenascin-C in the extracellular matrix promotes the selection of highly proliferative and tubulogenesis-defective endothelial cells

Energy Technology Data Exchange (ETDEWEB)

Alves, Tercia Rodrigues [Universidade do Estado do Rio de Janeiro (UERJ), Instituto de Biologia Roberto Alcantara Gomes, Departamento de Biologia Celular, Laboratorio de Biologia da Celula Endotelial e da Angiogenese (LabAngio), Rio de Janeiro (Brazil); Universidade Federal do Rio de Janeiro (UFRJ), Programa de Biologia Celular e do Desenvolvimento, Instituto de Ciencias Biomedicas, INNT/INCT/MCT, Rio de Janeiro (Brazil); Carvalho da Fonseca, Anna Carolina [Universidade Federal do Rio de Janeiro (UFRJ), Programa de Biologia Celular e do Desenvolvimento, Instituto de Ciencias Biomedicas, INNT/INCT/MCT, Rio de Janeiro (Brazil); Nunes, Sara Santana; Oliveira da Silva, Aline [Universidade do Estado do Rio de Janeiro (UERJ), Instituto de Biologia Roberto Alcantara Gomes, Departamento de Biologia Celular, Laboratorio de Biologia da Celula Endotelial e da Angiogenese (LabAngio), Rio de Janeiro (Brazil); Dubois, Luiz Gustavo Feijo; Faria, Jane; Kahn, Suzana Assad [Universidade Federal do Rio de Janeiro (UFRJ), Programa de Biologia Celular e do Desenvolvimento, Instituto de Ciencias Biomedicas, INNT/INCT/MCT, Rio de Janeiro (Brazil); Viana, Nathan Bessa [Universidade Federal do Rio de Janeiro, Laboratorio de Pincas Oticas, Coordenacao de Programas de Estudos Avancados, Instituto de Ciencias Biomedicas, Rio de Janeiro (Brazil); Universidade Federal do Rio de Janeiro, Instituto de Fisica, Rio de Janeiro (Brazil); Marcondes, Jorge [Universidade Federal do Rio de Janeiro, Hospital Universitario Clementino Fraga Filho, Servico de Neurocirurgia, Rio de Janeiro (Brazil); Legrand, Chantal [Institut Universitaire d' Hematologie, Universite Paris-Diderot, Paris 7, INSERM U553, Paris (France); Moura-Neto, Vivaldo [Universidade Federal do Rio de Janeiro (UFRJ), Programa de Biologia Celular e do Desenvolvimento, Instituto de Ciencias Biomedicas, INNT/INCT/MCT, Rio de Janeiro (Brazil); and others

2011-09-10

The extracellular matrix (ECM) contains important cues for tissue homeostasis and morphogenesis. The matricellular protein tenascin-C (TN-C) is overexpressed in remodeling tissues and cancer. In the present work, we studied the effect of different ECM-which exhibited a significant diversity in their TN-C content-in endothelial survival, proliferation and tubulogenic differentiation: autologous (endothelial) ECM devoid of TN-C, but bearing large amounts of FN; fibroblast ECM, bearing both high TN-C and FN contents; and finally, glioma-derived matrices, usually poor in FN, but very rich in TN-C. HUVECs initially adhered to the immobilized matrix produced by U373 MG glioma cells, but significantly detached and died by anoikis (50 to 80%) after 24 h, as compared with cells incubated with endothelial and fibroblast matrices. Surviving endothelial cells (20 to 50%) became up to 6-fold more proliferative and formed 74-97% less tube-like structures in vitro than cells grown on non-tumoral matrices. An antibody against the EGF-like repeats of tenascin-C (TN-C) partially rescued cells from the tubulogenic defect, indicating that this molecule is responsible for the selection of highly proliferative and tubulogenic defective endothelial cells. Interestingly, by using defined substrata, in conditions that mimic glioma and normal cell ECM composition, we observed that fibronectin (FN) modulates the TN-C-induced selection of endothelial cells. Our data show that TN-C is able to modulate endothelial branching morphogenesis in vitro and, since it is prevalent in matrices of injured and tumor tissues, also suggest a role for this protein in vascular morphogenesis, in these physiological contexts.

Tenascin-C in the extracellular matrix promotes the selection of highly proliferative and tubulogenesis-defective endothelial cells

International Nuclear Information System (INIS)

Alves, Tercia Rodrigues; Carvalho da Fonseca, Anna Carolina; Nunes, Sara Santana; Oliveira da Silva, Aline; Dubois, Luiz Gustavo Feijo; Faria, Jane; Kahn, Suzana Assad; Viana, Nathan Bessa; Marcondes, Jorge; Legrand, Chantal; Moura-Neto, Vivaldo

2011-01-01

The extracellular matrix (ECM) contains important cues for tissue homeostasis and morphogenesis. The matricellular protein tenascin-C (TN-C) is overexpressed in remodeling tissues and cancer. In the present work, we studied the effect of different ECM-which exhibited a significant diversity in their TN-C content-in endothelial survival, proliferation and tubulogenic differentiation: autologous (endothelial) ECM devoid of TN-C, but bearing large amounts of FN; fibroblast ECM, bearing both high TN-C and FN contents; and finally, glioma-derived matrices, usually poor in FN, but very rich in TN-C. HUVECs initially adhered to the immobilized matrix produced by U373 MG glioma cells, but significantly detached and died by anoikis (50 to 80%) after 24 h, as compared with cells incubated with endothelial and fibroblast matrices. Surviving endothelial cells (20 to 50%) became up to 6-fold more proliferative and formed 74-97% less tube-like structures in vitro than cells grown on non-tumoral matrices. An antibody against the EGF-like repeats of tenascin-C (TN-C) partially rescued cells from the tubulogenic defect, indicating that this molecule is responsible for the selection of highly proliferative and tubulogenic defective endothelial cells. Interestingly, by using defined substrata, in conditions that mimic glioma and normal cell ECM composition, we observed that fibronectin (FN) modulates the TN-C-induced selection of endothelial cells. Our data show that TN-C is able to modulate endothelial branching morphogenesis in vitro and, since it is prevalent in matrices of injured and tumor tissues, also suggest a role for this protein in vascular morphogenesis, in these physiological contexts.

Hepatocyte growth factor protects human endothelial cells against advanced glycation end products-induced apoposis

International Nuclear Information System (INIS)

Zhou Yijun; Wang Jiahe; Zhang Jin

2006-01-01

Advanced glycation end products (AGEs) form by a non-enzymatic reaction between reducing sugars and biological proteins, which play an important role in the pathogenesis of atherosclerosis. In this study, we assessed AGEs effects on human umbilical vein endothelial cells (HUVECs) growth, proliferation and apoptosis. Additionally, we investigated whether hepatocyte growth factor (HGF), an anti-apoptotic factor for endothelial cells, prevents AGEs-induced apoptosis of HUVECs. HUVECs were treated with AGEs in the presence or absence of HGF. Treatment of HUVECs with AGEs changed cell morphology, decreased cell viability, and induced DNA fragmentation, leading to apoptosis. Apoptosis was induced by AGEs in a dose- and time-dependent fashion. AGEs markedly elevated Bax and decreased NF-κB, but not Bcl-2 expression. Additionally, AGEs significantly inhibited cell growth through a pro-apoptotic action involving caspase-3 and -9 activations in HUVECs. Most importantly, pretreatment with HGF protected against AGEs-induced cytotoxicity in the endothelial cells. HGF significantly promoted the expression of Bcl-2 and NF-κB, while decreasing the activities of caspase-3 and -9 without affecting Bax level. Our data suggest that AGEs induce apoptosis in endothelial cells. HGF effectively attenuate AGEs-induced endothelial cell apoptosis. These findings provide new perspectives in the role of HGF in cardiovascular disease

Role of Heat Shock Protein 70 in Induction of Stress Fiber Formation in Rat Arterial Endothelial Cells in Response to Stretch Stress

International Nuclear Information System (INIS)

Luo, Shan-Shun; Sugimoto, Keiji; Fujii, Sachiko; Takemasa, Tohru; Fu, Song-Bin; Yamashita, Kazuo

2007-01-01

We investigated the mechanism by which endothelial cells (ECs) resist various forms of physical stress using an experimental system consisting of rat arterial EC sheets. Formation of actin stress fibers (SFs) and expression of endothelial heat-shock stress proteins (HSPs) in response to mechanical stretch stress were assessed by immunofluorescence microscopy. Stretch stimulation increased expression of HSPs 25 and 70, but not that of HSP 90. Treatment with SB203580, a p38 MAP kinase inhibitor that acts upstream of the HSP 25 activation cascade, or with geldanamycin, an inhibitor of HSP 90, had no effect on the SF formation response to mechanical stretch stress. In contrast, treatment with quercetin, an HSP 70 inhibitor, inhibited both upregulation of endothelial HSP 70 and formation of SFs in response to tensile stress. In addition, treatment of stretched ECs with cytochalasin D, which disrupts SF formation, did not adversely affect stretch-induced upregulation of endothelial HSP 70. Our data suggest that endothelial HSP 70 plays an important role in inducing SF formation in response to tensile stress

Nicotine stimulates urokinase-type plasminogen activator receptor expression and cell invasiveness through mitogen-activated protein kinase and reactive oxygen species signaling in ECV304 endothelial cells

Energy Technology Data Exchange (ETDEWEB)

Khoi, Pham Ngoc; Park, Jung Sun; Kim, Nam Ho; Jung, Young Do, E-mail: ydjung@chonnam.ac.kr

2012-03-01

Urokinase-type plasminogen activator receptor (uPAR) expression is elevated during inflammation, tissue remodeling and in many human cancers. This study investigated the effect of nicotine, a major alkaloid in tobacco, on uPAR expression and cell invasiveness in ECV304 endothelial cells. Nicotine stimulated uPAR expression in a dose-dependent manner and activated extracellular signal-regulated kinases-1/2 (Erk-1/2), c-Jun amino-terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK). Specific inhibitors of MEK-1 (PD98059) and JNK (SP600125) inhibited the nicotine-induced uPAR expression, while the p38 MAPK inhibitor SB203580 did not. Expression vectors encoding dominant negative MEK-1 (pMCL-K97M) and JNK (TAM67) also prevented nicotine-induced uPAR promoter activity. The intracellular hydrogen peroxide (H{sub 2}O{sub 2}) content was increased by nicotine treatment. The antioxidant N-acetylcysteine prevented nicotine-activated production of reactive oxygen species (ROS) and uPAR expression. Furthermore, exogenous H{sub 2}O{sub 2} increased uPAR mRNA expression. Deleted and site-directed mutagenesis demonstrated the involvement of the binding sites of transcription factor nuclear factor-kappaB (NF-κB) and activator protein (AP)-1 in the nicotine-induced uPAR expression. Studies with expression vectors encoding mutated NF-κB signaling molecules and AP-1 decoy confirmed that NF-κB and AP-1 were essential for the nicotine-stimulated uPAR expression. MAPK (Erk-1/2 and JNK) and ROS functioned as upstream signaling molecules in the activation of AP-1 and NF-κB, respectively. In addition, ECV304 endothelial cells treated with nicotine displayed markedly enhanced invasiveness, which was partially abrogated by uPAR neutralizing antibodies. The data indicate that nicotine induces uPAR expression via the MAPK/AP-1 and ROS/NF-κB signaling pathways and, in turn, stimulates invasiveness in human ECV304 endothelial cells. -- Highlights: ► Endothelial cells

Nicotine stimulates urokinase-type plasminogen activator receptor expression and cell invasiveness through mitogen-activated protein kinase and reactive oxygen species signaling in ECV304 endothelial cells

International Nuclear Information System (INIS)

Khoi, Pham Ngoc; Park, Jung Sun; Kim, Nam Ho; Jung, Young Do

2012-01-01

Urokinase-type plasminogen activator receptor (uPAR) expression is elevated during inflammation, tissue remodeling and in many human cancers. This study investigated the effect of nicotine, a major alkaloid in tobacco, on uPAR expression and cell invasiveness in ECV304 endothelial cells. Nicotine stimulated uPAR expression in a dose-dependent manner and activated extracellular signal-regulated kinases-1/2 (Erk-1/2), c-Jun amino-terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK). Specific inhibitors of MEK-1 (PD98059) and JNK (SP600125) inhibited the nicotine-induced uPAR expression, while the p38 MAPK inhibitor SB203580 did not. Expression vectors encoding dominant negative MEK-1 (pMCL-K97M) and JNK (TAM67) also prevented nicotine-induced uPAR promoter activity. The intracellular hydrogen peroxide (H 2 O 2 ) content was increased by nicotine treatment. The antioxidant N-acetylcysteine prevented nicotine-activated production of reactive oxygen species (ROS) and uPAR expression. Furthermore, exogenous H 2 O 2 increased uPAR mRNA expression. Deleted and site-directed mutagenesis demonstrated the involvement of the binding sites of transcription factor nuclear factor-kappaB (NF-κB) and activator protein (AP)-1 in the nicotine-induced uPAR expression. Studies with expression vectors encoding mutated NF-κB signaling molecules and AP-1 decoy confirmed that NF-κB and AP-1 were essential for the nicotine-stimulated uPAR expression. MAPK (Erk-1/2 and JNK) and ROS functioned as upstream signaling molecules in the activation of AP-1 and NF-κB, respectively. In addition, ECV304 endothelial cells treated with nicotine displayed markedly enhanced invasiveness, which was partially abrogated by uPAR neutralizing antibodies. The data indicate that nicotine induces uPAR expression via the MAPK/AP-1 and ROS/NF-κB signaling pathways and, in turn, stimulates invasiveness in human ECV304 endothelial cells. -- Highlights: ► Endothelial cells treated with nicotine

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

DEFF Research Database (Denmark)

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

1988-01-01

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

Mycoplasma suis infection results endothelial cell damage and activation: new insight into the cell tropism and pathogenicity of hemotrophic mycoplasma

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Sokoli Albina

2013-02-01

Full Text Available Abstract Hemotrophic mycoplasmas (HM are highly specialized red blood cell parasites that cause infectious anemia in a variety of mammals, including humans. To date, no in vitro cultivation systems for HM have been available, resulting in relatively little information about the pathogenesis of HM infection. In pigs, Mycoplasma suis-induced infectious anemia is associated with hemorrhagic diathesis, and coagulation dysfunction. However, intravasal coagulation and subsequent consumption coagulopathy can only partly explain the sequence of events leading to hemorrhagic diathesis manifesting as cyanosis, petechial bleeding, and ecchymosis, and to disseminated coagulation. The involvement of endothelial activation and damage in M. suis-associated pathogenesis was investigated using light and electron microscopy, immunohistochemistry, and cell sorting. M. suis interacted directly with endothelial cells in vitro and in vivo. Endothelial activation, widespread endothelial damage, and adherence of red blood cells to the endothelium were evident in M. suis-infected pigs. These alterations of the endothelium were accompanied by hemorrhage, intravascular coagulation, vascular occlusion, and massive morphological changes within the parenchyma. M. suis biofilm-like microcolonies formed on the surface of endothelial cells, and may represent a putative persistence mechanism of M. suis. In vitro analysis demonstrated that M. suis interacted with the endothelial cytoskeletal protein actin, and induced actin condensation and activation of endothelial cells, as determined by the up-regulation of ICAM, PECAM, E-selectin, and P-selectin. These findings demonstrate an additional cell tropism of HM for endothelial cells and suggest that M. suis interferes with the protective function of the endothelium, resulting in hemorrhagic diathesis.

Electronegative Low-Density Lipoprotein Increases C-Reactive Protein Expression in Vascular Endothelial Cells through the LOX-1 Receptor

OpenAIRE

Chu, Chih-Sheng; Wang, Yu-Chen; Lu, Long-Sheng; Walton, Brian; Yilmaz, H. Ramazan; Huang, Roger Y.; Sawamura, Tatsuya; Dixon, Richard A. F.; Lai, Wen-Ter; Chen, Chu-Huang; Lu, Jonathan

2013-01-01

Objectives Increased plasma C-reactive protein (CRP) levels are associated with the occurrence and severity of acute coronary syndrome. We investigated whether CRP can be generated in vascular endothelial cells (ECs) after exposure to the most electronegative subfraction of low-density lipoprotein (LDL), L5, which is atherogenic to ECs. Because L5 and CRP are both ligands for the lectin-like oxidized LDL receptor-1 (LOX-1), we also examined the role of LOX-1. Methods and Results Plasma LDL sa...

Telmisartan enhances mitochondrial activity and alters cellular functions in human coronary artery endothelial cells via AMP-activated protein kinase pathway.

Science.gov (United States)

Kurokawa, Hirofumi; Sugiyama, Seigo; Nozaki, Toshimitsu; Sugamura, Koichi; Toyama, Kensuke; Matsubara, Junichi; Fujisue, Koichiro; Ohba, Keisuke; Maeda, Hirofumi; Konishi, Masaaki; Akiyama, Eiichi; Sumida, Hitoshi; Izumiya, Yasuhiro; Yasuda, Osamu; Kim-Mitsuyama, Shokei; Ogawa, Hisao

2015-04-01

Mitochondrial dysfunction plays an important role in cellular senescence and impaired function of vascular endothelium, resulted in cardiovascular diseases. Telmisartan is a unique angiotensin II type I receptor blocker that has been shown to prevent cardiovascular events in high risk patients. AMP-activated protein kinase (AMPK) plays a critical role in mitochondrial biogenesis and endothelial function. This study assessed whether telmisartan enhances mitochondrial function and alters cellular functions via AMPK in human coronary artery endothelial cells (HCAECs). In cultured HCAECs, telmisartan significantly enhanced mitochondrial activity assessed by mitochondrial reductase activity and intracellular ATP production and increased the expression of mitochondria related genes. Telmisartan prevented cellular senescence and exhibited the anti-apoptotic and pro-angiogenic properties. The expression of genes related anti-oxidant and pro-angiogenic properties were increased by telmisartan. Telmisartan increased endothelial NO synthase and AMPK phosphorylation. Peroxisome proliferator-activated receptor gamma signaling was not involved in telmisartan-induced improvement of mitochondrial function. All of these effects were abolished by inhibition of AMPK. Telmisartan enhanced mitochondrial activity and exhibited anti-senescence effects and improving endothelial function through AMPK in HCAECs. Telmisartan could provide beneficial effects on vascular diseases via enhancement of mitochondrial activity and modulating endothelial function through AMPK activation. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Low density lipoprotein uptake by an endothelial-smooth muscle cell bilayer

International Nuclear Information System (INIS)

Alexander, J.J.; Miguel, R.; Graham, D.

1991-01-01

To study the interaction of endothelial and smooth muscle cells, and the means by which such interaction may affect lipid permeability of the arterial wall, cell bilayers were established by use of a transwell culture system. After confluent growth of both cell types had been achieved, iodine 125 bound to low-density lipoprotein (10 ng protein/ml) was added to the media of the upper well. After a 3-hour incubation period, the iodine 125-bound low-density lipoprotein content of the upper and lower media demonstrated an impedance to lipoprotein movement across the endothelial cell monolayer as compared to the bare porous polycarbonate filter of the transwell (p less than 10(-6)). The presence of smooth muscle cells in the bottom well significantly enhanced the permeability of the endothelial cell layer (p less than 10(-60)). This effect remained unchanged over a 9-day time course. Membrane binding and cellular uptake of low-density lipoprotein by endothelial cells was not altered by smooth muscle cells, indicating that this change in permeability could not be easily attributed to changes in receptor-mediated transport or transcytosis. Membrane binding (p less than 0.02) and cellular uptake (p less than 10(-6)) of low-density lipoprotein by smooth muscle cells in the bilayer, when adjusted for counts available in the smooth muscle cell media, were both reduced in the early incubation period as compared to isolated smooth muscle cells. The disproportionate reduction in uptake as compared to binding would suggest that this was not entirely a receptor-dependent process

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

Science.gov (United States)

Jeliazkova-Mecheva, Valentina V; Bobilya, Dennis J

2003-10-01

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

Cellular adhesion molecules on endothelial cells participate in radiation-mediated inflammation

International Nuclear Information System (INIS)

Hallahan, Dennis; Clark, Elizabeth T.; Kuchibhotla, Jaya; Gewertz, Bruce L.

1995-01-01

Purpose: The acute and subacute clinical manifestations of ionizing radiation mimic the inflammatory response to a number of stimuli. During the early stages of the inflammatory response, endothelial cells rapidly and transiently express a number of glycoproteins such as E-selectin, P-selectin, ICAM-1 and VCAM-1 which influence leucocyte adhesion. We quantified the expression of these cellular adhesion molecules (CAMs) in irradiated endothelial cells in order to determine whether these glycoproteins participate in radiation-mediated inflammation. Methods: Primary cultures of human umbilical vein endothelial cells (HUVEC) and HMEC cells were grown to 90% confluence and irradiated with a GE Maxitron x-ray generator. The cells were incubated with primary IgG1 antibody (mouse anti-human ICAM-1, VCAM-1, P-selectin and E-selectin and incubated with FITC-conjugated secondary antibody (goat anti-mouse IgG1). Fluorescence-activated cell sorting (FACS) analysis was utilized for quantitation of receptor expression of each CAM on irradiated endothelial cells. Electrophoretic mobility gel shift assays of nuclear protein extracts from irradiated HUVEC cells were performed using the E-selectin NFkB binding sequence (5'AGCTTAGAGGGGATTTCCGAGAGGA-3'). The E-selectin promoter was ligated to the growth hormone reporter. Plasmids pE-sel(-587 +35)GH or pE-sel(-587 +35)GH Δ NFκB (5 μg) was transfected into HMEC or HUVEC cells by use of lipofection. Transfectants were incubated for 16 h after transfection followed by treatment with 10 Gy (1 Gy/min, GE Maxitron) of ionizing radiation, and or with TNF or IL-1. Leukocyte adhesion to irradiated endothelial cells was quantified by HL-60 binding. Results: The log fluorescence of cells incubated with the antibody to E-selectin shifted by 32% at 4 h after irradiation. In comparison, a shift of 35% occurred 20 h after irradiation for cells incubated with the antibody to ICAM. However, there was no significant increase in P-selectin or VCAM

Radioprotection of mouse CNS endothelial cells in vivo

International Nuclear Information System (INIS)

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

1996-01-01

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

Internalization of proteinase 3 is concomitant with endothelial cell apoptosis and internalization of myeloperoxidase with generation of intracellular oxidants

NARCIS (Netherlands)

Yang, JJ; Preston, GA; Pendergraft, WF; Segelmark, M; Heeringa, P; Hogan, SL; Jennette, JC; Falk, RJ

The important issue addressed by the studies presented here is the mechanism of neutrophil-mediated damage to endothelial and epithelial cells during inflammation. Binding of neutrophil-released granule proteins to endothelial cells may be involved in vascular damage in patients with inflammatory

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

Science.gov (United States)

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

2017-01-01

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

Cellular Dewetting: Opening of Macroapertures in Endothelial Cells

Science.gov (United States)

Gonzalez-Rodriguez, David; Maddugoda, Madhavi P.; Stefani, Caroline; Janel, Sebastien; Lafont, Frank; Cuvelier, Damien; Lemichez, Emmanuel; Brochard-Wyart, Françoise

2012-05-01

Pathogenic bacteria can cross from blood vessels to host tissues by opening transendothelial cell macroapertures (TEMs). To induce TEM opening, bacteria intoxicate endothelial cells with proteins that disrupt the contractile cytoskeletal network. Cell membrane tension is no longer resisted by contractile fibers, leading to the opening of TEMs. Here we model the opening of TEMs as a new form of dewetting. While liquid dewetting is irreversible, we show that cellular dewetting is transient. Our model predicts the minimum radius for hole nucleation, the maximum TEM size, and the dynamics of TEM opening, in good agreement with experimental data. The physical model is then coupled with biological experimental data to reveal that the protein missing in metastasis (MIM) controls the line tension at the rim of the TEM and opposes its opening.

Corneal endothelial expansion promoted by human bone marrow mesenchymal stem cell-derived conditioned medium.

Directory of Open Access Journals (Sweden)

Makiko Nakahara

Full Text Available Healthy corneal endothelium is essential for maintaining corneal clarity, as the damage of corneal endothelial cells and loss of cell count causes severe visual impairment. Corneal transplantation is currently the only therapy for severe corneal disorders. The greatly limited proliferative ability of human corneal endothelial cells (HCECs, even in vitro, has challenged researchers to establish efficient techniques for the cultivating HCECs, a pivotal issue for clinical applications. The aim of this study was to evaluate conditioned medium (CM obtained from human bone marrow-derived mesenchymal stem cells (MSCs (MSC-CM for use as a consistent expansion protocol of HCECs. When HCECs were maintained in the presence of MSC-CM, cell morphology assumed a hexagonal shape similar to corneal endothelial cells in vivo, as opposed to the irregular cell shape observed in control cultures in the absence of MSC-CM. They also maintained the functional protein phenotypes; ZO-1 and Na(+/K(+-ATPase were localized at the intercellular adherent junctions and pump proteins of corneal endothelium were accordingly expressed. In comparison to the proliferative potential observed in the control cultures, HCECs maintained in MSC-CM were found to have more than twice as many Ki67-positive cells and a greatly increased incorporation of BrdU into DNA. MSC-CM further facilitated the cell migration of HCECs. Lastly, the mechanism of cell proliferation mediated by MSC-CM was investigated, and phosphorylation of Akt and ERK1/2 was observed in HCECs after exposure to MSC-CM. The inhibitor to PI 3-kinase maintained the level of p27(Kip1 for up to 24 hours and greatly blocked the expression of cyclin D1 and D3 during the early G1 phase, leading to the reduction of cell density. These findings indicate that MSC-CM not only stimulates the proliferation of HCECs by regulating the G1 proteins of the cell cycle but also maintains the characteristic differentiated phenotypes necessary

Adhesion and endothelialization of endothelial cells on the surface of endovascular stents by the novel rotational culture of cells

International Nuclear Information System (INIS)

Tang Chaojun; Wang Guixue; Cao Yi; Wu Xue; Xie Xiang; Xiao Li

2008-01-01

Recent researches indicate that the initial event in the implantation of endovascular stents involves mechanical injury to the vessel wall. Confluent endothelialization of vascular grafts in vitro before implantation has been suggested as a way to reduce injury of the blood vessel. The purpose of this study is to establish a useful way to improve the adhesion of endothelial cells and accelerate endothelialization on the surface of endovascular stents by a novel rotational culture device. Numerical simulation was used to predict the shear stress on the surface of stents. The number of cellular adhesion was calculated by cell counting, the cell growth was observed by scanning electron microscope and fluorescence microscope. Numerical simulation results showed that the stents was exposed to shear stress of 2.66 x 10 -3 to 8.88 x 10 -2 Pa. Rotational culture of human umbilical vein endothelial cells could enhance the adhesion of cells and accelerate endothelialization on the surface of stents when the culture conditions for EC adhesion were intermediate rotation speed, higher dynamic incubation times, lower cell densities

Importance of mitochondrial calcium uniporter in high glucose-induced endothelial cell dysfunction.

Science.gov (United States)

Chen, Wei; Yang, Jie; Chen, Shuhua; Xiang, Hong; Liu, Hengdao; Lin, Dan; Zhao, Shaoli; Peng, Hui; Chen, Pan; Chen, Alex F; Lu, Hongwei

2017-11-01

Mitochondrial Ca 2+ overload is implicated in hyperglycaemia-induced endothelial cell dysfunction, but the key molecular events responsible remain unclear. We examined the involvement of mitochondrial calcium uniporter, which mediates mitochondrial Ca 2+ uptake, in endothelial cell dysfunction resulting from high-glucose treatment. Human umbilical vein endothelial cells were exposed to various glucose concentrations and to high glucose (30 mM) following mitochondrial calcium uniporter inhibition or activation with ruthenium red and spermine, respectively. Subsequently, mitochondrial calcium uniporter and mitochondrial calcium uniporter regulator 1 messenger RNA and protein expression was measured by real-time polymerase chain reaction and western blotting. Ca 2+ concentrations were analysed by laser confocal microscopy, and cytoplasmic and mitochondrial oxidative stress was detected using 2',7'-dichlorofluorescein diacetate and MitoSOX Red, respectively. Apoptosis was assessed by annexin V-fluorescein isothiocyanate/propidium iodide staining, and a wound-healing assay was performed using an in vitro model. High glucose markedly upregulated mitochondrial calcium uniporter and mitochondrial calcium uniporter regulator 1 messenger RNA expression, as well as protein production, in a dose- and time-dependent manner with a maximum effect demonstrated at 72 h and 30 mM glucose concentration. Moreover, high-glucose treatment significantly raised both mitochondrial and cytoplasmic Ca 2+ and reactive oxygen species levels, increased apoptosis and compromised wound healing (all p calcium uniporter, respectively. Mitochondrial calcium uniporter plays an important role in hyperglycaemia-induced endothelial cell dysfunction and may constitute a therapeutic target to reduce vascular complications in diabetes.

Irradiation-induced up-regulation of HLA-E on macrovascular endothelial cells confers protection against killing by activated natural killer cells.

Directory of Open Access Journals (Sweden)

Isabelle Riederer

Full Text Available BACKGROUND: Apart from the platelet/endothelial cell adhesion molecule 1 (PECAM-1, CD31, endoglin (CD105 and a positive factor VIII-related antigen staining, human primary and immortalized macro- and microvascular endothelial cells (ECs differ in their cell surface expression of activating and inhibitory ligands for natural killer (NK cells. Here we comparatively study the effects of irradiation on the phenotype of ECs and their interaction with resting and activated NK cells. METHODOLOGY/PRINCIPAL FINDINGS: Primary macrovascular human umbilical vein endothelial cells (HUVECs only express UL16 binding protein 2 (ULBP2 and the major histocompatibility complex (MHC class I chain-related protein MIC-A (MIC-A as activating signals for NK cells, whereas the corresponding immortalized EA.hy926 EC cell line additionally present ULBP3, membrane heat shock protein 70 (Hsp70, intercellular adhesion molecule ICAM-1 (CD54 and HLA-E. Apart from MIC-B, the immortalized human microvascular endothelial cell line HMEC, resembles the phenotype of EA.hy926. Surprisingly, primary HUVECs are more sensitive to Hsp70 peptide (TKD plus IL-2 (TKD/IL-2-activated NK cells than their immortalized EC counterpatrs. This finding is most likely due to the absence of the inhibitory ligand HLA-E, since the activating ligands are shared among the ECs. The co-culture of HUVECs with activated NK cells induces ICAM-1 (CD54 and HLA-E expression on the former which drops to the initial low levels (below 5% when NK cells are removed. Sublethal irradiation of HUVECs induces similar but less pronounced effects on HUVECs. Along with these findings, irradiation also induces HLA-E expression on macrovascular ECs and this correlates with an increased resistance to killing by activated NK cells. Irradiation had no effect on HLA-E expression on microvascular ECs and the sensitivity of these cells to NK cells remained unaffected. CONCLUSION/SIGNIFICANCE: These data emphasize that an irradiation

A novel role for inhibitor of apoptosis (IAP) proteins as regulators of endothelial barrier function by mediating RhoA activation.

Science.gov (United States)

Hornburger, Michael C; Mayer, Bettina A; Leonhardt, Stefanie; Willer, Elisabeth A; Zahler, Stefan; Beyerle, Andrea; Rajalingam, Krishnaraj; Vollmar, Angelika M; Fürst, Robert

2014-04-01

Inhibitor of apoptosis (IAP) proteins, such as XIAP or cIAP1/2, are important regulators of apoptosis in cancer cells, and IAP antagonists are currently evaluated as antitumor agents. Beyond their function in cancer cells, this study demonstrates a novel role of IAPs as regulators of vascular endothelial permeability. Two structurally different IAP antagonists, ABT and Smac085, as well as silencing of IAPs, reduced the thrombin receptor-activating peptide (TRAP)-induced barrier dysfunction in human endothelial cells as assessed by measuring macromolecular permeability or transendothelial electrical resistance. ABT diminished thrombin-evoked stress fiber formation, activation of myosin light chain 2, and disassembly of adherens junctions independent of calcium signaling, protein kinase C, and mitogen-activated protein kinases. Interestingly, ABT and silencing of IAPs, in particular XIAP, reduced the TRAP-evoked RhoA activation, whereas Rac1 was not affected. XIAP and, to a lesser extent, cIAP1 were found to directly interact with RhoA independently of the RhoA activation status. Under cell-free conditions, XIAP did not induce an ubiquitination of RhoA. In summary, our work discloses IAPs as crucial regulators of endothelial permeability and suggests IAP inhibition as interesting approach for the prevention of endothelial barrier dysfunction.

Tumor and Endothelial Cell Hybrids Participate in Glioblastoma Vasculature

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Soufiane El Hallani

2014-01-01

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

Expansion and maintenance of human embryonic stem cell–derived endothelial cells by TGFβ inhibition is Id1 dependent

Science.gov (United States)

James, Daylon; Nam, Hyung-song; Seandel, Marco; Nolan, Daniel; Janovitz, Tyler; Tomishima, Mark; Studer, Lorenz; Lee, Gabsang; Lyden, David; Benezra, Robert; Zaninovic, Nikica; Rosenwaks, Zev; Rabbany, Sina Y; Rafii, Shahin

2010-01-01

Previous efforts to differentiate human embryonic stem cells (hESCs) into endothelial cells have not achieved sustained expansion and stability of vascular cells. To define vasculogenic developmental pathways and enhance differentiation, we used an endothelial cell–specific VE-cadherin promoter driving green fluorescent protein (GFP) (hVPr-GFP) to screen for factors that promote vascular commitment. In phase 1 of our method, inhibition of transforming growth factor (TGF)β at day 7 of differentiation increases hVPr-GFP+ cells by tenfold. In phase 2, TGFβ inhibition maintains the proliferation and vascular identity of purified endothelial cells, resulting in a net 36-fold expansion of endothelial cells in homogenous monolayers, which exhibited a transcriptional profile of Id1highVEGFR2highVE-cadherin+ ephrinB2+. Using an Id1-YFP hESC reporter line, we showed that TGFβ inhibition sustains Id1 expression in hESC-derived endothelial cells and that Id1 is required for increased proliferation and preservation of endothelial cell commitment. Our approach provides a serum-free method for differentiation and long-term maintenance of hESC-derived endothelial cells at a scale relevant to clinical application. PMID:20081865

The role of shear stress and altered tissue properties on endothelial to mesenchymal transformation and tumor-endothelial cell interaction.

Science.gov (United States)

Mina, Sara G; Huang, Peter; Murray, Bruce T; Mahler, Gretchen J

2017-07-01

Tumor development is influenced by stromal cells in aspects including invasion, growth, angiogenesis, and metastasis. Activated fibroblasts are one group of stromal cells involved in cancer metastasis, and one source of activated fibroblasts is endothelial to mesenchymal transformation (EndMT). EndMT begins when the endothelial cells delaminate from the cell monolayer, lose cell-cell contacts, lose endothelial markers such as vascular endothelial-cadherin (VE-cadherin), gain mesenchymal markers like alpha-smooth muscle actin (α-SMA), and acquire mesenchymal cell-like properties. A three-dimensional (3D) culture microfluidic device was developed for investigating the role of steady low shear stress (1 dyne/cm 2 ) and altered extracellular matrix (ECM) composition and stiffness on EndMT. Shear stresses resulting from fluid flow within tumor tissue are relevant to both cancer metastasis and treatment effectiveness. Low and oscillatory shear stress rates have been shown to enhance the invasion of metastatic cancer cells through specific changes in actin and tubulin remodeling. The 3D ECM within the device was composed of type I collagen and glycosaminoglycans (GAGs), hyaluronic acid and chondroitin sulfate. An increase in collagen and GAGs has been observed in the solid tumor microenvironment and has been correlated with poor prognosis in many different cancer types. In this study, it was found that ECM composition and low shear stress upregulated EndMT, including upregulation of mesenchymal-like markers (α-SMA and Snail) and downregulated endothelial marker protein and gene expression (VE-cadherin). Furthermore, this novel model was utilized to investigate the role of EndMT in breast cancer cell proliferation and migration. Cancer cell spheroids were embedded within the 3D ECM of the microfluidic device. The results using this device show for the first time that the breast cancer spheroid size is dependent on shear stress and that the cancer cell migration rate

Endothelial induced EMT in breast epithelial cells with stem cell properties.

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Valgardur Sigurdsson

Full Text Available Epithelial to mesenchymal transition (EMT is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M derived from D492, show reduced expression of keratins, a switch from E-Cadherin (E-Cad to N-Cadherin (N-Cad and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44(high/CD24(low ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D492 was partially blocked by inhibition of HGF signaling. Basal-like breast cancer, a vascular rich cancer with stem cell properties and adverse prognosis has been linked with EMT. We immunostained several basal-like breast cancer samples for endothelial and EMT markers. Cancer cells close to the vascular rich areas show no or decreased expression of E-Cad and increased N-Cad expression suggesting EMT. Collectively, we have shown in a 3D culture model that endothelial cells are potent inducers of EMT in breast epithelial cells with stem cell properties. Furthermore, we demonstrate that basal-like breast cancer contains cells with an EMT phenotype, most prominently close to vascular rich areas of these tumors. We conclude that endothelial cells are potent inducers of EMT and may play a role in progression of basal-like breast cancer.

Endothelial induced EMT in breast epithelial cells with stem cell properties.

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Sigurdsson, Valgardur; Hilmarsdottir, Bylgja; Sigmundsdottir, Hekla; Fridriksdottir, Agla J R; Ringnér, Markus; Villadsen, Rene; Borg, Ake; Agnarsson, Bjarni A; Petersen, Ole William; Magnusson, Magnus K; Gudjonsson, Thorarinn

2011-01-01

Epithelial to mesenchymal transition (EMT) is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M) derived from D492, show reduced expression of keratins, a switch from E-Cadherin (E-Cad) to N-Cadherin (N-Cad) and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44(high)/CD24(low) ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D492 was partially blocked by inhibition of HGF signaling. Basal-like breast cancer, a vascular rich cancer with stem cell properties and adverse prognosis has been linked with EMT. We immunostained several basal-like breast cancer samples for endothelial and EMT markers. Cancer cells close to the vascular rich areas show no or decreased expression of E-Cad and increased N-Cad expression suggesting EMT. Collectively, we have shown in a 3D culture model that endothelial cells are potent inducers of EMT in breast epithelial cells with stem cell properties. Furthermore, we demonstrate that basal-like breast cancer contains cells with an EMT phenotype, most prominently close to vascular rich areas of these tumors. We conclude that endothelial cells are potent inducers of EMT and may play a role in progression of basal-like breast cancer.

Requirement of phosphorylatable endothelial nitric oxide synthase at Ser-1177 for vasoinhibin-mediated inhibition of endothelial cell migration and proliferation in vitro.

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García, Celina; Nuñez-Anita, Rosa Elvira; Thebault, Stéphanie; Arredondo Zamarripa, David; Jeziorsky, Michael C; Martínez de la Escalera, Gonzalo; Clapp, Carmen

2014-03-01

Endothelial nitric oxide synthase (eNOS)-derived nitric oxide is a major vasorelaxing factor and a mediator of vasopermeability and angiogenesis. Vasoinhibins, a family of antiangiogenic prolactin fragments that include 16 K prolactin, block most eNOS-mediated vascular effects. Vasoinhibins activate protein phosphatase 2A, causing eNOS inactivation through dephosphorylation of eNOS at serine residue 1179 in bovine endothelial cells and thereby blocking vascular permeability. In this study, we examined whether human eNOS phosphorylation at S1177 (analogous to bovine S1179) influences other actions of vasoinhibins. Bovine umbilical vein endothelial cells were stably transfected with human wild-type eNOS (WT) or with phospho-mimetic (S1177D) or non-phosphorylatable (S1177A) eNOS mutants. Vasoinhibins inhibited the increases in eNOS activity, migration, and proliferation following the overexpression of WT eNOS but did not affect these responses in cells expressing S1177D and S1177A eNOS mutants. We conclude that eNOS inhibition by dephosphorylation of S1177 is fundamental for the inhibition of endothelial cell migration and proliferation by vasoinhibins.

In vitro analysis of human periodontal microvascular endothelial cells.

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Tsubokawa, Mizuki; Sato, Soh

2014-08-01

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

Hypertonic saline impedes tumor cell-endothelial cell interaction by reducing adhesion molecule and laminin expression.

LENUS (Irish Health Repository)

Shields, Conor J

2012-02-03

BACKGROUND: Hypertonic saline infusion dampens inflammatory responses and suppresses neutrophil-endothelial interaction by reducing adhesion molecule expression. This study tested the hypothesis that hypertonic saline attenuates tumor cell adhesion to the endothelium through a similar mechanism. METHODS: Human colon cancer cells (LS174T) were transfected with green fluorescent protein and exposed to lipopolysaccharide, tumor necrosis factor-alpha, and interleukin-6 under hypertonic and isotonic conditions for 1 and 4 hours. Confluent human umbilical vein endothelial cells were similarly exposed. Cellular apoptosis and expression of adhesion molecules and laminin were measured by flow cytometry. Tumor cell adhesion to endothelium and laminin was assessed with fluorescence microscopy. Data are represented as mean +\\/- standard error of mean, and an ANOVA test was performed to gauge statistical significance, with P <.05 considered significant. RESULTS: Hypertonic exposure significantly reduced tumor cell adhesion despite the presence of the perioperative cell stressors (42 +\\/- 2.9 vs 172.5 +\\/- 12.4, P <.05), attenuated tumor cell beta-1 integrin (14.43 vs 23.84, P <.05), and endothelial cell laminin expression (22.78 +\\/- 2.2 vs 33.74 +\\/- 2.4, P <.05), but did not significantly alter cell viability. CONCLUSION: Hypertonic saline significantly attenuates tumor cell adhesion to endothelium by inhibiting adhesion molecule and laminin expression. This may halt the metastatic behavior of tumor cells shed at surgery.

Oxidative stress induced pulmonary endothelial cell proliferation is ...

African Journals Online (AJOL)

Cellular hyper-proliferation, endothelial dysfunction and oxidative stress are hallmarks of the pathobiology of pulmonary hypertension. Indeed, pulmonary endothelial cells proliferation is susceptible to redox state modulation. Some studies suggest that superoxide stimulates endothelial cell proliferation while others have ...

Bacterial Cellulose Shifts Transcriptome and Proteome of Cultured Endothelial Cells Towards Native Differentiation.

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Feil, Gerhard; Horres, Ralf; Schulte, Julia; Mack, Andreas F; Petzoldt, Svenja; Arnold, Caroline; Meng, Chen; Jost, Lukas; Boxleitner, Jochen; Kiessling-Wolf, Nicole; Serbest, Ender; Helm, Dominic; Kuster, Bernhard; Hartmann, Isabel; Korff, Thomas; Hahne, Hannes

2017-09-01

Preserving the native phenotype of primary cells in vitro is a complex challenge. Recently, hydrogel-based cellular matrices have evolved as alternatives to conventional cell culture techniques. We developed a bacterial cellulose-based aqueous gel-like biomaterial, dubbed Xellulin, which mimics a cellular microenvironment and seems to maintain the native phenotype of cultured and primary cells. When applied to human umbilical vein endothelial cells (HUVEC), it allowed the continuous cultivation of cell monolayers for more than one year without degradation or dedifferentiation. To investigate the impact of Xellulin on the endothelial cell phenotype in detail, we applied quantitative transcriptomics and proteomics and compared the molecular makeup of native HUVEC, HUVEC on collagen-coated Xellulin and collagen-coated cell culture plastic (polystyrene).Statistical analysis of 12,475 transcripts and 7831 proteins unveiled massive quantitative differences of the compared transcriptomes and proteomes. K -means clustering followed by network analysis showed that HUVEC on plastic upregulate transcripts and proteins controlling proliferation, cell cycle and protein biosynthesis. In contrast, HUVEC on Xellulin maintained, by and large, the expression levels of genes supporting their native biological functions and signaling networks such as integrin, receptor tyrosine kinase MAP/ERK and PI3K signaling pathways, while decreasing the expression of proliferation associated proteins. Moreover, CD34-an endothelial cell differentiation marker usually lost early during cell culture - was re-expressed within 2 weeks on Xellulin but not on plastic. And HUVEC on Xellulin showed a significantly stronger functional responsiveness to a prototypic pro-inflammatory stimulus than HUVEC on plastic.Taken together, this is one of the most comprehensive transcriptomic and proteomic studies of native and propagated HUVEC, which underscores the importance of the morphology of the cellular

The Effects of Inhaled Nickel Nanoparticles on Murine Endothelial Progenitor Cells

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Liberda, Eric N.

Introduction. Particulate air pollution, specifically nickel found on or in particulate matter, has been associated with an increased risk of mortality in human population studies and can cause increases in vascular inflammation, generate reactive oxygen species, alter vasomotor tone, and potentiate atherosclerosis in murine exposures. With the discovery of endothelial progenitor cells (EPCs), a door has been opened which may explain these observed cardiovascular effects associated with inhaled air particles and nickel exposure. In order to further quantify the effects of inhaled nickel nanoparticles and attempt to elucidate how the observed findings from other studies may occur, several whole body inhalation exposure experiments to nickel nanoparticles were performed. Methods. Following whole body exposure to approximately 500mug/m3 of nickel nanoparticles for 5 hrs, bone marrow EPCs from C57BL/6 mice were isolated. EPCs were harvested for their RNA or used in a variety of assays including chemotaxis, tube formation, and proliferation. Gene expression was assessed for important receptors involved in EPC mobilization and homing using RT-PCR methods. EPCs, circulating endothelial progenitor cells, circulating endothelial cells (CECs), and endothelial microparticles (EMPs) were quantified on a BD FACSCalibur to examine endothelial damage and repair associated with the inhalation exposure. Plasma proteins were assessed using the 2D DIGE proteomic approach and commercially available ELISAs. Results and Conclusions. Exposure to inhaled nickel nanoparticles significantly increased both bone marrow EPCs as well as their levels in circulation. CECs were significantly upregulated suggesting that endothelial damage occurred due to the exposure. There was no significant difference in EMPs between the two groups. Tube formation and chemotaxis, but not proliferation, of bone marrow EPCs was impaired in the nickel nanoparticle exposed group. This decrease in EPC function

Loss of 51chromium, lactate dehydrogenase, and 111indium as indicators of endothelial cell injury

International Nuclear Information System (INIS)

Chopra, J.; Joist, J.H.; Webster, R.O.

1987-01-01

Injury to endothelial cells appears to be an important initial event in the pathogenesis of many diseases such as acute lung injury, venous and arterial thromboembolism, and atherosclerosis. Different methods for detecting damage to cultured endothelial cells have been described. However, their relative sensitivity as markers of endothelial cell damage has not been adequately determined. We compared the loss of 51 Chromium ( 51 Cr), the cytoplasmic enzyme lactate dehydrogenase (LDH), and 111 Indium ( 111 In) from endothelial cells upon exposure to several injurious agents. Cultured bovine pulmonary artery endothelial cells in confluent monolayers were labeled with 51 Cr or 111 Inoxine and exposed to increasing concentrations of the nonionic detergent, Triton X-100 (0.2 to 1%), hydrogen peroxide (1 to 500 microM), or neutrophils stimulated with phorbol myristate acetate. With all forms of injury, loss of 51 Cr occurred earlier and to a greater extent than LDH loss which in turn was greater than loss of 111 In. Substantial loss of 51 Cr was observed in the absence of appreciable ultrastructural damage to endothelial cell external membranes. The findings may reflect the relative ease with which small molecules such as adenine nucleotides ( 51 Cr-labeled) escape whereas larger molecules such as LDH and proteins binding 111 In are retained intracellularly. Thus, 51 Cr loss appears to be a more sensitive indicator of sublytic endothelial cell injury than either 111 In or LDH release

Magnetizable stent-grafts enable endothelial cell capture

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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.

CMTM3 (CKLF-Like Marvel Transmembrane Domain 3) Mediates Angiogenesis by Regulating Cell Surface Availability of VE-Cadherin in Endothelial Adherens Junctions.

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Chrifi, Ihsan; Louzao-Martinez, Laura; Brandt, Maarten; van Dijk, Christian G M; Burgisser, Petra; Zhu, Changbin; Kros, Johan M; Duncker, Dirk J; Cheng, Caroline

2017-06-01

Decrease in VE-cadherin adherens junctions reduces vascular stability, whereas disruption of adherens junctions is a requirement for neovessel sprouting during angiogenesis. Endocytosis plays a key role in regulating junctional strength by altering bioavailability of cell surface proteins, including VE-cadherin. Identification of new mediators of endothelial endocytosis could enhance our understanding of angiogenesis. Here, we assessed the function of CMTM3 (CKLF-like MARVEL transmembrane domain 3), which we have previously identified as highly expressed in Flk1 + endothelial progenitor cells during embryonic development. Using a 3-dimensional coculture of human umbilical vein endothelial cells-GFP (green fluorescent protein) and pericytes-RFP (red fluorescent protein), we demonstrated that siRNA-mediated CMTM3 silencing in human umbilical vein endothelial cells impairs angiogenesis. In vivo CMTM3 inhibition by morpholino injection in developing zebrafish larvae confirmed that CMTM3 expression is required for vascular sprouting. CMTM3 knockdown in human umbilical vein endothelial cells does not affect proliferation or migration. Intracellular staining demonstrated that CMTM3 colocalizes with early endosome markers EEA1 (early endosome marker 1) and Clathrin + vesicles and with cytosolic VE-cadherin in human umbilical vein endothelial cells. Adenovirus-mediated CMTM3 overexpression enhances endothelial endocytosis, shown by an increase in Clathrin + , EEA1 + , Rab11 + , Rab5 + , and Rab7 + vesicles. CMTM3 overexpression enhances, whereas CMTM3 knockdown decreases internalization of cell surface VE-cadherin in vitro. CMTM3 promotes loss of endothelial barrier function in thrombin-induced responses, shown by transendothelial electric resistance measurements in vitro. In this study, we have identified a new regulatory function for CMTM3 in angiogenesis. CMTM3 is involved in VE-cadherin turnover and is a regulator of the cell surface pool of VE-cadherin. Therefore, CMTM

Angiogenesis interactome and time course microarray data reveal the distinct activation patterns in endothelial cells.

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Liang-Hui Chu

Full Text Available Angiogenesis involves stimulation of endothelial cells (EC by various cytokines and growth factors, but the signaling mechanisms are not completely understood. Combining dynamic gene expression time-course data for stimulated EC with protein-protein interactions associated with angiogenesis (the "angiome" could reveal how different stimuli result in different patterns of network activation and could implicate signaling intermediates as points for control or intervention. We constructed the protein-protein interaction networks of positive and negative regulation of angiogenesis comprising 367 and 245 proteins, respectively. We used five published gene expression datasets derived from in vitro assays using different types of blood endothelial cells stimulated by VEGFA (vascular endothelial growth factor A. We used the Short Time-series Expression Miner (STEM to identify significant temporal gene expression profiles. The statistically significant patterns between 2D fibronectin and 3D type I collagen substrates for telomerase-immortalized EC (TIME show that different substrates could influence the temporal gene activation patterns in the same cell line. We investigated the different activation patterns among 18 transmembrane tyrosine kinase receptors, and experimentally measured the protein level of the tyrosine-kinase receptors VEGFR1, VEGFR2 and VEGFR3 in human umbilical vein EC (HUVEC and human microvascular EC (MEC. The results show that VEGFR1-VEGFR2 levels are more closely coupled than VEGFR1-VEGFR3 or VEGFR2-VEGFR3 in HUVEC and MEC. This computational methodology can be extended to investigate other molecules or biological processes such as cell cycle.

Citrus Polyphenol Hesperidin Stimulates Production of Nitric Oxide in Endothelial Cells while Improving Endothelial Function and Reducing Inflammatory Markers in Patients with Metabolic Syndrome

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Rizza, Stefano; Muniyappa, Ranganath; Iantorno, Micaela; Kim, Jeong-a; Chen, Hui; Pullikotil, Philomena; Senese, Nicoletta; Tesauro, Manfredi; Lauro, Davide; Cardillo, Carmine

2011-01-01

Context: Hesperidin, a citrus flavonoid, and its metabolite hesperetin may have vascular actions relevant to their health benefits. Molecular and physiological mechanisms of hesperetin actions are unknown. Objective: We tested whether hesperetin stimulates production of nitric oxide (NO) from vascular endothelium and evaluated endothelial function in subjects with metabolic syndrome on oral hesperidin therapy. Design, Setting, and Interventions: Cellular mechanisms of action of hesperetin were evaluated in bovine aortic endothelial cells (BAEC) in primary culture. A randomized, placebo-controlled, double-blind, crossover trial examined whether oral hesperidin administration (500 mg once daily for 3 wk) improves endothelial function in individuals with metabolic syndrome (n = 24). Main Outcome Measure: We measured the difference in brachial artery flow-mediated dilation between placebo and hesperidin treatment periods. Results: Treatment of BAEC with hesperetin acutely stimulated phosphorylation of Src, Akt, AMP kinase, and endothelial NO synthase to produce NO; this required generation of H2O2. Increased adhesion of monocytes to BAEC and expression of vascular cell adhesion molecule-1 in response to TNF-α treatment was reduced by pretreatment with hesperetin. In the clinical study, when compared with placebo, hesperidin treatment increased flow-mediated dilation (10.26 ± 1.19 vs. 7.78 ± 0.76%; P = 0.02) and reduced concentrations of circulating inflammatory biomarkers (high-sensitivity C-reactive protein, serum amyloid A protein, soluble E-selectin). Conclusions: Novel mechanisms for hesperetin action in endothelial cells inform effects of oral hesperidin treatment to improve endothelial dysfunction and reduce circulating markers of inflammation in our exploratory clinical trial. Hesperetin has vasculoprotective actions that may explain beneficial cardiovascular effects of citrus consumption. PMID:21346065

The challenging environment on board the International Space Station affects endothelial cell function by triggering oxidative stress through thioredoxin interacting protein overexpression: the ESA-SPHINX experiment.

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Versari, Silvia; Longinotti, Giulia; Barenghi, Livia; Maier, Jeanette Anne Marie; Bradamante, Silvia

2013-11-01

Exposure to microgravity generates alterations that are similar to those involved in age-related diseases, such as cardiovascular deconditioning, bone loss, muscle atrophy, and immune response impairment. Endothelial dysfunction is the common denominator. To shed light on the underlying mechanism, we participated in the Progress 40P mission with Spaceflight of Human Umbilical Vein Endothelial Cells (HUVECs): an Integrated Experiment (SPHINX), which consisted of 12 in-flight and 12 ground-based control modules and lasted 10 d. Postflight microarray analysis revealed 1023 significantly modulated genes, the majority of which are involved in cell adhesion, oxidative phosphorylation, stress responses, cell cycle, and apoptosis. Thioredoxin-interacting protein was the most up-regulated (33-fold), heat-shock proteins 70 and 90 the most down-regulated (5.6-fold). Ion channels (TPCN1, KCNG2, KCNJ14, KCNG1, KCNT1, TRPM1, CLCN4, CLCA2), mitochondrial oxidative phosphorylation, and focal adhesion were widely affected. Cytokine detection in the culture media indicated significant increased secretion of interleukin-1α and interleukin-1β. Nitric oxide was found not modulated. Our data suggest that in cultured HUVECs, microgravity affects the same molecular machinery responsible for sensing alterations of flow and generates a prooxidative environment that activates inflammatory responses, alters endothelial behavior, and promotes senescence.

Theobroma cacao increases cells viability and reduces IL-6 and sVCAM-1 level in endothelial cells induced by plasma from preeclamptic patients.

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Rahayu, Budi; Baktiyani, Siti Candra Windu; Nurdiana, Nurdiana

2016-01-01

This study aims to investigate whether an ethanolic extract of Theobroma cacao bean is able to increase cell viability and decrease IL-6 and sVCAM-1 in endothelial cells induced by plasma from preeclamptic patients. Endothelial cells were obtained from human umbilical vascular endothelial cells. At confluency, endothelial cells were divided into six groups, which included control (untreated), endothelial cells exposed to plasma from normal pregnancy, endothelial cells exposed to 2% plasma from preeclamptic patients (PP), endothelial cells exposed to PP in the presence of ethanolic extract of T. cacao (PP+TC) at the following three doses: 25, 50, and 100 ppm. The analysis was performed in silico using the Hex 8.0, LigPlus and LigandScout 3.1 software. Analysis on IL-6 and sVCAM-1 levels were done by enzyme linked immunosorbent assay (ELISA). We found that seven of them could bind to the protein NFκB (catechin, leucoanthocyanidin, niacin, phenylethylamine, theobromine, theophylline, and thiamin). This increase in IL-6 was significantly (Pcacao extract. Plasma from PP significantly increased sVCAM-1 levels compared to untreated cells. This increase in sVCAM-1 was significantly attenuated by all doses of the extract. In conclusion, T. cacao extract prohibits the increase in IL-6 and sVCAM-1 in endothelial cells induced by plasma from preeclamptic patients. Therefore this may provide a herbal therapy for attenuating the endothelial dysfunction found in preeclampsia. Copyright © 2016 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved.

Endothelial cell permeability during hantavirus infection involves factor XII-dependent increased activation of the kallikrein-kinin system.

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Shannon L Taylor

Full Text Available Hemorrhagic fever with renal syndrome (HFRS and hantavirus pulmonary syndrome (HPS are diseases caused by hantavirus infections and are characterized by vascular leakage due to alterations of the endothelial barrier. Hantavirus-infected endothelial cells (EC display no overt cytopathology; consequently, pathogenesis models have focused either on the influx of immune cells and release of cytokines or on increased degradation of the adherens junction protein, vascular endothelial (VE-cadherin, due to hantavirus-mediated hypersensitization of EC to vascular endothelial growth factor (VEGF. To examine endothelial leakage in a relevant in vitro system, we co-cultured endothelial and vascular smooth muscle cells (vSMC to generate capillary blood vessel-like structures. In contrast to results obtained in monolayers of cultured EC, we found that despite viral replication in both cell types as well as the presence of VEGF, infected in vitro vessels neither lost integrity nor displayed evidence of VE-cadherin degradation. Here, we present evidence for a novel mechanism of hantavirus-induced vascular leakage involving activation of the plasma kallikrein-kinin system (KKS. We show that incubation of factor XII (FXII, prekallikrein (PK, and high molecular weight kininogen (HK plasma proteins with hantavirus-infected EC results in increased cleavage of HK, higher enzymatic activities of FXIIa/kallikrein (KAL and increased liberation of bradykinin (BK. Measuring cell permeability in real-time using electric cell-substrate impedance sensing (ECIS, we identified dramatic increases in endothelial cell permeability after KKS activation and liberation of BK. Furthermore, the alterations in permeability could be prevented using inhibitors that directly block BK binding, the activity of FXIIa, or the activity of KAL. Lastly, FXII binding and autoactivation is increased on the surface of hantavirus-infected EC. These data are the first to demonstrate KKS activation

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

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

2013-01-17

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

Multidrug Resistance-associated Protein-1 (MRP-1)-dependent Glutathione Disulfide (GSSG) Efflux as a Critical Survival Factor for Oxidant-enriched Tumorigenic Endothelial Cells.

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Gordillo, Gayle M; Biswas, Ayan; Khanna, Savita; Spieldenner, James M; Pan, Xueliang; Sen, Chandan K

2016-05-06

Endothelial cell tumors are the most common soft tissue tumors in infants. Tumor-forming endothelial (EOMA) cells are able to escape cell death fate despite excessive nuclear oxidant burden. Our previous work recognized perinuclear Nox-4 as a key contributor to EOMA growth. The objective of this work was to characterize the mechanisms by which EOMA cells evade oxidant toxicity and thrive. In EOMA cells, compared with in the cytosol, the nuclear GSSG/GSH ratio was 5-fold higher. Compared to the ratio observed in healthy murine aortic endothelial (MAE) cells, GSSG/GSH was over twice as high in EOMA cells. Multidrug resistance-associated protein-1 (MRP-1), an active GSSG efflux mechanism, showed 2-fold increased activity in EOMA compared with MAE cells. Hyperactive YB-1 and Ape/Ref-1 were responsible for high MRP-1 expression in EOMA. Proximity ligand assay demonstrated MRP-1 and YB-1 binding. Such binding enabled the nuclear targeting of MRP-1 in EOMA in a leptomycin-B-sensitive manner. MRP-1 inhibition as well as knockdown trapped nuclear GSSG, causing cell death of EOMA. Disulfide loading of cells by inhibition of GSSG reductase (bischoloronitrosourea) or thioredoxin reductase (auranofin) was effective in causing EOMA death as well. In sum, EOMA cells survive a heavy oxidant burden by rapid efflux of GSSG, which is lethal if trapped within the cell. A hyperactive MRP-1 system for GSSG efflux acts as a critical survival factor for these cells, making it a potential target for EOMA therapeutics. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Multidrug Resistance-associated Protein-1 (MRP-1)-dependent Glutathione Disulfide (GSSG) Efflux as a Critical Survival Factor for Oxidant-enriched Tumorigenic Endothelial Cells*

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Gordillo, Gayle M.; Biswas, Ayan; Khanna, Savita; Spieldenner, James M.; Pan, Xueliang; Sen, Chandan K.

2016-01-01

Endothelial cell tumors are the most common soft tissue tumors in infants. Tumor-forming endothelial (EOMA) cells are able to escape cell death fate despite excessive nuclear oxidant burden. Our previous work recognized perinuclear Nox-4 as a key contributor to EOMA growth. The objective of this work was to characterize the mechanisms by which EOMA cells evade oxidant toxicity and thrive. In EOMA cells, compared with in the cytosol, the nuclear GSSG/GSH ratio was 5-fold higher. Compared to the ratio observed in healthy murine aortic endothelial (MAE) cells, GSSG/GSH was over twice as high in EOMA cells. Multidrug resistance-associated protein-1 (MRP-1), an active GSSG efflux mechanism, showed 2-fold increased activity in EOMA compared with MAE cells. Hyperactive YB-1 and Ape/Ref-1 were responsible for high MRP-1 expression in EOMA. Proximity ligand assay demonstrated MRP-1 and YB-1 binding. Such binding enabled the nuclear targeting of MRP-1 in EOMA in a leptomycin-B-sensitive manner. MRP-1 inhibition as well as knockdown trapped nuclear GSSG, causing cell death of EOMA. Disulfide loading of cells by inhibition of GSSG reductase (bischoloronitrosourea) or thioredoxin reductase (auranofin) was effective in causing EOMA death as well. In sum, EOMA cells survive a heavy oxidant burden by rapid efflux of GSSG, which is lethal if trapped within the cell. A hyperactive MRP-1 system for GSSG efflux acts as a critical survival factor for these cells, making it a potential target for EOMA therapeutics. PMID:26961872

The hemostatic agent ethamsylate enhances P-selectin membrane expression in human platelets and cultured endothelial cells.

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Alvarez-Guerra, Miriam; Hernandez, Maria Rosa; Escolar, Ginés; Chiavaroli, Carlo; Garay, Ricardo P; Hannaert, Patrick

2002-09-15

Ethamsylate possesses antihemorrhagic properties, but whether or not it directly activates blood platelets is unclear. Here we investigated the platelet activation potential of ethamsylate, by measuring membrane P-selectin expression with flow cytometry in human whole blood and also by immunofluorescence imaging of isolated human platelets. Moreover, we measured membrane P-selectin expression in the SV40-transformed aortic rat endothelial cell line (SVAREC) and 14C-ethamsylate membrane binding and/or uptake in platelets and endothelial cells. Whole blood flow cytometry showed a modest, but statistically significant increase by ethamsylate in the percentage of platelets expressing P-selectin (from 2% to 4-5%, p ethamsylate tested (1 microM), with maximal enhancement of P-selectin expression (75-90%) at 10 microM ethamsylate. Similar results were obtained in SVAREC endothelial cells. 14C-ethamsylate specifically bound to platelets and endothelial cell membranes, without significant uptake into the cell interior. In conclusion, ethamsylate enhances membrane P-selectin expression in human platelets and in cultured endothelial cells. Ethamsylate specifically binds to some protein receptor in platelet and endothelial cell membranes, receptor which can signal for membrane P-selectin expression. These results support the view that ethamsylate acts on the first step of hemostasis, by improving platelet adhesiveness and restoring capillary resistance. Copyright 2002 Elsevier Science Ltd.

Melatonin prevents human pancreatic carcinoma cell PANC-1-induced human umbilical vein endothelial cell proliferation and migration by inhibiting vascular endothelial growth factor expression.

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Cui, Peilin; Yu, Minghua; Peng, Xingchun; Dong, Lv; Yang, Zhaoxu

2012-03-01

Melatonin is an important natural oncostatic agent, and our previous studies have found its inhibitory action on tumor angiogenesis, but the mechanism remains unclear. It is well known that vascular endothelial growth factor (VEGF) plays key roles in tumor angiogenesis and has become an important target for antitumor therapy. Pancreatic cancer is a representative of the most highly vascularized and angiogenic solid tumors, which responds poorly to chemotherapy and radiation. Thus, seeking new treatment strategies targeting which have anti-angiogenic capability is urgent in clinical practice. In this study, a co-culture system between human umbilical vein endothelial cells (HUVECs) and pancreatic carcinoma cells (PANC-1) was used to investigate the direct effect of melatonin on the tumor angiogenesis and its possible action on VEGF expression. We found HUVECs exhibited an increased cell proliferation and cell migration when co-cultured with PANC-1 cells, but the process was prevented when melatonin added to the incubation medium. Melatonin at concentrations of 1 μm and 1 mm inhibited the cell proliferation and migration of HUVECs and also decreased both the VEGF protein secreted to the cultured medium and the protein produced by the PANC-1 cells. In addition, the VEGF mRNA expression was also down-regulated by melatonin. Taken together, our present study shows that melatonin at pharmacological concentrations inhibited the elevated cell proliferation and cell migration of HUVECs stimulated by co-culturing them with PANC-1 cells; this was associated with a suppression of VEGF expression in PANC-1 cells. © 2011 John Wiley & Sons A/S.

Curcumin modulates endothelial permeability and monocyte transendothelial migration by affecting endothelial cell dynamics.

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Monfoulet, Laurent-Emmanuel; Mercier, Sylvie; Bayle, Dominique; Tamaian, Radu; Barber-Chamoux, Nicolas; Morand, Christine; Milenkovic, Dragan

2017-11-01

Curcumin is a phenolic compound that exhibits beneficial properties for cardiometabolic health. We previously showed that curcumin reduced the infiltration of immune cells into the vascular wall and prevented atherosclerosis development in mice. This study aimed to investigate the effect of curcumin on monocyte adhesion and transendothelial migration (TEM) and to decipher the underlying mechanisms of these actions. Human umbilical vein endothelial cells (HUVECs) were exposed to curcumin (0.5-1μM) for 3h prior to their activation by Tumor Necrosis Factor alpha (TNF-α). Endothelial permeability, monocyte adhesion and transendothelial migration assays were conducted under static condition and shear stress that mimics blood flow. We further investigated the impact of curcumin on signaling pathways and on the expression of genes using macroarrays. Pre-exposure of endothelial cells to curcumin reduced monocyte adhesion and their transendothelial migration in both static and shear stress conditions. Curcumin also prevented changes in both endothelial permeability and the area of HUVECs when induced by TNF-α. We showed that curcumin modulated the expression of 15 genes involved in the control of cytoskeleton and endothelial junction dynamic. Finally, we showed that curcumin inhibited NF-κB signaling likely through an antagonist interplay with several kinases as suggested by molecular docking analysis. Our findings demonstrate the ability of curcumin to reduce monocyte TEM through a multimodal regulation of the endothelial cell dynamics with a potential benefit on the vascular endothelial function barrier. Copyright © 2017 Elsevier Inc. All rights reserved.

A novel immunotoxin reveals a new role for CD321 in endothelial cells.

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Takeshi Fukuhara

Full Text Available There are currently several antibody therapies that directly target tumors, and antibody-drug conjugates represent a novel moiety as next generation therapeutics. Here, we used a unique screening probe, DT3C, to identify functional antibodies that recognized surface molecules and functional epitopes, and which provided toxin delivery capability. Accordingly, we generated the 90G4 antibody, which induced DT3C-dependent cytotoxicity in endothelial cells. Molecular analysis revealed that 90G4 recognized CD321, a protein localized at tight junctions. Although CD321 plays a pivotal role in inflammation and lymphocyte trans-endothelial migration, little is known about its mechanism of action in endothelial cells. Targeting of CD321 by the 90G4 immunotoxin induced cell death. Moreover, 90G4 immunotoxin caused cytotoxicity primarily in migratory endothelial cells, but not in those forming sheets, suggesting a critical role for CD321 in tumor angiogenesis. We also found that hypoxia triggered redistribution of CD321 to a punctate localization on the basal side of cells, resulting in functional impairment of tight junctions and increased motility. Thus, our findings raise the intriguing possibility that endothelial CD321 presented cellular localization in tight junction as well as multifunctional dynamics in several conditions, leading to illuminate the importance of widely-expressed CD321 as a potential target for antitumor therapy.

Qidantongmai Protects Endothelial Cells Against Hypoxia-Induced ...

African Journals Online (AJOL)

induced damage. The ability of QDTM to modulate the serum VEGF-A level may play an important role in its effects on endothelial cells. Key words: Traditional Chinese Medicine, human umbilical vein endothelial cells, hypoxia, VEGF ...

Role of PD 0332991 on the Proliferation and Apoptosis of Vascular Endothelial Cells

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Chenlong ZHAO

2018-05-01

Full Text Available Background and objective Angiogenesis is an important process in the development of tumor. PD 0332991, a cell cycle inhibitor, can specifically inhibit CD4/6 phosphorylation and cell cycle progression. In xeongraft mice models, PD 0332991 treated mice had significantly decreased angiogenesis and vascular density compared with the control group, but the mechanism remains unknown. The purpose of this study is to investigate the role and molecular mechanism of PD 0332991 on vascular endothelial cells. Methods EA.hy926 cells, a kind of vascular endothelial cell, were used as the research model. The effects of PD 0332991 on the activity and proliferation of EA.hy926 cells were detected by the MTT, EdU assays. Wound-healing assays and transwell assays were used to determine the effects of PD 0332991 on the mobility of EA.hy926. The influence of PD 0332991 on cell cycle and apoptosis of endothelial cells was tested by flow cytometry, and the Western blot was applied to observe the expression of cell cycle related proteins in EA.hy926 cells treated by PD 0332991. Results PD 0332991 significantly inhibited the proliferation and mobility of EA.hy926 cells, caused cell cycle arrest and apoptosis. At the same time, PD 0332991 inhibited the expression of CDK4/6 and phosphorylation of Rb, and thus inhibited the cell cycle progression of EA.hy926 cells. Conclusion PD 0332991 can inhibit the proliferation and activity of endothelial cells and induces apoptosis.

Isolation and characterization of human umbilical cord-derived endothelial colony-forming cells

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Zhang, Hao; Tao, Yanling; Ren, Saisai; Liu, Haihui; Zhou, Hui; Hu, Jiangwei; Tang, Yongyong; Zhang, Bin; Chen, Hu

2017-01-01

Endothelial colony-forming cells (ECFCs) are a population of endothelial progenitor cells (EPCs) that display robust proliferative potential and vessel-forming capability. Previous studies have demonstrated that a limited number of ECFCs may be obtained from adult bone marrow, peripheral blood and umbilical cord (UC) blood. The present study describes an effective method for isolating ECFCs from human UC. The ECFCs derived from human UC displayed the full properties of EPCs. Analysis of the growth kinetics, cell cycle and colony-forming ability of the isolated human UC-ECFCs indicated that the cells demonstrated properties of stem cells, including relative stability and rapid proliferation in vitro. Gene expression of Fms related tyrosine kinase 1, kinase insert domain receptor, vascular endothelial cadherin, cluster of differentiation (CD)31, CD34, epidermal growth factor homology domains-2, von Willebrand factor and endothelial nitric oxide synthase was assessed by reverse transcription-polymerase chain reaction. The cells were positive for CD34, CD31, CD73, CD105 and vascular endothelial growth factor receptor-2, and negative for CD45, CD90 and human leukocyte antigen-antigen D related protein according to flow cytometry. 1,1′-dioctadecyl-3,3,3′,3′-tetra-methyl-indocarbocyanine perchlorate-labeled acetylated low-density lipoprotein and fluorescein isothiocyanate-Ulex europaeus-l were used to verify the identity of the UC-ECFCs. Matrigel was used to investigate tube formation capability. The results demonstrated that the reported technique is a valuable method for isolating human UC-ECFCs, which have potential for use in vascular regeneration. PMID:29067104

Regulation of brain capillary endothelial cells by P2Y receptors coupled to Ca2+, phospholipase C and mitogen-activated protein kinase.

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Albert, J L; Boyle, J P; Roberts, J A; Challiss, R A; Gubby, S E; Boarder, M R

1997-11-01

1. The blood-brain barrier is formed by capillary endothelial cells and is regulated by cell-surface receptors, such as the G protein-coupled P2Y receptors for nucleotides. Here we investigated some of the characteristics of control of brain endothelial cells by these receptors, characterizing the phospholipase C and Ca2+ response and investigating the possible involvement of mitogen-activated protein kinases (MAPK). 2. Using an unpassaged primary culture of rat brain capillary endothelial cells we showed that ATP, UTP and 2-methylthio ATP (2MeSATP) give similar and substantial increases in cytosolic Ca2+, with a rapid rise to peak followed by a slower decline towards basal or to a sustained plateau. Removal of extracellular Ca2+ had little effect on the peak Ca2+-response, but resulted in a more rapid decline to basal. There was no response to alpha,beta-MethylATP (alpha,beta MeATP) in these unpassaged cells, but a response to this P2X agonist was seen after a single passage. 3. ATP (log EC50 -5.1+/-0.2) also caused an increase in the total [3H]-inositol (poly)phosphates ([3H]-InsPx) in the presence of lithium with a rank order of agonist potency of ATP=UTP=UDP>ADP, with 2MeSATP and alpha,beta MeATP giving no detectable response. 4. Stimulating the cells with ATP or UTP gave a rapid rise in the level of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), with a peak at 10 s followed by a decline to a sustained plateau phase. 2MeSATP gave no detectable increase in the level of Ins(1,4,5)P3. 5. None of the nucleotides tested affected basal cyclic AMP, while ATP and ATPgammaS, but not 2MeSATP, stimulated cyclic AMP levels in the presence of 5 microM forskolin. 6. Both UTP and ATP stimulated tyrosine phosphorylation of p42 and p44 mitogen-activated protein kinase (MAPK), while 2MeSATP gave a smaller increase in this index of MAPK activation. By use of a peptide kinase assay, UTP gave a substantial increase in MAPK activity with a concentration-dependency consistent with

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

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

2016-04-15

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

Endothelial cell-derived microparticles induce plasmacytoid dendritic cell maturation: potential implications in inflammatory diseases.

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Angelot, Fanny; Seillès, Estelle; Biichlé, Sabeha; Berda, Yael; Gaugler, Béatrice; Plumas, Joel; Chaperot, Laurence; Dignat-George, Françoise; Tiberghien, Pierre; Saas, Philippe; Garnache-Ottou, Francine

2009-11-01

Increased circulating endothelial microparticles, resulting from vascular endothelium dysfunction, and plasmacytoid dendritic cell activation are both encountered in common inflammatory disorders. The aim of our study was to determine whether interactions between endothelial microparticles and plasmacytoid dendritic cells could contribute to such pathologies. Microparticles generated from endothelial cell lines, platelets or activated T cells were incubated with human plasmacytoid dendritic cells sorted from healthy donor blood or with monocyte-derived dendritic cells. Dendritic cell maturation was evaluated by flow cytometry, cytokine secretion as well as naive T-cell activation and polarization. Labeled microparticles were also used to study cellular interactions. Endothelial microparticles induced plasmacytoid dendritic cell maturation. In contrast, conventional dendritic cells were resistant to endothelial microparticle-induced maturation. In addition to upregulation of co-stimulatory molecules, endothelial microparticle-matured plasmacytoid dendritic cells secreted inflammatory cytokines (interleukins 6 and 8, but no interferon-alpha) and also induced allogeneic naive CD4(+) T cells to proliferate and to produce type 1 cytokines such as interferon-gamma and tumor necrosis factor-alpha. Endothelial microparticle endocytosis by plasmacytoid dendritic cells appeared to be required for plasmacytoid dendritic cell maturation. Importantly, the ability of endothelial microparticles to induce plasmacytoid dendritic cells to mature was specific as microparticles derived from activated T cells or platelets (the major source of circulating microparticules in healthy subjects) did not induce such plasmacytoid dendritic cell maturation. Our data show that endothelial microparticles specifically induce plasmacytoid dendritic cell maturation and production of inflammatory cytokines. This novel activation pathway may be implicated in various inflammatory disorders and

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

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

2015-01-01

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

Heart-type fatty-acid-binding protein (FABP3 is a lysophosphatidic acid-binding protein in human coronary artery endothelial cells

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Ryoko Tsukahara

2014-01-01

Full Text Available Fatty-acid-binding protein 3, muscle and heart (FABP3, also known as heart-type FABP, is a member of the family of intracellular lipid-binding proteins. It is a small cytoplasmic protein with a molecular mass of about 15 kDa. FABPs are known to be carrier proteins for transporting fatty acids and other lipophilic substances from the cytoplasm to the nucleus, where these lipids are released to a group of nuclear receptors such as peroxisome proliferator-activated receptors (PPARs. In this study, using lysophosphatidic acid (LPA-coated agarose beads, we have identified FABP3 as an LPA carrier protein in human coronary artery endothelial cells (HCAECs. Administration of LPA to HCAECs resulted in a dose-dependent increase in PPARγ activation. Furthermore, the LPA-induced PPARγ activation was abolished when the FABP3 expression was reduced using small interfering RNA (siRNA. We further show that the nuclear fraction of control HCAECs contained a significant amount of exogenously added LPA, whereas FABP3 siRNA-transfected HCAECs had a decreased level of LPA in the nucleus. Taken together, these results suggest that FABP3 governs the transcriptional activities of LPA by targeting them to cognate PPARγ in the nucleus.

Protein Kinase-C Beta Contributes to Impaired Endothelial Insulin Signaling in Humans with Diabetes Mellitus

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Tabit, Corey E; Shenouda, Sherene M; Holbrook, Monica; Fetterman, Jessica L; Kiani, Soroosh; Frame, Alissa A; Kluge, Matthew A; Held, Aaron; Dohadwala, Mustali; Gokce, Noyan; Farb, Melissa; Rosenzweig, James; Ruderman, Neil; Vita, Joseph A; Hamburg, Naomi M

2013-01-01

Background Abnormal endothelial function promotes atherosclerotic vascular disease in diabetes. Experimental studies indicate that disruption of endothelial insulin signaling through the activity of protein kinase C-β (PKCβ) and nuclear factor κB (NFκB) reduces nitric oxide availability. We sought to establish whether similar mechanisms operate in the endothelium in human diabetes mellitus. Methods and Results We measured protein expression and insulin response in freshly isolated endothelial cells from patients with Type 2 diabetes mellitus (n=40) and non-diabetic controls (n=36). Unexpectedly, we observed 1.7-fold higher basal endothelial nitric oxide synthase (eNOS) phosphorylation at serine 1177 in patients with diabetes (P=0.007) without a difference in total eNOS expression. Insulin stimulation increased eNOS phosphorylation in non-diabetic subjects but not in diabetic patients (P=0.003) consistent with endothelial insulin resistance. Nitrotyrosine levels were higher in diabetic patients indicating endothelial oxidative stress. PKCβ expression was higher in diabetic patients and was associated with lower flow-mediated dilation (r=−0.541, P=0.02) Inhibition of PKCβ with LY379196 reduced basal eNOS phosphorylation and improved insulin-mediated eNOS activation in patients with diabetes. Endothelial NFκB activation was higher in diabetes and was reduced with PKCβ inhibition. Conclusions We provide evidence for the presence of altered eNOS activation, reduced insulin action and inflammatory activation in the endothelium of patients with diabetes. Our findings implicate PKCβ activity in endothelial insulin resistance. PMID:23204109

Fibrinogen-Induced Streptococcus mutans Biofilm Formation and Adherence to Endothelial Cells

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Telma Blanca Lombardo Bedran

2013-01-01

Full Text Available Streptococcus mutans, the predominant bacterial species associated with dental caries, can enter the bloodstream and cause infective endocarditis. The aim of this study was to investigate S. mutans biofilm formation and adherence to endothelial cells induced by human fibrinogen. The putative mechanism by which biofilm formation is induced as well as the impact of fibrinogen on S. mutans resistance to penicillin was also evaluated. Bovine plasma dose dependently induced biofilm formation by S. mutans. Of the various plasma proteins tested, only fibrinogen promoted the formation of biofilm in a dose-dependent manner. Scanning electron microscopy observations revealed the presence of complex aggregates of bacterial cells firmly attached to the polystyrene support. S. mutans in biofilms induced by the presence of fibrinogen was markedly resistant to the bactericidal effect of penicillin. Fibrinogen also significantly increased the adherence of S. mutans to endothelial cells. Neither S. mutans cells nor culture supernatants converted fibrinogen into fibrin. However, fibrinogen is specifically bound to the cell surface of S. mutans and may act as a bridging molecule to mediate biofilm formation. In conclusion, our study identified a new mechanism promoting S. mutans biofilm formation and adherence to endothelial cells which may contribute to infective endocarditis.

Activation of protein kinase C and disruption of endothelial monolayer integrity by sodium arsenite-Potential mechanism in the development of atherosclerosis

International Nuclear Information System (INIS)

Pereira, Flavia E.; Coffin, J. Douglas; Beall, Howard D.

2007-01-01

Arsenic exposure has been shown to exacerbate atherosclerosis, beginning with activation of the endothelium that lines the vessel wall. Endothelial barrier integrity is maintained by proteins of the adherens junction (AJ) such as vascular endothelial cadherin (VE-cadherin) and β-catenin and their association with the actin cytoskeleton. In the present study, human aortic endothelial cells (HAECs) were exposed to 1, 5 and 10 μM sodium arsenite [As(III)] for 1, 6, 12 and 24 h, and the effects on endothelial barrier integrity were determined. Immunofluorescence studies revealed formation of actin stress fibers and non-uniform VE-cadherin and β-catenin staining at cell-cell junctions that were concentration- and time-dependent. Intercellular gaps were observed with a measured increase in endothelial permeability. In addition, concentration-dependent increases in tyrosine phosphorylation (PY) of β-catenin and activation of protein kinase Cα (PKCα) were observed. Inhibition of PKCα restored VE-cadherin and β-catenin staining at cell-cell junctions and abolished the As(III)-induced formation of actin stress fibers and intercellular gaps. Endothelial permeability and PY of β-catenin were also reduced to basal levels. These results demonstrate that As(III) induces activation of PKCα, which leads to increased PY of β-catenin downstream of PKCα activation. Phosphorylation of β-catenin plausibly severs the association of VE-cadherin and β-catenin, which along with formation of actin stress fibers, results in intercellular gap formation and increased endothelial permeability. To the best of our knowledge, this is the first report demonstrating that As(III) causes a loss of endothelial monolayer integrity, which potentially could contribute to the development of atherosclerosis

PX-18 Protects Human Saphenous Vein Endothelial Cells under Arterial Blood Pressure.

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Kupreishvili, Koba; Stooker, Wim; Emmens, Reindert W; Vonk, Alexander B A; Sipkens, Jessica A; van Dijk, Annemieke; Eijsman, Leon; Quax, Paul H; van Hinsbergh, Victor W M; Krijnen, Paul A J; Niessen, Hans W M

2017-07-01

Arterial blood pressure-induced shear stress causes endothelial cell apoptosis and inflammation in vein grafts after coronary artery bypass grafting. As the inflammatory protein type IIA secretory phospholipase A 2 (sPLA 2 -IIA) has been shown to progress atherosclerosis, we hypothesized a role for sPLA 2 -IIA herein. The effects of PX-18, an inhibitor of both sPLA 2 -IIA and apoptosis, on residual endothelium and the presence of sPLA 2 -IIA were studied in human saphenous vein segments (n = 6) perfused at arterial blood pressure with autologous blood for 6 hrs. The presence of PX-18 in the perfusion blood induced a significant 20% reduction in endothelial cell loss compared to veins perfused without PX18, coinciding with significantly reduced sPLA 2 -IIA levels in the media of the vein graft wall. In addition, PX-18 significantly attenuated caspase-3 activation in human umbilical vein endothelial cells subjected to shear stress via mechanical stretch independent of sPLA 2 -IIA. In conclusion, PX-18 protects saphenous vein endothelial cells from arterial blood pressure-induced death, possibly also independent of sPLA 2 -IIA inhibition. Copyright © 2017 Elsevier Inc. All rights reserved.

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

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

2017-08-04

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

Differential Gene Expression of Primary Cultured Lymphatic and Blood Vascular Endothelial Cells

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Gregory M. Nelson

2007-12-01

Full Text Available Blood vascular endothelial cells (BECs and the developmentally related lymphatic endothelial cells (LECs create complementary, yet distinct vascular networks. Each endothelial cell type interacts with flowing fluid and circulating cells, yet each vascular system has evolved specialized gene expression programs and thus both cell types display different phenotypes. BECs and LECs express distinct genes that are unique to their specific vascular microenvironment. Tumors also take advantage of the molecules that are expressed in these vascular systems to enhance their metastatic potential. We completed transcriptome analyses on primary cultured LECs and BECs, where each comparative set was isolated from the same individual. Differences were resolved in the expression of several major categories, such as cell adhesion molecules (CAMs, cytokines, cytokine receptors. We have identified new molecules that are associated with BECs (e.g., claudin-9, CXCL11, neurexin-1, neurexin-2, the neuronal growth factor regulator-1 and LECs (e.g., claudin-7, CD58, hyaluronan and proteoglycan link protein 1 (HAPLN1, the poliovirus receptor-related 3 molecule that may lead to novel therapeutic treatments for diseases of lymphatic or blood vessels, including metastasis of cancer to lymph nodes or distant organs.

Exosomes from metastatic cancer cells transfer amoeboid phenotype to non-metastatic cells and increase endothelial permeability: their emerging role in tumor heterogeneity.

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Schillaci, Odessa; Fontana, Simona; Monteleone, Francesca; Taverna, Simona; Di Bella, Maria Antonietta; Di Vizio, Dolores; Alessandro, Riccardo

2017-07-05

The goal of this study was to understand if exosomes derived from high-metastatic cells may influence the behavior of less aggressive cancer cells and the properties of the endothelium. We found that metastatic colon cancer cells are able to transfer their amoeboid phenotype to isogenic primary cancer cells through exosomes, and that this morphological transition is associated with the acquisition of a more aggressive behavior. Moreover, exosomes from the metastatic line (SW620Exos) exhibited higher ability to cause endothelial hyperpermeability than exosomes from the non metastatic line (SW480Exos). SWATH-based quantitative proteomic analysis highlighted that SW620Exos are significantly enriched in cytoskeletal-associated proteins including proteins activating the RhoA/ROCK pathway, known to induce amoeboid properties and destabilization of endothelial junctions. In particular, thrombin was identified as a key mediator of the effects induced by SW620Exos in target cells, in which we also found a significant increase of RhoA activity. Overall, our results demonstrate that in a heterogeneous context exosomes released by aggressive sub-clones can contribute to accelerate tumor progression by spreading malignant properties that affect both the tumor cell plasticity and the endothelial cell behavior.

The regulatory mechanism of Hsp90α secretion from endothelial cells and its role in angiogenesis during wound healing

International Nuclear Information System (INIS)

Song, Xiaomin; Luo, Yongzhang

2010-01-01

Research highlights: → Growth factors such as bFGF, VEGF, PDGF and SDF-1 stimulate Hsp90α secretion from endothelial cells. → Secreted Hsp90α localizes on the leading edge of activated endothelial cells. → Secreted Hsp90α promotes angiogenesis in wound healing. -- Abstract: Heat shock protein 90α (Hsp90α) is a ubiquitously expressed molecular chaperone, which is essential for the maintenance of eukaryote homeostasis. Hsp90α can also be secreted extracellularly and is associated with several physiological and pathological processes including wound healing, cancer, infectious diseases and diabetes. Angiogenesis, defined as the sprouting of new blood vessels from pre-existing capillaries via endothelial cell proliferation and migration, commonly occurs in and contributes to the above mentioned processes. However, the secretion of Hsp90α from endothelial cells and also its function in angiogenesis are still unclear. Here we investigated the role of extracellular Hsp90α in angiogenesis using dermal endothelial cells in vitro and a wound healing model in vivo. We find that the secretion of Hsp90α but not Hsp90β is increased in activated endothelial cells with the induction of angiogenic factors and matrix proteins. Secreted Hsp90α localizes on the leading edge of endothelial cells and promotes their angiogenic activities, whereas Hsp90α neutralizing antibodies reverse the effect. Furthermore, using a mouse skin wound healing model in vivo, we demonstrate that extracellular Hsp90α localizes on blood vessels in granulation tissues of wounded skin and promotes angiogenesis during wound healing. Taken together, our study reveals that Hsp90α can be secreted by activated endothelial cells and is a positive regulator of angiogenesis, suggesting the potential application of Hsp90α as a stimulator for wound repair.

The regulatory mechanism of Hsp90{alpha} secretion from endothelial cells and its role in angiogenesis during wound healing

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Song, Xiaomin [National Engineering Laboratory for Anti-tumor Protein Therapeutics, Tsinghua University, Beijing 100084 (China); Beijing Key Laboratory for Protein Therapeutics, Tsinghua University, Beijing 100084 (China); Cancer Biology Laboratory, School of Life Sciences, Tsinghua University, Beijing 100084 (China); Luo, Yongzhang, E-mail: yluo@tsinghua.edu.cn [National Engineering Laboratory for Anti-tumor Protein Therapeutics, Tsinghua University, Beijing 100084 (China); Beijing Key Laboratory for Protein Therapeutics, Tsinghua University, Beijing 100084 (China); Cancer Biology Laboratory, School of Life Sciences, Tsinghua University, Beijing 100084 (China)

2010-07-16

Research highlights: {yields} Growth factors such as bFGF, VEGF, PDGF and SDF-1 stimulate Hsp90{alpha} secretion from endothelial cells. {yields} Secreted Hsp90{alpha} localizes on the leading edge of activated endothelial cells. {yields} Secreted Hsp90{alpha} promotes angiogenesis in wound healing. -- Abstract: Heat shock protein 90{alpha} (Hsp90{alpha}) is a ubiquitously expressed molecular chaperone, which is essential for the maintenance of eukaryote homeostasis. Hsp90{alpha} can also be secreted extracellularly and is associated with several physiological and pathological processes including wound healing, cancer, infectious diseases and diabetes. Angiogenesis, defined as the sprouting of new blood vessels from pre-existing capillaries via endothelial cell proliferation and migration, commonly occurs in and contributes to the above mentioned processes. However, the secretion of Hsp90{alpha} from endothelial cells and also its function in angiogenesis are still unclear. Here we investigated the role of extracellular Hsp90{alpha} in angiogenesis using dermal endothelial cells in vitro and a wound healing model in vivo. We find that the secretion of Hsp90{alpha} but not Hsp90{beta} is increased in activated endothelial cells with the induction of angiogenic factors and matrix proteins. Secreted Hsp90{alpha} localizes on the leading edge of endothelial cells and promotes their angiogenic activities, whereas Hsp90{alpha} neutralizing antibodies reverse the effect. Furthermore, using a mouse skin wound healing model in vivo, we demonstrate that extracellular Hsp90{alpha} localizes on blood vessels in granulation tissues of wounded skin and promotes angiogenesis during wound healing. Taken together, our study reveals that Hsp90{alpha} can be secreted by activated endothelial cells and is a positive regulator of angiogenesis, suggesting the potential application of Hsp90{alpha} as a stimulator for wound repair.

Activation of endothelial cells after exposure to ambient ultrafine particles: The role of NADPH oxidase

International Nuclear Information System (INIS)

Mo Yiqun; Wan Rong; Chien Sufan; Tollerud, David J.; Zhang Qunwei

2009-01-01

Several studies have shown that ultrafine particles (UFPs) may pass from the lungs to the circulation because of their very small diameter, and induce lung oxidative stress with a resultant increase in lung epithelial permeability. The direct effects of UFPs on vascular endothelium remain unknown. We hypothesized that exposure to UFPs leads to endothelial cell O 2 ·- generation via NADPH oxidase and results in activation of endothelial cells. Our results showed that UFPs, at a non-toxic dose, induced reactive oxygen species (ROS) generation in mouse pulmonary microvascular endothelial cells (MPMVEC) that was inhibited by pre-treatment with the ROS scavengers or inhibitors, but not with the mitochondrial inhibitor, rotenone. UFP-induced ROS generation in MPMVEC was abolished by p67 phox siRNA transfection and UFPs did not cause ROS generation in MPMVEC isolated from gp91 phox knock-out mice. UFP-induced ROS generation in endothelial cells was also determined in vivo by using a perfused lung model with imaging. Moreover, Western blot and immunofluorescence staining results showed that MPMVEC treated with UFPs resulted in the translocation of cytosolic proteins of NADPH oxidase, p47 phox , p67 phox and rac 1, to the plasma membrane. These results demonstrate that NADPH oxidase in the pulmonary endothelium is involved in ROS generation following exposure to UFPs. To investigate the activation of endothelial cells by UFP-induced oxidative stress, we determined the activation of the mitogen-activated protein kinases (MAPKs) in MPMVEC. Our results showed that exposure of MPMVEC to UFPs caused increased phosphorylation of p38 and ERK1/2 MAPKs that was blocked by pre-treatment with DPI or p67 phox siRNA. Exposure of MPMVEC obtained from gp91 phox knock-out mice to UFPs did not cause increased phosphorylation of p38 and ERK1/2 MAPKs. These findings confirm that UFPs can cause endothelial cells to generate ROS directly via activation of NADPH oxidase. UFP-induced ROS lead to

Nonparenchymal cells cultivated from explants of fibrotic liver resemble endothelial and smooth muscle cells from blood vessel walls

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Voss, B.; Rauterberg, J.; Pott, G.; Brehmer, U.; Allam, S.; Lehmann, R.; von Bassewitz, D.B.

1982-01-01

Tissue specimens from human fibrotic liver obtained by needle biopsy were cultured. Two cell types emerged from the tissue explants. From their morphology and biosynthetic products they resembled smooth muscle cells and endothelial cells from blood vessel walls. In the endothelial cells, factor VIII-associated protein was demonstrated by indirect immunofluorescence. Synthesis of collagen types I and III, basement membrane collagen types IV and V, and fibronectin by both cell types was observed by immunofluorescence microscopy. Homogeneous cultures of smooth muscle cells were observed in subcultures. After incubation with [ 14 C]glycine, collagen was isolated and characterized by CM cellulose chromatography, and consisted mainly of types I and III. These data suggest involvement of mesenchymal cells in hepatic fibrosis; they presumably originate from blood vessel or sinusoidal walls

Modulation of cGMP by human HO-1 retrovirus gene transfer in pulmonary microvessel endothelial cells.

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Abraham, Nader G; Quan, Shuo; Mieyal, Paul A; Yang, Liming; Burke-Wolin, Theresa; Mingone, Christopher J; Goodman, Alvin I; Nasjletti, Alberto; Wolin, Michael S

2002-11-01

Carbon monoxide (CO) stimulates guanylate cyclase (GC) and increases guanosine 3',5'-cyclic monophosphate (cGMP) levels. We transfected rat-lung pulmonary endothelial cells with a retrovirus-mediated human heme oxygenase (hHO)-1 gene. Pulmonary cells that expressed hHO-1 exhibited a fourfold increase in HO activity associated with decreases in the steady-state levels of heme and cGMP without changes in soluble GC (sGC) and endothelial nitric oxide synthase (NOS) proteins or basal nitrite production. Heme elicited significant increases in CO production and intracellular cGMP levels in both pulmonary endothelial and pulmonary hHO-1-expressing cells. N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, significantly decreased cGMP levels in heme-treated pulmonary endothelial cells but not heme-treated hHO-1-expressing cells. In the presence of exogenous heme, CO and cGMP levels in hHO-1-expressing cells exceeded the corresponding levels in pulmonary endothelial cells. Acute exposure of endothelial cells to SnCl2, which is an inducer of HO-1, increased cGMP levels, whereas chronic exposure decreased heme and cGMP levels. These results indicate that prolonged overexpression of HO-1 ultimately decreases sGC activity by limiting the availability of cellular heme. Heme activates sGC and enhances cGMP levels via a mechanism that is largely insensitive to NOS inhibition.

Intravenous Lipid Infusion Induces Endoplasmic Reticulum Stress in Endothelial Cells and Blood Mononuclear Cells of Healthy Adults.

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Tampakakis, Emmanouil; Tabit, Corey E; Holbrook, Monika; Linder, Erika A; Berk, Brittany D; Frame, Alissa A; Bretón-Romero, Rosa; Fetterman, Jessica L; Gokce, Noyan; Vita, Joseph A; Hamburg, Naomi M

2016-01-11

Endoplasmic reticulum (ER) stress and the subsequent unfolded protein response may initially be protective, but when prolonged, have been implicated in atherogenesis in diabetic conditions. Triglycerides and free fatty acids (FFAs) are elevated in patients with diabetes and may contribute to ER stress. We sought to evaluate the effect of acute FFA elevation on ER stress in endothelial and circulating white cells. Twenty-one healthy subjects were treated with intralipid (20%; 45 mL/h) plus heparin (12 U/kg/h) infusion for 5 hours. Along with increased triglyceride and FFA levels, intralipid/heparin infusion reduced the calf reactive hyperemic response without a change in conduit artery flow-mediated dilation consistent with microvascular dysfunction. To investigate the short-term effects of elevated triglycerides and FFA, we measured markers of ER stress in peripheral blood mononuclear cells (PBMCs) and vascular endothelial cells (VECs). In VECs, activating transcription factor 6 (ATF6) and phospho-inositol requiring kinase 1 (pIRE1) proteins were elevated after infusion (both P<0.05). In PBMCs, ATF6 and spliced X-box-binding protein 1 (XBP-1) gene expression increased by 2.0- and 2.5-fold, respectively (both P<0.05), whereas CHOP and GADD34 decreased by ≈67% and 74%, respectively (both P<0.01). ATF6 and pIRE1 protein levels also increased (both P<0.05), and confocal microscopy revealed the nuclear localization of ATF6 after infusion, suggesting activation. Along with microvascular dysfunction, intralipid infusion induced an early protective ER stress response evidenced by activation of ATF6 and IRE1 in both leukocytes and endothelial cells. Our results suggest a potential link between metabolic disturbances and ER stress that may be relevant to vascular disease. © 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

Serum of patients with antiphospholipid syndrome induces adhesion molecules in endothelial cells.

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Engel, Bettina; Müller, Gregor; Roch, Beate; Schröder, Hans-Egbert; Aringer, Martin; Bornstein, Stefan R; Morawietz, Henning

2017-11-01

The antiphospholipid syndrome (APS) is a systemic auto-immune disease with an unclear pathophysiology. The aim of our study was to understand the development of APS on a cellular level. Therefore, we analyzed the influence of human serum of APS patients on endothelial expression of specific genes and proteins in comparison to a control group. In this study, we analyzed the expression of ICAM-1, VCAM-1, E-selectin and annexin V in primary cultures of human umbilical vein endothelial cells (HUVEC) in response to 10% (v/v) serum of control patients (n = 6), patients with systemic lupus erythematosus (SLE) and no APS (n = 4) or APS patients (n = 9) for 24 h. Total RNA was prepared from confluent endothelial cell layers and mRNA expression of ICAM-1, VCAM-1 and E-selectin was analyzed by reverse transcription polymerase-chain reaction (RT-PCR). The protein expression was determined by Western blot. Serum protein concentrations of soluble forms of adhesion molecules sICAM-1 and sVCAM-1 were quantified by ELISA. Gene expression data were correlated with clinical parameters. The mRNA expression of ICAM-1 was increased in cells incubated with serum from APS patients (166 ± 22% of control; P = 0.023). Serum of patients with (SLE)/no APS caused a 1.4-fold higher ICAM-1 mRNA level. Western blot analysis showed an increase in protein expression of adhesion molecules ICAM-1 (260 ± 49%; P = 0.011) and VCAM-1 (357 ± 97%; P = 0.023) in cells that were incubated with serum from APS patients. Plasma analysis showed elevated levels of sVCAM-1 in APS patients (189 ± 34%; P = 0.045) compared to the levels measured in the control group. The sVCAM-1 plasma level was correlating with the frequency of abortions. An augmented expression of endothelial adhesion molecules is involved in the pathophysiology of patients with antiphospholipid syndrome. Copyright © 2017 Elsevier B.V. All rights reserved.

Glucagon-Like Peptide 1 Prevents Reactive Oxygen Species-Induced Endothelial Cell Senescence Through the Activation of Protein Kinase A

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Oeseburg, Hisko; de Boer, Rudolf A.; Buikema, Hendrik; van der Harst, Pim; van Gilst, Wiek H.; Sillje, Herman H. W.

Objective-Endothelial cell senescence is an important contributor to vascular aging and is increased under diabetic conditions. Here we investigated whether the antidiabetic hormone glucagon-like peptide 1 (GLP-1) could prevent oxidative stress-induced cellular senescence in endothelial cells.

Signaling hierarchy regulating human endothelial cell development

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Our present knowledge of the regulation of mammalian endothelial cell differentiation has been largely derived from studies of mouse embryonic development. However, unique mechanisms and hierarchy of signals that govern human endothelial cell development are unknown and, thus, explored in these stud...

Animal study on transplantation of human umbilical vein endothelial cells for corneal endothelial decompensation

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Li Cui

2014-06-01

Full Text Available AIM: To explore the feasibility of culturing human umbilical vein endothelial cells(HUVECon acellular corneal stroma and performing the posterior lamellar endothelial keratoplasty(PLEKtreating corneal endothelial decompensation.METHODS: Thirty New-Zealand rabbits were divided into three groups randomly, 10 rabbits for experimental group, 10 for stroma group and 10 for control group. Corneal endothelial cells were removed to establish animal model of corneal endothelial failure. PLEK was performed on the rabbits of experimental group and stroma group, and nothing was transplantated onto the rabbits of control group with the deep layer excised only. Postoperative observation was taken for 3mo. The degree of corneal edema and central corneal thickness were recorded for statistical analysis.RESULTS: Corneas in experimental group were relieved in edema obviously compared with that in stroma group and the control group, and showed increased transparency 7d after the operation. The average density of endothelial cells was 2 026.4±129.3cells/mm2, and average central corneal thickness was 505.2±25.4μm in experimental group, while 1 535.6±114.5μm in stroma group and 1 493.5±70.2μm in control group 3mo after operation.CONCLUSION:We achieved preliminary success in our study that culturing HUVEC on acellular corneal stroma and performing PLEK for corneal endothelial decompensation. HUVEC transplanted could survive in vivo, and have normal biological function of keeping cornea transparent. This study provides a new idea and a new way clinically for the treatment of corneal endothelial diseases.

Radiation-induced VEGF-C expression and endothelial cell proliferation in lung cancer

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Chen, Yu-Hsuan [National Taiwan University Hospital, Department of Oncology, Taipei (China); National Taiwan University, Pharmacological Institute, College of Medicine, Taipei (China); Pan, Shiow-Lin; Wang, Jing-Chi; Teng, Che-Ming [National Taiwan University, Pharmacological Institute, College of Medicine, Taipei (China); Kuo, Sung-Hsin [National Taiwan University Hospital, Department of Oncology, Taipei (China); National Taiwan University College of Medicine, Department of Internal Medicine, Taipei (China); Cheng, Jason Chia-Hsien [National Taiwan University Hospital, Department of Oncology, Taipei (China); National Taiwan University College of Medicine, Graduate Institute of Clinical Medicine, Taipei (China)

2014-12-15

The present study was undertaken to investigate whether radiation induces the expression of vascular endothelial growth factor C (VEGF-C) through activation of the PI3K/Akt/mTOR pathway,subsequently affecting endothelial cells. Radiotherapy-induced tumor micro-lymphatic vessel density (MLVD) was determined in a lung cancer xenograft model established in SCID mice. The protein expression and phosphorylation of members of the PI3K/Akt/mTOR pathway and VEGF-C secretion and mRNA expression in irradiated lung cancer cells were assessed by Western blot analysis, enzyme-linked immunosorbent assays (ELISAs), and reverse transcriptase-polymerase chain reaction (RT-PCR). Moreover, specific chemical inhibitors were used to evaluate the role of the PI3K/Akt/mTOR signaling pathway. Conditioned medium (CM) from irradiated control-siRNA or VEGF-C-siRNA-expressing A549 cells was used to evaluate the proliferation of endothelial cells by the MTT assay. Radiation increased VEGF-C expression in a dose-dependent manner over time at the protein but not at the mRNA level. Radiation also up-regulated the phosphorylation of Akt, mTOR, 4EBP, and eIF4E, but not of p70S6K. Radiation-induced VEGF-C expression was down-regulated by LY294002 and rapamycin (both p < 0.05). Furthermore, CM from irradiated A549 cells enhanced human umbilical vein endothelial cell (HUVEC) and lymphatic endothelial cell (LEC) proliferation, which was not observed with CM from irradiated VEGF-C-siRNA-expressing A549 cells. Radiation-induced activation of the PI3K/Akt/mTOR signaling pathway increases VEGF-C expression in lung cancer cells, thereby promoting endothelial cell proliferation. (orig.) [German] Die vorliegende Studie untersucht, ob die Strahlung die Expression von VEGF-C (vascular endothelial growth factor C) mittels Aktivierung des PI3K/Akt/mTOR-Signalwegs induziert und anschliessend die endothelialen Zellen beeinflusst. Die durch Strahlentherapie induzierte Mikrolymphgefaessdichte (MLVD) im Tumor wurde in

Date syrup-derived polyphenols attenuate angiogenic responses and exhibits anti-inflammatory activity mediated by vascular endothelial growth factor and cyclooxygenase-2 expression in endothelial cells.

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Taleb, Hajer; Morris, R Keith; Withycombe, Cathryn E; Maddocks, Sarah E; Kanekanian, Ara D

2016-07-01

Bioactive components such as polyphenols, present in many plants, are purported to have anti-inflammatory and antiangiogenic properties. Date syrup, produced from date fruit of the date palm tree, has traditionally been used to treat a wide range of diseases with etiologies involving angiogenesis and inflammation. It was hypothesized that polyphenols in date syrup reduce angiogenic responses such as cell migration, tube formation, and matrix metalloproteinase activity in an inflammatory model by exhibiting anti-inflammatory activity mediated by vascular endothelial growth factor (VEGF) and the prostaglandin enzyme cyclooxygenase-2 (COX-2) in endothelial cells. Date syrup polyphenols at 60 and 600μg/mL reduced inflammation and suppressed several stages of angiogenesis, including endothelial cell migration, invasion, matrix metalloproteinase activity, and tube formation, without evidence of cytotoxicity. VEGF and COX-2 expression induced by tumor necrosis factor-alpha at both gene expression and protein level was significantly reduced by date syrup polyphenols in comparison to untreated cells. In conclusion, polyphenols in date syrup attenuated angiogenic responses and exhibited anti-inflammatory activity mediated by VEGF and COX-2 expression in endothelial cells. Copyright © 2016 Elsevier Inc. All rights reserved.

Laminin Peptide-Immobilized Hydrogels Modulate Valve Endothelial Cell Hemostatic Regulation.

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Liezl Rae Balaoing

Full Text Available Valve endothelial cells (VEC have unique phenotypic responses relative to other types of vascular endothelial cells and have highly sensitive hemostatic functions affected by changes in valve tissues. Furthermore, effects of environmental factors on VEC hemostatic function has not been characterized. This work used a poly(ethylene glycol diacrylate (PEGDA hydrogel platform to evaluate the effects of substrate stiffness and cell adhesive ligands on VEC phenotype and expression of hemostatic genes. Hydrogels of molecular weights (MWs 3.4, 8, and 20 kDa were polymerized into platforms of different rigidities and thiol-modified cell adhesive peptides were covalently bound to acrylate groups on the hydrogel surfaces. The peptide RKRLQVQLSIRT (RKR is a syndecan-1 binding ligand derived from laminin, a trimeric protein and a basement membrane matrix component. Conversely, RGDS is an integrin binding peptide found in many extracellular matrix (ECM proteins including fibronectin, fibrinogen, and von Willebrand factor (VWF. VECs adhered to and formed a stable monolayer on all RKR-coated hydrogel-MW combinations. RGDS-coated platforms supported VEC adhesion and growth on RGDS-3.4 kDa and RGDS-8 kDa hydrogels. VECs cultured on the softer RKR-8 kDa and RKR-20 kDa hydrogel platforms had significantly higher gene expression for all anti-thrombotic (ADAMTS-13, tissue factor pathway inhibitor, and tissue plasminogen activator and thrombotic (VWF, tissue factor, and P-selectin proteins than VECs cultured on RGDS-coated hydrogels and tissue culture polystyrene controls. Stimulated VECs promoted greater platelet adhesion than non-stimulated VECs on their respective culture condition; yet stimulated VECs on RGDS-3.4 kDa gels were not as responsive to stimulation relative to the RKR-gel groups. Thus, the syndecan binding, laminin-derived peptide promoted stable VEC adhesion on the softer hydrogels and maintained VEC phenotype and natural hemostatic function. In

Human trophoblast-derived hydrogen sulfide stimulates placental artery endothelial cell angiogenesis.

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Chen, Dong-Bao; Feng, Lin; Hodges, Jennifer K; Lechuga, Thomas J; Zhang, Honghai

2017-09-01

Endogenous hydrogen sulfide (H2S), mainly synthesized by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH), has been implicated in regulating placental angiogenesis; however, the underlying mechanisms are unknown. This study was to test a hypothesis that trophoblasts synthesize H2S to promote placental angiogenesis. Human choriocarcinoma-derived BeWo cells expressed both CBS and CTH proteins, while the first trimester villous trophoblast-originated HTR-8/SVneo cells expressed CTH protein only. The H2S producing ability of BeWo cells was significantly inhibited by either inhibitors of CBS (carboxymethyl hydroxylamine hemihydrochloride, CHH) or CTH (β-cyano-L-alanine, BCA) and that in HTR-8/SVneo cells was inhibited by CHH only. H2S donors stimulated cell proliferation, migration, and tube formation in ovine placental artery endothelial cells (oFPAECs) as effectively as vascular endothelial growth factor. Co-culture with BeWo and HTR-8/SVneo cells stimulated oFPAEC migration, which was inhibited by CHH or BCA in BeWo but CHH only in HTR-8/SVneo cells. Primary human villous trophoblasts (HVT) were more potent than trophoblast cell lines in stimulating oFPAEC migration that was inhibited by CHH and CHH/BCA combination in accordance with its H2S synthesizing activity linked to CBS and CTH expression patterns. H2S donors activated endothelial nitric oxide synthase (NOS3), v-AKT murine thymoma viral oncogene homolog 1 (AKT1), and extracellular signal-activated kinase 1/2 (mitogen-activated protein kinase 3/1, MAPK3/1) in oFPAECs. H2S donor-induced NOS3 activation was blocked by AKT1 but not MAPK3/1 inhibition. In keeping with our previous studies showing a crucial role of AKT1, MAPK3/1, and NOS3/NO in placental angiogenesis, these data show that trophoblast-derived endogenous H2S stimulates placental angiogenesis, involving activation of AKT1, NOS3/NO, and MAPK3/1. © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study

Nucleolin down-regulation is involved in ADP-induced cell cycle arrest in S phase and cell apoptosis in vascular endothelial cells.

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Wenmeng Wang

Full Text Available High concentration of extracellular ADP has been reported to induce cell apoptosis, but the molecular mechanisms remain not fully elucidated. In this study, we found by serendipity that ADP treatment of human umbilical vein endothelial cells (HUVEC and human aortic endothelial cells (HAEC down-regulated the protein level of nucleolin in a dose- and time-dependent manner. ADP treatment did not decrease the transcript level of nucloelin, suggesting that ADP might induce nucleolin protein degradation. HUVEC and HAEC expressed ADP receptor P2Y13 receptor, but did not express P2Y1 or P2Y12 receptors. However, P2Y1, 12, 13 receptor antagonists MRS2179, PSB0739, MRS2211 did not inhibit ADP-induced down-regulation of nucleolin. Moreover, MRS2211 itself down-regulated nucleolin protein level. In addition, 2-MeSADP, an agonist for P2Y1, 12 and 13 receptors, did not down-regulate nucleolin protein. These results suggested that ADP-induced nucleolin down-regulation was not due to the activation of P2Y1, 12, or 13 receptors. We also found that ADP treatment induced cell cycle arrest in S phase, cell apoptosis and cell proliferation inhibition via nucleolin down-regulation. The over-expression of nucleolin by gene transfer partly reversed ADP-induced cell cycle arrest, cell apoptosis and cell proliferation inhibition. Furthermore, ADP sensitized HUVEC to cisplatin-induced cell death by the down-regulation of Bcl-2 expression. Taken together, we found, for the first time to our knowledge, a novel mechanism by which ADP regulates cell proliferation by induction of cell cycle arrest and cell apoptosis via targeting nucelolin.

Transport of lipoprotein lipase across endothelial cells

International Nuclear Information System (INIS)

Saxena, U.; Klein, M.G.; Goldberg, I.J.

1991-01-01

Lipoprotein lipase (LPL), synthesized in muscle and fat, hydrolyzes plasma triglycerides primarily while bound to luminal endothelial cell surfaces. To obtain information about the movement of LPL from the basal to the luminal endothelial cell surface, the authors studied the transport of purified bovine milk LPL across bovine aortic endothelial cell monolayers. 125 I-labeled LPL ( 125 I-LPL) added to the basal surface of the monolayers was detected on the apical side of the cells in two compartments: (1) in the medium of the upper chamber, and (2) bound to the apical cell surface. The amount of 125 I-LPL on the cell surface, but not in the medium, reached saturation with time and LPL dose. Catalytically active LPL was transported to the apical surface but very little LPL activity appeared in the medium. Heparinase treatment of the basal cell surface and addition of dextran sulfate to the lower chamber decreased the amount of 125 I-LPL appearing on the apical surface. Similarly, the presence of increasing molar ratios of oleic acid/bovine serum albumin at the basal surface decreased the transport of active LPL across the monolayer. Thus, a saturable transport system, which requires haparan sulfate proteoglycans and is inhibited by high concentrations of free fatty acids on the basal side of the cells, appears to exist for passage of enzymatically active LPL across endothelial cells. They postulate that regulation of LPL transport to the endothelial luminal surface modulates the physiologically active pool of LPL in vivo

Microfluidically supported biochip design for culture of endothelial cell layers with improved perfusion conditions.

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Raasch, Martin; Rennert, Knut; Jahn, Tobias; Peters, Sven; Henkel, Thomas; Huber, Otmar; Schulz, Ingo; Becker, Holger; Lorkowski, Stefan; Funke, Harald; Mosig, Alexander

2015-03-02

Hemodynamic forces generated by the blood flow are of central importance for the function of endothelial cells (ECs), which form a biologically active cellular monolayer in blood vessels and serve as a selective barrier for macromolecular permeability. Mechanical stimulation of the endothelial monolayer induces morphological remodeling in its cytoskeleton. For in vitro studies on EC biology culture devices are desirable that simulate conditions of flow in blood vessels and allow flow-based adhesion/permeability assays under optimal perfusion conditions. With this aim we designed a biochip comprising a perfusable membrane that serves as cell culture platform multi-organ-tissue-flow (MOTiF biochip). This biochip allows an effective supply with nutrition medium, discharge of catabolic cell metabolites and defined application of shear stress to ECs under laminar flow conditions. To characterize EC layers cultured in the MOTiF biochip we investigated cell viability, expression of EC marker proteins and cell adhesion molecules of ECs dynamically cultured under low and high shear stress, and compared them with an endothelial culture in established two-dimensionally perfused flow chambers and under static conditions. We show that ECs cultured in the MOTiF biochip form a tight EC monolayer with increased cellular density, enhanced cell layer thickness, presumably as the result of a rapid and effective adaption to shear stress by remodeling of the cytoskeleton. Moreover, endothelial layers in the MOTiF biochip express higher amounts of EC marker proteins von-Willebrand-factor and PECAM-1. EC layers were highly responsive to stimulation with TNFα as detected at the level of ICAM-1, VCAM-1 and E-selectin expression and modulation of endothelial permeability in response to TNFα/IFNγ treatment under flow conditions. Compared to static and two-dimensionally perfused cell culture condition we consider MOTiF biochips as a valuable tool for studying EC biology in vitro under

In vitro differentiation of human skin-derived multipotent stromal cells into putative endothelial-like cells

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Vishnubalaji Radhakrishnan

2012-01-01

Full Text Available Abstract Background Multipotent stem cells have been successfully isolated from various tissues and are currently utilized for tissue-engineering and cell-based therapies. Among the many sources, skin has recently emerged as an attractive source for multipotent cells because of its abundance. Recent literature showed that skin stromal cells (SSCs possess mesoderm lineage differentiation potential; however, the endothelial differentiation and angiogenic potential of SSC remains elusive. In our study, SSCs were isolated from human neonatal foreskin (hNFSSCs and adult dermal skin (hADSSCs using explants cultures and were compared with bone marrow (hMSC-TERT and adipose tissue-derived mesenchymal stem cells (hADMSCs for their potential differentiation into osteoblasts, adipocytes, and endothelial cells. Results Concordant with previous studies, both MSCs and SSCs showed similar morphology, surface protein expression, and were able to differentiate into osteoblasts and adipocytes. Using an endothelial induction culture system combined with an in vitro matrigel angiogenesis assay, hNFSSCs and hADSSCs exhibited the highest tube-forming capability, which was similar to those formed by human umbilical vein endothelial cells (HUVEC, with hNFSSCs forming the most tightly packed, longest, and largest diameter tubules among the three cell types. CD146 was highly expressed on hNFSSCs and HUVEC followed by hADSSCs, and hMSC-TERT, while its expression was almost absent on hADMSCs. Similarly, higher vascular density (based on the expression of CD31, CD34, vWF, CD146 and SMA was observed in neonatal skin, followed by adult dermal skin and adipose tissue. Thus, our preliminary data indicated a plausible relationship between vascular densities, and the expression of CD146 on multipotent cells derived from those tissues. Conclusions Our data is the first to demonstrate that human dermal skin stromal cells can be differentiated into endothelial lineage. Hence, SSCs

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

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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.

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

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Meina Shi

2015-01-01

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

Mannose 6-phosphate receptor and sortilin mediated endocytosis of α-galactosidase A in kidney endothelial cells

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Prabakaran, Thaneas; Nielsen, Rikke Skovgaard; Satchell, Simon C

2012-01-01

endothelial cells, in order to clarify if the recombinant enzyme is targeted to the lysosomes via the universal mannose 6-phosphate receptor (M6PR) and possibly other receptors. Immunohistochemical localization of infused recombinant α-Gal A in a renal biopsy from a classic Fabry disease patient showed...... that recombinant protein localize in the endothelial cells of the kidney. Affinity purification studies using α-Gal A resins identified M6PR and sortilin as α-Gal A receptors in cultured glomerular endothelial cells. Immunohistochemical analyses of normal human kidney with anti-sortilin and anti-M6PR showed...... that sortilin and M6PR were expressed in the endothelium of smaller and larger vessels. Uptake studies in cultured glomerular endothelial cells of α-Gal A labeled with fluorescence and (125)I showed by inhibition with RAP and M6P that sortilin and M6PR mediated uptake of α-Gal A. Biacore studies revealed that α...

Functional convergence of Akt protein with VEGFR-1 in human endothelial progenitor cells exposed to sera from patient with type 2 diabetes mellitus.

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Hassanpour, Mehdi; Rezabakhsh, Aysa; Rahbarghazi, Reza; Nourazarian, Alireza; Nouri, Mohammad; Avci, Çığır Biray; Ghaderi, Shahrooz; Alidadyani, Neda; Bagca, Bakiye Goker; Bagheri, Hesam Saghaei

2017-11-01

Diabetes mellitus type 2 predisposes patients to various microvascular complications. In the current experiment, the potent role of diabetes mellitus was investigated on the content of VEGFR-1, -2, Tie-1 and -2, and Akt in human endothelial progenitor cells. The gene expression profile of mTOR and Hedgehog signaling pathways were measured by PCR array. The possible crosstalk between RTKs, mTOR and Hedgehog signaling was also studied by bioinformatic analysis. Endothelial progenitor cells were incubated with serum from normal and diabetic for 7days. Compared to non-treated cells, diabetic serum-induced cell apoptosis (~2-fold) and prohibited cell migration toward bFGF (p1). ELISA analysis showed that diabetes exposed cells had increased abundance of Tie-1, -2 and VEGFR-2 and reduced amount of VEGFR-1 (p1) in diabetic cells. Western blotting showed a marked reduction in the protein level of Akt after cells exposure to serum from diabetic subjects (p1). PCR array revealed a significant stimulation of both mTOR and Hedgehog signaling pathways in diabetic cells (p1, -2 and Tie-2, but not Tie-1, are master regulators of angiogenesis. There is a crosstalk between RTKs and mTOR signaling by involving P62, GABARAPL1, and HTT genes. It seems that physical interaction and co-expression of Akt decreased the level of VEGFR-1 in diabetic cells. Regarding data from the present experiment, diabetic serum contributed to uncontrolled induction of both mTOR and Hedgehog signaling in endothelial progenitor cells. Diabetes mellitus induces mTOR pathway by involving receptor tyrosine kinases while Hedgehog stimulation is independent of these receptors. Copyright © 2017 Elsevier Inc. All rights reserved.

The Phosphatase PTP-PEST/PTPN12 Regulates Endothelial Cell Migration and Adhesion, but Not Permeability, and Controls Vascular Development and Embryonic Viability*

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Souza, Cleiton Martins; Davidson, Dominique; Rhee, Inmoo; Gratton, Jean-Philippe; Davis, Elaine C.; Veillette, André

2012-01-01

Protein-tyrosine phosphatase (PTP)-PEST (PTPN12) is ubiquitously expressed. It is essential for normal embryonic development and embryonic viability in mice. Herein we addressed the involvement of PTP-PEST in endothelial cell functions using a combination of genetic and biochemical approaches. By generating primary endothelial cells from an inducible PTP-PEST-deficient mouse, we found that PTP-PEST is not needed for endothelial cell differentiation and proliferation or for the control of endothelial cell permeability. Nevertheless, it is required for integrin-mediated adhesion and migration of endothelial cells. PTP-PEST-deficient endothelial cells displayed increased tyrosine phosphorylation of Cas, paxillin, and Pyk2, which were previously also implicated in integrin functions. By eliminating PTP-PEST in endothelial cells in vivo, we obtained evidence that expression of PTP-PEST in endothelial cells is required for normal vascular development and embryonic viability. Therefore, PTP-PEST is a key regulator of integrin-mediated functions in endothelial cells seemingly through its capacity to control Cas, paxillin, and Pyk2. This function explains at least in part the essential role of PTP-PEST in embryonic development and viability. PMID:23105101

Protective effect of atorvastatin on radiation-induced vascular endothelial cell injury in vitro

International Nuclear Information System (INIS)

Ran Xinze; Zong Zhaowen; Liu Dengqun; Su Yongping; Zheng Huaien; Ran Xi; Xiang Guiming

2010-01-01

Vascular endothelial cells are very sensitive to ionizing radiation, and it is important to develop effective prevent agents and measures in radiation exposure protection. In the present study, the protective effects of atorvastatin on irradiated human umbilical vein endothelial cells (HUVEC) and the possible mechanisms were explored. Cultured HUVEC were treated by atorvastatin at a final concentration of 10 μmol/ml for 10 minutes, and then irradiated at a dose of 2 Gy or 25 Gy. Twenty-four hours after irradiation, apoptosis of HUVEC was monitored by flow cytometry, and the expression of thrombomodulin (TM) and protein C activation in HUVEC was respectively assessed by flow cytometry and spectrophotometry. After treatment with atorvastatin for 24 h, the rate of cell apoptosis decreased by 6% and 16% in cells irradiated with 2 Gy and 25 Gy, respectively. TM expression increased by 77%, 59%, and 61% in untreated cells, 2 Gy irradiation-treated cells, and 25 Gy irradiation-treated cells, respectively. The protein C levels in 2 Gy and 25 Gy irradiation-treated cells were reduced by 23% and 34% when compared with untreated cells, but up-regulated by 79% and 76% when compared with cells which were irradiated and treated with atorvastatin. In conclusion, these data indicate that atorvastatin exerts protective effects on irradiated HUVEC by reducing apoptosis by up-regulating TM expression and enhancing protein C activation in irradiated HUVEC. (author)

Protective effect of atorvastatin on radiation-induced vascular endothelial cell injury in vitro

Energy Technology Data Exchange (ETDEWEB)

Xinze, Ran; Zhaowen, Zong; Dengqun, Liu; Yongping, Su; Huaien, Zheng [College of Preventive Medicine, Third Military Medical Univ., Chongqing (China); Xi, Ran; Guiming, Xiang [Xinqiao Hospital, Third Military Medical Univ., Chongqing (China)

2010-09-15

Vascular endothelial cells are very sensitive to ionizing radiation, and it is important to develop effective prevent agents and measures in radiation exposure protection. In the present study, the protective effects of atorvastatin on irradiated human umbilical vein endothelial cells (HUVEC) and the possible mechanisms were explored. Cultured HUVEC were treated by atorvastatin at a final concentration of 10 {mu}mol/ml for 10 minutes, and then irradiated at a dose of 2 Gy or 25 Gy. Twenty-four hours after irradiation, apoptosis of HUVEC was monitored by flow cytometry, and the expression of thrombomodulin (TM) and protein C activation in HUVEC was respectively assessed by flow cytometry and spectrophotometry. After treatment with atorvastatin for 24 h, the rate of cell apoptosis decreased by 6% and 16% in cells irradiated with 2 Gy and 25 Gy, respectively. TM expression increased by 77%, 59%, and 61% in untreated cells, 2 Gy irradiation-treated cells, and 25 Gy irradiation-treated cells, respectively. The protein C levels in 2 Gy and 25 Gy irradiation-treated cells were reduced by 23% and 34% when compared with untreated cells, but up-regulated by 79% and 76% when compared with cells which were irradiated and treated with atorvastatin. In conclusion, these data indicate that atorvastatin exerts protective effects on irradiated HUVEC by reducing apoptosis by up-regulating TM expression and enhancing protein C activation in irradiated HUVEC. (author)

Identification and functional analysis of endothelial tip cell-enriched genes.

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del Toro, Raquel; Prahst, Claudia; Mathivet, Thomas; Siegfried, Geraldine; Kaminker, Joshua S; Larrivee, Bruno; Breant, Christiane; Duarte, Antonio; Takakura, Nobuyuki; Fukamizu, Akiyoshi; Penninger, Josef; Eichmann, Anne

2010-11-11

Sprouting of developing blood vessels is mediated by specialized motile endothelial cells localized at the tips of growing capillaries. Following behind the tip cells, endothelial stalk cells form the capillary lumen and proliferate. Expression of the Notch ligand Delta-like-4 (Dll4) in tip cells suppresses tip cell fate in neighboring stalk cells via Notch signaling. In DLL4(+/-) mouse mutants, most retinal endothelial cells display morphologic features of tip cells. We hypothesized that these mouse mutants could be used to isolate tip cells and so to determine their genetic repertoire. Using transcriptome analysis of retinal endothelial cells isolated from DLL4(+/-) and wild-type mice, we identified 3 clusters of tip cell-enriched genes, encoding extracellular matrix degrading enzymes, basement membrane components, and secreted molecules. Secreted molecules endothelial-specific molecule 1, angiopoietin 2, and apelin bind to cognate receptors on endothelial stalk cells. Knockout mice and zebrafish morpholino knockdown of apelin showed delayed angiogenesis and reduced proliferation of stalk cells expressing the apelin receptor APJ. Thus, tip cells may regulate angiogenesis via matrix remodeling, production of basement membrane, and release of secreted molecules, some of which regulate stalk cell behavior.

Reduced endothelial thioredoxin-interacting protein protects arteries from damage induced by metabolic stress in vivo.

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Bedarida, Tatiana; Domingues, Alison; Baron, Stephanie; Ferreira, Chrystophe; Vibert, Francoise; Cottart, Charles-Henry; Paul, Jean-Louis; Escriou, Virginie; Bigey, Pascal; Gaussem, Pascale; Leguillier, Teddy; Nivet-Antoine, Valerie

2018-06-01

Although thioredoxin-interacting protein (TXNIP) is involved in a variety of biologic functions, the contribution of endothelial TXNIP has not been well defined. To investigate the endothelial function of TXNIP, we generated a TXNIP knockout mouse on the Cdh5-cre background (TXNIP fl/fl cdh5 cre ). Control (TXNIP fl/fl ) and TXNIP fl/fl cdh5 cre mice were fed a high protein-low carbohydrate (HP-LC) diet for 3 mo to induce metabolic stress. We found that TXNIP fl/fl and TXNIP fl/fl cdh5 cre mice on an HP-LC diet displayed impaired glucose tolerance and dyslipidemia concretizing the metabolic stress induced. We evaluated the impact of this metabolic stress on mice with reduced endothelial TXNIP expression with regard to arterial structure and function. TXNIP fl/fl cdh5 cre mice on an HP-LC diet exhibited less endothelial dysfunction than littermate mice on an HP-LC diet. These mice were protected from decreased aortic medial cell content, impaired aortic distensibility, and increased plasminogen activator inhibitor 1 secretion. This protective effect came with lower oxidative stress and lower inflammation, with a reduced NLRP3 inflammasome expression, leading to a decrease in cleaved IL-1β. We also show the major role of TXNIP in inflammation with a knockdown model, using a TXNIP-specific, small interfering RNA included in a lipoplex. These findings demonstrate a key role for endothelial TXNIP in arterial impairments induced by metabolic stress, making endothelial TXNIP a potential therapeutic target.-Bedarida, T., Domingues, A., Baron, S., Ferreira, C., Vibert, F., Cottart, C.-H., Paul, J.-L., Escriou, V., Bigey, P., Gaussem, P., Leguillier, T., Nivet-Antoine, V. Reduced endothelial thioredoxin-interacting protein protects arteries from damage induced by metabolic stress in vivo.

Decellularized matrix from tumorigenic human mesenchymal stem cells promotes neovascularization with galectin-1 dependent endothelial interaction.

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Jorge S Burns

Full Text Available BACKGROUND: Acquisition of a blood supply is fundamental for extensive tumor growth. We recently described vascular heterogeneity in tumours derived from cell clones of a human mesenchymal stem cell (hMSC strain (hMSC-TERT20 immortalized by retroviral vector mediated human telomerase (hTERT gene expression. Histological analysis showed that cells of the most vascularized tumorigenic clone, -BD11 had a pericyte-like alpha smooth muscle actin (ASMA+ and CD146+ positive phenotype. Upon serum withdrawal in culture, -BD11 cells formed cord-like structures mimicking capillary morphogenesis. In contrast, cells of the poorly tumorigenic clone, -BC8 did not stain for ASMA, tumours were less vascularized and serum withdrawal in culture led to cell death. By exploring the heterogeneity in hMSC-TERT20 clones we aimed to understand molecular mechanisms by which mesenchymal stem cells may promote neovascularization. METHODOLOGY/PRINCIPAL FINDINGS: Quantitative qRT-PCR analysis revealed similar mRNA levels for genes encoding the angiogenic cytokines VEGF and Angiopoietin-1 in both clones. However, clone-BD11 produced a denser extracellular matrix that supported stable ex vivo capillary morphogenesis of human endothelial cells and promoted in vivo neovascularization. Proteomic characterization of the -BD11 decellularized matrix identified 50 extracellular angiogenic proteins, including galectin-1. siRNA knock down of galectin-1 expression abrogated the ex vivo interaction between decellularized -BD11 matrix and endothelial cells. More stable shRNA knock down of galectin-1 expression did not prevent -BD11 tumorigenesis, but greatly reduced endothelial migration into -BD11 cell xenografts. CONCLUSIONS: Decellularized hMSC matrix had significant angiogenic potential with at least 50 angiogenic cell surface and extracellular proteins, implicated in attracting endothelial cells, their adhesion and activation to form tubular structures. hMSC -BD11 surface galectin-1

Decellularized Matrix from Tumorigenic Human Mesenchymal Stem Cells Promotes Neovascularization with Galectin-1 Dependent Endothelial Interaction

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Burns, Jorge S.; Kristiansen, Malthe; Kristensen, Lars P.; Larsen, Kenneth H.; Nielsen, Maria O.; Christiansen, Helle; Nehlin, Jan; Andersen, Jens S.; Kassem, Moustapha

2011-01-01

Background Acquisition of a blood supply is fundamental for extensive tumor growth. We recently described vascular heterogeneity in tumours derived from cell clones of a human mesenchymal stem cell (hMSC) strain (hMSC-TERT20) immortalized by retroviral vector mediated human telomerase (hTERT) gene expression. Histological analysis showed that cells of the most vascularized tumorigenic clone, -BD11 had a pericyte-like alpha smooth muscle actin (ASMA+) and CD146+ positive phenotype. Upon serum withdrawal in culture, -BD11 cells formed cord-like structures mimicking capillary morphogenesis. In contrast, cells of the poorly tumorigenic clone, -BC8 did not stain for ASMA, tumours were less vascularized and serum withdrawal in culture led to cell death. By exploring the heterogeneity in hMSC-TERT20 clones we aimed to understand molecular mechanisms by which mesenchymal stem cells may promote neovascularization. Methodology/Principal Findings Quantitative qRT-PCR analysis revealed similar mRNA levels for genes encoding the angiogenic cytokines VEGF and Angiopoietin-1 in both clones. However, clone-BD11 produced a denser extracellular matrix that supported stable ex vivo capillary morphogenesis of human endothelial cells and promoted in vivo neovascularization. Proteomic characterization of the -BD11 decellularized matrix identified 50 extracellular angiogenic proteins, including galectin-1. siRNA knock down of galectin-1 expression abrogated the ex vivo interaction between decellularized -BD11 matrix and endothelial cells. More stable shRNA knock down of galectin-1 expression did not prevent -BD11 tumorigenesis, but greatly reduced endothelial migration into -BD11 cell xenografts. Conclusions Decellularized hMSC matrix had significant angiogenic potential with at least 50 angiogenic cell surface and extracellular proteins, implicated in attracting endothelial cells, their adhesion and activation to form tubular structures. hMSC -BD11 surface galectin-1 expression was

Cafestol Inhibits Cyclic-Strain-Induced Interleukin-8, Intercellular Adhesion Molecule-1, and Monocyte Chemoattractant Protein-1 Production in Vascular Endothelial Cells

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Hao, Wen-Rui; Sung, Li-Chin; Chen, Chun-Chao; Chen, Jin-Jer

2018-01-01

Moderate coffee consumption is inversely associated with cardiovascular disease mortality; however, mechanisms underlying this causal effect remain unclear. Cafestol, a diterpene found in coffee, has various properties, including an anti-inflammatory property. This study investigated the effect of cafestol on cyclic-strain-induced inflammatory molecule secretion in vascular endothelial cells. Cells were cultured under static or cyclic strain conditions, and the secretion of inflammatory molecules was determined using enzyme-linked immunosorbent assay. The effects of cafestol on mitogen-activated protein kinases (MAPK), heme oxygenase-1 (HO-1), and sirtuin 1 (Sirt1) signaling pathways were examined using Western blotting and specific inhibitors. Cafestol attenuated cyclic-strain-stimulated intercellular adhesion molecule-1 (ICAM-1), monocyte chemoattractant protein- (MCP-) 1, and interleukin- (IL-) 8 secretion. Cafestol inhibited the cyclic-strain-induced phosphorylation of extracellular signal-regulated kinase and p38 MAPK. By contrast, cafestol upregulated cyclic-strain-induced HO-1 and Sirt1 expression. The addition of zinc protoporphyrin IX, sirtinol, or Sirt1 silencing (transfected with Sirt1 siRNA) significantly attenuated cafestol-mediated modulatory effects on cyclic-strain-stimulated ICAM-1, MCP-1, and IL-8 secretion. This is the first study to report that cafestol inhibited cyclic-strain-induced inflammatory molecule secretion, possibly through the activation of HO-1 and Sirt1 in endothelial cells. The results provide valuable insights into molecular pathways that may contribute to the effects of cafestol. PMID:29854096

Insulin resistance in vascular endothelial cells promotes intestinal tumour formation

DEFF Research Database (Denmark)

Wang, X; Häring, M-F; Rathjen, Thomas

2017-01-01

in vascular endothelial cells. Strikingly, these mice had 42% more intestinal tumours than controls, no change in tumour angiogenesis, but increased expression of vascular cell adhesion molecule-1 (VCAM-1) in primary culture of tumour endothelial cells. Insulin decreased VCAM-1 expression and leukocyte...... adhesion in quiescent tumour endothelial cells with intact insulin receptors and partly prevented increases in VCAM-1 and leukocyte adhesion after treatment with tumour necrosis factor-α. Knockout of insulin receptors in endothelial cells also increased leukocyte adhesion in mesenteric venules...

VE-cadherin cleavage by LasB protease from Pseudomonas aeruginosa facilitates type III secretion system toxicity in endothelial cells.

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Guillaume Golovkine

2014-03-01

Full Text Available Infection of the vascular system by Pseudomonas aeruginosa (Pa occurs during bacterial dissemination in the body or in blood-borne infections. Type 3 secretion system (T3SS toxins from Pa induce a massive retraction when injected into endothelial cells. Here, we addressed the role of type 2 secretion system (T2SS effectors in this process. Mutants with an inactive T2SS were much less effective than wild-type strains at inducing cell retraction. Furthermore, secretomes from wild-types were sufficient to trigger cell-cell junction opening when applied to cells, while T2SS-inactivated mutants had minimal activity. Intoxication was associated with decreased levels of vascular endothelial (VE-cadherin, a homophilic adhesive protein located at endothelial cell-cell junctions. During the process, the protein was cleaved in the middle of its extracellular domain (positions 335 and 349. VE-cadherin attrition was T3SS-independent but T2SS-dependent. Interestingly, the epithelial (E-cadherin was unaffected by T2SS effectors, indicating that this mechanism is specific to endothelial cells. We showed that one of the T2SS effectors, the protease LasB, directly affected VE-cadherin proteolysis, hence promoting cell-cell junction disruption. Furthermore, mouse infection with Pa to induce acute pneumonia lead to significant decreases in lung VE-cadherin levels, whereas the decrease was minimal with T2SS-inactivated or LasB-deleted mutant strains. We conclude that the T2SS plays a pivotal role during Pa infection of the vascular system by breaching the endothelial barrier, and propose a model in which the T2SS and the T3SS cooperate to intoxicate endothelial cells.

Endothelial Cells Control Pancreatic Cell Fate at Defined Stages through EGFL7 Signaling

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Der-I Kao

2015-02-01

Full Text Available Although endothelial cells have been shown to affect mouse pancreatic development, their precise function in human development remains unclear. Using a coculture system containing human embryonic stem cell (hESC-derived progenitors and endothelial cells, we found that endothelial cells play a stage-dependent role in pancreatic development, in which they maintain pancreatic progenitor (PP self-renewal and impair further differentiation into hormone-expressing cells. The mechanistic studies suggest that the endothelial cells act through the secretion of EGFL7. Consistently, endothelial overexpression of EGFL7 in vivo using a transgenic mouse model resulted in an increase of PP proliferation rate and a decrease of differentiation toward endocrine cells. These studies not only identified the role of EGFL7 as the molecular handle involved in the crosstalk between endothelium and pancreatic epithelium, but also provide a paradigm for using hESC stepwise differentiation to dissect the stage-dependent roles of signals controlling organogenesis.

Extraembryonic origin of circulating endothelial cells.

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Luc Pardanaud

Full Text Available Circulating endothelial cells (CEC are contained in the bone marrow and peripheral blood of adult humans and participate to the revascularization of ischemic tissues. These cells represent attractive targets for cell or gene therapy aimed at improving ischemic revascularization or inhibition of tumor angiogenesis. The embryonic origin of CEC has not been addressed previously. Here we use quail-chick chimeras to study CEC origin and participation to the developing vasculature. CEC are traced with different markers, in particular the QH1 antibody recognizing only quail endothelial cells. Using yolk-sac chimeras, where quail embryos are grafted onto chick yolk sacs and vice-versa, we show that CEC are generated in the yolk sac. These cells are mobilized during wound healing, demonstrating their participation to angiogenic repair processes. Furthermore, we found that the allantois is also able to give rise to CEC in situ. In contrast to the yolk sac and allantois, the embryo proper does not produce CEC. Our results show that CEC exclusively originate from extra-embryonic territories made with splanchnopleural mesoderm and endoderm, while definitive hematopoietic stem cells and endothelial cells are of intra-embryonic origin.

Extraembryonic origin of circulating endothelial cells.

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Pardanaud, Luc; Eichmann, Anne

2011-01-01

Circulating endothelial cells (CEC) are contained in the bone marrow and peripheral blood of adult humans and participate to the revascularization of ischemic tissues. These cells represent attractive targets for cell or gene therapy aimed at improving ischemic revascularization or inhibition of tumor angiogenesis. The embryonic origin of CEC has not been addressed previously. Here we use quail-chick chimeras to study CEC origin and participation to the developing vasculature. CEC are traced with different markers, in particular the QH1 antibody recognizing only quail endothelial cells. Using yolk-sac chimeras, where quail embryos are grafted onto chick yolk sacs and vice-versa, we show that CEC are generated in the yolk sac. These cells are mobilized during wound healing, demonstrating their participation to angiogenic repair processes. Furthermore, we found that the allantois is also able to give rise to CEC in situ. In contrast to the yolk sac and allantois, the embryo proper does not produce CEC. Our results show that CEC exclusively originate from extra-embryonic territories made with splanchnopleural mesoderm and endoderm, while definitive hematopoietic stem cells and endothelial cells are of intra-embryonic origin.

Effect of cAMP derivates on assembly and maintenance of tight junctions in human umbilical vein endothelial cells

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Beese Michaela

2010-09-01

Full Text Available Abstract Background Endothelial tight and adherens junctions control a variety of physiological processes like adhesion, paracellular transport of solutes or trafficking of activated leukocytes. Formation and maintenance of endothelial junctions largely depend on the microenvironment of the specific vascular bed and on interactions of the endothelium with adjacent cell types. Consequently, primary cultures of endothelial cells often lose their specific junctional pattern and fail to establish tight monolayer in vitro. This is also true for endothelial cells isolated from the vein of human umbilical cords (HUVEC which are widely used as model for endothelial cell-related studies. Results We here compared the effect of cyclic 3'-5'-adenosine monophosphate (cAMP and its derivates on formation and stabilization of tight junctions and on alterations in paracellular permeability in HUVEC. We demonstrated by light and confocal laser microscopy that for shorter time periods the sodium salt of 8-bromoadenosine-cAMP (8-Br-cAMP/Na and for longer incubation periods 8-(4-chlorophenylthio-cAMP (pCPT-cAMP exerted the greatest effects of all compounds tested here on formation of continuous tight junction strands in HUVEC. We further demonstrated that although all compounds induced protein kinase A-dependent expression of the tight junction proteins claudin-5 and occludin only pCPT-cAMP slightly enhanced paracellular barrier functions. Moreover, we showed that pCPT-cAMP and 8-Br-cAMP/Na induced expression and membrane translocation of tricellulin. Conclusions pCPT-cAMP and, to a lesser extend, 8-Br-cAMP/Na improved formation of continuous tight junction strands and decreased paracellular permeability in primary HUVEC. We concluded that under these conditions HUVEC represent a feasible in vitro model to study formation and disassembly of endothelial tight junctions and to characterize tight junction-associated proteins

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

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

2013-10-09

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

Endothelial galectin-1 binds to specific glycans on nipah virus fusion protein and inhibits maturation, mobility, and function to block syncytia formation.

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Omai B Garner

2010-07-01

Full Text Available Nipah virus targets human endothelial cells via NiV-F and NiV-G envelope glycoproteins, resulting in endothelial syncytia formation and vascular compromise. Endothelial cells respond to viral infection by releasing innate immune effectors, including galectins, which are secreted proteins that bind to specific glycan ligands on cell surface glycoproteins. We demonstrate that galectin-1 reduces NiV-F mediated fusion of endothelial cells, and that endogenous galectin-1 in endothelial cells is sufficient to inhibit syncytia formation. Galectin-1 regulates NiV-F mediated cell fusion at three distinct points, including retarding maturation of nascent NiV-F, reducing NiV-F lateral mobility on the plasma membrane, and directly inhibiting the conformational change in NiV-F required for triggering fusion. Characterization of the NiV-F N-glycome showed that the critical site for galectin-1 inhibition is rich in glycan structures known to bind galectin-1. These studies identify a unique set of mechanisms for regulating pathophysiology of NiV infection at the level of the target cell.

Angiogenesis in mucous retention cyst: a human in vivo-like model of endothelial cell differentiation in mucous substrate.

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Swelam, Wael; Ida-Yonemochi, Hiroko; Saku, Takashi

2005-01-01

Mucous retention cysts contain a mucous pool in the lumina, in which pure angiogenic processes are occasionally observed. By using this unique human material, our aim was to understand the in vivo angiogenic process. Fifteen surgical tissue samples of mucous retention cysts of the lip were examined for expression of vascular endothelial markers and extracellular matrix molecules by immunohistochemistry and in situ hybridization (ISH). Endothelial cells forming new vascular channels showed immunopositivities for CD31, CD34, vascular endothelial growth factor (VEGF), and von Willebrand factor (vWF). These newly formed capillaries were surrounded by tenascin-positive matrices and further by a dense infiltration of CD68-positive cells with foamy to epitheloid appearances. Some of these cells were simultaneously positive for CD34, VEGF, and one of its receptors, Flk-1, and they showed definite mRNA as well as protein signals for tenascin. In addition, these cells often tended to be aligned, which suggested tubule formation. The results suggest that monocyte/macrophage lineage cells are a major source for endothelial cells at least in mucous retention cysts and that tenascin produced by those cells plays an important role in differentiation of endothelial cells.

Regulation of human heme oxygenase in endothelial cells by using sense and antisense retroviral constructs.

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Quan, S; Yang, L; Abraham, N G; Kappas, A

2001-10-09

Our objective was to determine whether overexpression and underexpression of human heme oxygenase (HHO)-1 could be controlled on a long-term basis by introduction of the HO-1 gene in sense (S) and antisense (AS) orientation with an appropriate vector into endothelial cells. Retroviral vector (LXSN) containing viral long terminal repeat promoter-driven human HO-1 S (LSN-HHO-1) and LXSN vectors containing HHO-1 promoter (HOP)-controlled HHO-1 S and AS (LSN-HOP-HHO-1 and LSN-HOP-HHO-1-AS) sequences were constructed and used to transfect rat lung microvessel endothelial cells (RLMV cells) and human dermal microvessel endothelial cells (HMEC-1 cells). RLMV cells transduced with HHO-1 S expressed human HO-1 mRNA and HO-1 protein associated with elevation in total HO activity compared with nontransduced cells. Vector-mediated expression of HHO-1 S or AS under control of HOP resulted in effective production of HO-1 or blocked induction of endogenous human HO-1 in HMEC-1 cells, respectively. Overexpression of HO-1 AS was associated with a long-term decrease (45%) of endogenous HO-1 protein and an increase (167%) in unmetabolized exogenous heme in HMEC-1 cells. Carbon monoxide (CO) production in HO-1 S- or AS-transduced HMEC-1 cells after heme treatment was increased (159%) or decreased (50%), respectively, compared with nontransduced cells. HO-2 protein levels did not change. These findings demonstrate that HHO-1 S and AS retroviral constructs are functional in enhancing and reducing HO activity, respectively, and thus can be used to regulate cellular heme levels, the activity of heme-dependent enzymes, and the rate of heme catabolism to CO and bilirubin.

Human β-Defensin 3 Reduces TNF-α-Induced Inflammation and Monocyte Adhesion in Human Umbilical Vein Endothelial Cells

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Tianying Bian

2017-01-01

Full Text Available The aim of this study was to investigate the role of human β-defensin 3 (hBD3 in the initiation stage of atherosclerosis with human umbilical vein endothelial cells (HUVECs triggered by tumor necrosis factor- (TNF- α. The effects of hBD3 on TNF-α-induced endothelial injury and inflammatory response were evaluated. Our data revealed that first, hBD3 reduced the production of interleukin-6 (IL-6, IL-8, monocyte chemoattractant protein-1 (MCP-1, and macrophage migration inhibitory factor (MIF in HUVECs in a dose-dependent manner. In addition, hBD3 significantly prevented intracellular reactive oxygen species (ROS production by HUVECs. Second, western blot analysis demonstrated that hBD3 dose-dependently suppressed the protein levels of intracellular adhesion molecule-1 (ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1 in TNF-α-induced HUVECs. As a result, hBD3 inhibited monocyte adhesion to TNF-α-treated endothelial cells. Additionally, hBD3 suppressed TNF-α-induced F-actin reorganization in HUVECs. Third, hBD3 markedly inhibited NF-κB activation by decreasing the phosphorylation of IKK-α/β, IκB, and p65 subunit within 30 min. Moreover, the phosphorylation of p38 and c-Jun N-terminal protein kinase (JNK in the mitogen-activated protein kinase (MAPK pathway were also inhibited by hBD3 in HUVECs. In conclusion, hBD3 exerts anti-inflammatory and antioxidative effects in endothelial cells in response to TNF-α by inhibiting NF-κB and MAPK signaling.

Bevacizumab inhibits proliferation of choroidal endothelial cells by regulation of the cell cycle.

Science.gov (United States)

Rusovici, Raluca; Patel, Chirag J; Chalam, Kakarla V

2013-01-01

The purpose of this study was to evaluate cell cycle changes in choroidal endothelial cells treated with varying doses of bevacizumab in the presence of a range of concentrations of vascular endothelial growth factor (VEGF). Bevacizumab, a drug widely used in the treatment of neovascular age-related macular degeneration, choroidal neovascularization, and proliferative diabetic retinopathy, neutralizes all isoforms of VEGF. However, the effect of intravitreal administration of bevacizumab on the choroidal endothelial cell cycle has not been established. Monkey choroidal endothelial (RF/6A) cells were treated with VEGF 50 ng/mL and escalating doses of bevacizumab 0.1-2 mg/mL for 72 hours. Cell cycle changes in response to bevacizumab were analyzed by flow cytometry and propidium iodide staining. Cell proliferation was measured using the WST-1 assay. Morphological changes were recorded by bright field cell microscopy. Bevacizumab inhibited proliferation of choroidal endothelial cells by stabilization of the cell cycle in G0/G1 phase. Cell cycle analysis of VEGF-enriched choroidal endothelial cells revealed a predominant increase in the G2/M population (21.84%, P, 0.01) and a decrease in the G0/G1 phase population (55.08%, P, 0.01). Addition of escalating doses of bevacizumab stabilized VEGF-enriched cells in the G0/G1 phase (55.08%, 54.49%, 56.3%, and 64% [P, 0.01]) and arrested proliferation by inhibiting the G2/M phase (21.84%, 21.46%, 20.59%, 20.94%, and 16.1% [P, 0.01]). The increase in G0/G1 subpopulation in VEGF-enriched and bevacizumab-treated cells compared with VEGF-enriched cells alone was dose-dependent. Bevacizumab arrests proliferation of VEGF-enriched choroidal endothelial cells by stabilizing the cell cycle in the G0/G1 phase and inhibiting the G2/M phase in a dose-dependent fashion.

Chorein Sensitivity of Actin Polymerization, Cell Shape and Mechanical Stiffness of Vascular Endothelial Cells

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Ioana Alesutan

2013-09-01

Full Text Available Background/Aims: Endothelial cell stiffness plays a key role in endothelium-dependent control of vascular tone and arterial blood pressure. Actin polymerization and distribution of microfilaments is essential for mechanical cell stiffness. Chorein, a protein encoded by the VPS13A gene, defective in chorea-acanthocytosis (ChAc, is involved in neuronal cell survival as well as cortical actin polymerization of erythrocytes and blood platelets. Chorein is expressed in a wide variety of further cells, yet nothing is known about the impact of chorein on cells other than neurons, erythrocytes and platelets. The present study explored whether chorein is expressed in human umbilical vein endothelial cells (HUVECs and addressed the putative role of chorein in the regulation of cytoskeletal architecture, stiffness and survival of those cells. Methods: In HUVECs with or without silencing of the VPS13A gene, VPS13A mRNA expression was determined utilizing quantitative RT-PCR, cytoskeletal organization visualized by confocal microscopy, G/F actin ratio and phosphorylation status of focal adhesion kinase quantified by western blotting, cell death determined by flow cytometry, mechanical properties studied by atomic force microscopy (AFM and cell morphology analysed by scanning ion conductance microscopy (SICM. Results: VPS13A mRNA expression was detectable in HUVECs. Silencing of the VPS13A gene attenuated the filamentous actin network, decreased the ratio of soluble G-actin over filamentous F-actin, reduced cell stiffness and changed cell morphology as compared to HUVECs silenced with negative control siRNA. These effects were paralleled by a significant decrease in FAK phosphorylation following VPS13A silencing. Moreover, silencing of the VPS13A gene increased caspase 3 activity and induced necrosis in HUVECs. Conclusions: Chorein is a novel regulator of cytoskeletal architecture, cell shape, mechanical stiffness and survival of vascular endothelial cells.

Disturbance of copper homeostasis is a mechanism for homocysteine-induced vascular endothelial cell injury.

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Daoyin Dong

Full Text Available Elevation of serum homocysteine (Hcy levels is a risk factor for cardiovascular diseases. Previous studies suggested that Hcy interferes with copper (Cu metabolism in vascular endothelial cells. The present study was undertaken to test the hypothesis that Hcy-induced disturbance of Cu homeostasis leads to endothelial cell injury. Exposure of human umbilical vein endothelial cells (HUVECs to concentrations of Hcy at 0.01, 0.1 or 1 mM resulted in a concentration-dependent decrease in cell viability and an increase in necrotic cell death. Pretreatment of the cells with a final concentration of 5 µM Cu in cultures prevented the effects of Hcy. Hcy decreased intracellular Cu concentrations. HPLC-ICP-MS analysis revealed that Hcy caused alterations in the distribution of intracellular Cu; more Cu was redistributed to low molecular weight fractions. ESI-Q-TOF detected the formation of Cu-Hcy complexes. Hcy also decreased the protein levels of Cu chaperone COX17, which was accompanied by a decrease in the activity of cytochrome c oxidase (CCO and a collapse of mitochondrial membrane potential. These effects of Hcy were all preventable by Cu pretreatment. The study thus demonstrated that Hcy disturbs Cu homeostasis and limits the availability of Cu to critical molecules such as COX17 and CCO, leading to mitochondrial dysfunction and endothelial cell injury.

Sodium caprate transiently opens claudin-5-containing barriers at tight junctions of epithelial and endothelial cells

DEFF Research Database (Denmark)

Del Vecchio, Giovanna; Tscheik, Christian; Tenz, Kareen

2012-01-01

Claudin-5 is a tight junction (TJ) protein which limits the diffusion of small hydrophilic molecules. Thus, it represents a potential pharmacological target to improve drug delivery to the tissues protected by claudin-5-dependent barriers. Sodium caprate is known as an absorption enhancer which...... opens the paracellular space acting on TJ proteins and actin cytoskeleton. Its action on claudin-5 is not understood so far. Epithelial and endothelial systems were used to evaluate the effect of caprate on claudin-5 in TJ-free cells and on claudin-5 fully integrated in TJ. To this aim, confocal...... of endothelial and epithelial cells. In conclusion, the study further elucidates the cellular effects of caprate at the tight junctions....

Elevated capillary tube hematocrit reflects degradation of endothelial cell glycocalyx by oxidized LDL

NARCIS (Netherlands)

Constantinescu, A. A.; Vink, H.; Spaan, J. A.

2001-01-01

Proteoglycans and plasma proteins bound to the endothelial cell glycocalyx are essential for vascular function, but at the same time, they lower capillary tube hematocrit by reducing capillary volume available to flowing blood. Because oxidized low-density lipoproteins (oxLDL) reduce the effective

Identification of MicroRNA-124 as a Major Regulator of Enhanced Endothelial Cell Glycolysis in Pulmonary Arterial Hypertension via PTBP1 (Polypyrimidine Tract Binding Protein) and Pyruvate Kinase M2.

Science.gov (United States)

Caruso, Paola; Dunmore, Benjamin J; Schlosser, Kenny; Schoors, Sandra; Dos Santos, Claudia; Perez-Iratxeta, Carol; Lavoie, Jessie R; Zhang, Hui; Long, Lu; Flockton, Amanda R; Frid, Maria G; Upton, Paul D; D'Alessandro, Angelo; Hadinnapola, Charaka; Kiskin, Fedir N; Taha, Mohamad; Hurst, Liam A; Ormiston, Mark L; Hata, Akiko; Stenmark, Kurt R; Carmeliet, Peter; Stewart, Duncan J; Morrell, Nicholas W

2017-12-19

Pulmonary arterial hypertension (PAH) is characterized by abnormal growth and enhanced glycolysis of pulmonary artery endothelial cells. However, the mechanisms underlying alterations in energy production have not been identified. Here, we examined the miRNA and proteomic profiles of blood outgrowth endothelial cells (BOECs) from patients with heritable PAH caused by mutations in the bone morphogenetic protein receptor type 2 ( BMPR2 ) gene and patients with idiopathic PAH to determine mechanisms underlying abnormal endothelial glycolysis. We hypothesized that in BOECs from patients with PAH, the downregulation of microRNA-124 (miR-124), determined with a tiered systems biology approach, is responsible for increased expression of the splicing factor PTBP1 (polypyrimidine tract binding protein), resulting in alternative splicing of pyruvate kinase muscle isoforms 1 and 2 (PKM1 and 2) and consequently increased PKM2 expression. We questioned whether this alternative regulation plays a critical role in the hyperglycolytic phenotype of PAH endothelial cells. Heritable PAH and idiopathic PAH BOECs recapitulated the metabolic abnormalities observed in pulmonary artery endothelial cells from patients with idiopathic PAH, confirming a switch from oxidative phosphorylation to aerobic glycolysis. Overexpression of miR-124 or siRNA silencing of PTPB1 restored normal proliferation and glycolysis in heritable PAH BOECs, corrected the dysregulation of glycolytic genes and lactate production, and partially restored mitochondrial respiration. BMPR2 knockdown in control BOECs reduced the expression of miR-124, increased PTPB1 , and enhanced glycolysis. Moreover, we observed reduced miR-124, increased PTPB1 and PKM2 expression, and significant dysregulation of glycolytic genes in the rat SUGEN-hypoxia model of severe PAH, characterized by reduced BMPR2 expression and endothelial hyperproliferation, supporting the relevance of this mechanism in vivo. Pulmonary vascular and

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

Obesity-Induced Endoplasmic Reticulum Stress Causes Lung Endothelial Dysfunction and Promotes Acute Lung Injury.

Science.gov (United States)

Shah, Dilip; Romero, Freddy; Guo, Zhi; Sun, Jianxin; Li, Jonathan; Kallen, Caleb B; Naik, Ulhas P; Summer, Ross

2017-08-01

Obesity is a significant risk factor for acute respiratory distress syndrome. The mechanisms underlying this association are unknown. We recently showed that diet-induced obese mice exhibit pulmonary vascular endothelial dysfunction, which is associated with enhanced susceptibility to LPS-induced acute lung injury. Here, we demonstrate that lung endothelial dysfunction in diet-induced obese mice coincides with increased endoplasmic reticulum (ER) stress. Specifically, we observed enhanced expression of the major sensors of misfolded proteins, including protein kinase R-like ER kinase, inositol-requiring enzyme α, and activating transcription factor 6, in whole lung and in primary lung endothelial cells isolated from diet-induced obese mice. Furthermore, we found that primary lung endothelial cells exposed to serum from obese mice, or to saturated fatty acids that mimic obese serum, resulted in enhanced expression of markers of ER stress and the induction of other biological responses that typify the lung endothelium of diet-induced obese mice, including an increase in expression of endothelial adhesion molecules and a decrease in expression of endothelial cell-cell junctional proteins. Similar changes were observed in lung endothelial cells and in whole-lung tissue after exposure to tunicamycin, a compound that causes ER stress by blocking N-linked glycosylation, indicating that ER stress causes endothelial dysfunction in the lung. Treatment with 4-phenylbutyric acid, a chemical protein chaperone that reduces ER stress, restored vascular endothelial cell expression of adhesion molecules and protected against LPS-induced acute lung injury in diet-induced obese mice. Our work indicates that fatty acids in obese serum induce ER stress in the pulmonary endothelium, leading to pulmonary endothelial cell dysfunction. Our work suggests that reducing protein load in the ER of pulmonary endothelial cells might protect against acute respiratory distress syndrome in obese

Isolation and characterization of vascular endothelial cells derived from fetal tooth buds of miniature swine.

Science.gov (United States)

Nasu, Masanori; Nakahara, Taka; Tominaga, Noriko; Tamaki, Yuichi; Ide, Yoshiaki; Tachibana, Toshiaki; Ishikawa, Hiroshi

2013-03-01

The aim of the present study was to isolate endothelial cells from tooth buds (unerupted deciduous teeth) of miniature swine. Mandibular molar tooth buds harvested from swine fetuses at fetal days 90-110 were cultured in growth medium supplemented with 15% fetal bovine serum in 100-mm culture dishes until the primary cells outgrown from the tooth buds reached confluence. A morphologically defined set of pavement-shaped primary cells were picked up manually with filter paper containing trypsin/ethylenediamine tetraacetic acid solution and transferred to a separate dish. A characterization of the cellular characteristics and a functional analysis of the cultured cells at passages 3 to 5 were performed using immunofluorescence, a reverse transcriptase polymerase chain reaction assay, a tube formation assay, and transmission electron microscopy. The isolated cells grew in a pavement arrangement and showed the characteristics of contact inhibition upon reaching confluence. The population doubling time was ~48 h at passage 3. As shown by immunocytostaining and western blotting with specific antibodies, the cells produced the endothelial marker proteins such as vascular endothelial cadherin, von Willebrand factor, and vascular endothelial growth factor receptor-2. Observation with time-lapse images showed that small groups of cells aggregated and adhered to each other to form tube-like structures. Moreover, as revealed through transmission electron microscopy, these adherent cells had formed junctional complexes. These endothelial cells from the tooth buds of miniature swine are available as cell lines for studies on tube formation and use in regenerative medical science.

5-Hydroxytryptamine 4 Receptor in the Endothelial Cells

DEFF Research Database (Denmark)

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

2006-01-01

39 5-HYDROXYTRYPTAMINE 4 RECEPTOR IN THE ENDOTHELIAL CELLS. J. Profirovic, I. Vardya, T. Voyno-Yasenetskaya, Department of Pharmacology, University of Illinois at Chicago, Chicago, IL. Serotonin (5-hydroxytryptamine [5-HT]) is an important neurotransmitter that regulates multiple events in the ce......39 5-HYDROXYTRYPTAMINE 4 RECEPTOR IN THE ENDOTHELIAL CELLS. J. Profirovic, I. Vardya, T. Voyno-Yasenetskaya, Department of Pharmacology, University of Illinois at Chicago, Chicago, IL. Serotonin (5-hydroxytryptamine [5-HT]) is an important neurotransmitter that regulates multiple events...... 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...

The endothelial protein C receptor rs867186-GG genotype is associated with increased soluble EPCR and could mediate protection against severe malaria

DEFF Research Database (Denmark)

Shabani, Estela; Opoka, Robert O; Bangirana, Paul

2016-01-01

The endothelial protein C receptor (EPCR) appears to play an important role in Plasmodium falciparum endothelial cell binding in severe malaria (SM). Despite consistent findings of elevated soluble EPCR (sEPCR) in other infectious diseases, field studies to date have provided conflicting data abo...

Vascular endothelial growth factor modified macrophages transdifferentiate into endothelial-like cells and decrease foam cell formation.

Science.gov (United States)

Yan, Dan; He, Yujuan; Dai, Jun; Yang, Lili; Wang, Xiaoyan; Ruan, Qiurong

2017-06-30

Macrophages are largely involved in the whole process of atherosclerosis from an initiation lesion to an advanced lesion. Endothelial disruption is the initial step and macrophage-derived foam cells are the hallmark of atherosclerosis. Promotion of vascular integrity and inhibition of foam cell formation are two important strategies for preventing atherosclerosis. How can we inhibit even the reverse negative role of macrophages in atherosclerosis? The present study was performed to investigate if overexpressing endogenous human vascular endothelial growth factor (VEGF) could facilitate transdifferentiation of macrophages into endothelial-like cells (ELCs) and inhibit foam cell formation. We demonstrated that VEGF-modified macrophages which stably overexpressed human VEGF (hVEGF 165 ) displayed a high capability to alter their phenotype and function into ELCs in vitro Exogenous VEGF could not replace endogenous VEGF to induce the transdifferentiation of macrophages into ELCs in vitro We further showed that VEGF-modified macrophages significantly decreased cytoplasmic lipid accumulation after treatment with oxidized LDL (ox-LDL). Moreover, down-regulation of CD36 expression in these cells was probably one of the mechanisms of reduction in foam cell formation. Our results provided the in vitro proof of VEGF-modified macrophages as atheroprotective therapeutic cells by both promotion of vascular repair and inhibition of foam cell formation. © 2017 The Author(s).

Inhibition of endothelial cell expression of plasminogen activator inhibitor type-1 by gemfibrozil.

Science.gov (United States)

Fujii, S; Sawa, H; Sobel, B E

1993-10-18

Increased concentrations of plasminogen activator inhibitor type-1 (PAI-1) in plasma are associated with impaired fibrinolysis and venous and arterial thrombo-embolic disease. In pilot studies designed to identify pharmacologic approaches capable of diminishing such increases, we found that gemfibrozil attenuated the stimulation of synthesis of PAI-1 in a human, immortal, hepatoma cell line (Hep G2) induced by platelets. The present study was performed to determine whether it exerts analogous effects in non-immortal endothelial cells and whether it may therefore facilitate fibrinolysis locally in vivo. Human umbilical vein endothelial cells were incubated with pharmacologic concentrations of gemfibrozil. Gemfibrozil, 100 microM, suppressed basal PAI-1 production by 15% and attenuated the augmentation of synthesis of PAI-1 induced by lysates from platelets (4 x 10(7)/ml) by 36% over 24 h without inhibiting overall protein synthesis. In addition, the increases in PAI-1 mRNA otherwise induced by platelet lysates over 6 h were suppressed by 49% (Northern blots) without any demonstrable change in the intracellular half-life of PAI-1 mRNA. Pulse-chase experiments demonstrated diminution of PAI-1 protein synthesis in parallel with the changes observed in PAI-1 mRNA. To determine whether these effects of gemfibrozil on endothelial cells in vitro were paralleled by consistent changes in the concentrations of PAI-1 in plasma in vivo, we studied rabbits with induced carotid artery thrombosis and thrombolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

MRP4 knockdown enhances migration, suppresses apoptosis, and produces aggregated morphology in human retinal vascular endothelial cells

Energy Technology Data Exchange (ETDEWEB)

Tagami, Mizuki [Department of Surgery Related, Division of Ophthalmology, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017 (Japan); Kusuhara, Sentaro, E-mail: kusu@med.kobe-u.ac.jp [Department of Surgery Related, Division of Ophthalmology, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017 (Japan); Imai, Hisanori [Department of Surgery Related, Division of Ophthalmology, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017 (Japan); Uemura, Akiyoshi [Department of Surgery Related, Division of Ophthalmology, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017 (Japan); Department of Vascular Biology, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017 (Japan); Honda, Shigeru; Tsukahara, Yasutomo; Negi, Akira [Department of Surgery Related, Division of Ophthalmology, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017 (Japan)

2010-10-01

Research highlights: {yields} Exogenous VEGF decreases MRP4 expression in a dose-dependent manner. {yields} MRP4 knockdown leads to enhanced cell migration. {yields} MRP4 knockdown suppresses caspase-3-mediated cell apoptosis. {yields} MRP4 knockdown produces cell assembly and cell aggregation. -- Abstract: The multidrug resistance protein (MRP) MRP4/ABCC4 is an ATP-binding cassette transporter that actively effluxes endogenous and xenobiotic substrates out of cells. In the rodent retina, Mrp4 mRNA and protein are exclusively expressed in vascular endothelial cells, but the angiogenic properties of Mrp4 are poorly understood so far. This study aims to explore the angiogenic properties of MRP4 in human retinal microvascular endothelial cells (HRECs) utilizing the RNA interference (RNAi) technique. MRP4 expression was decreased at the mRNA and protein levels after stimulation with exogenous vascular endothelial growth factor in a dose-dependent manner. RNAi-mediated MRP4 knockdown in HRECs do not affect cell proliferation but enhances cell migration. Moreover, cell apoptosis induced by serum starvation was less prominent in MRP4 siRNA-treated HRECs as compared to control siRNA-treated HRECs. In a Matrigel-based tube-formation assay, although MRP4 knockdown did not lead to a significant change in the total tube length, MRP4 siRNA-treated HRECs assembled and aggregated into a massive tube-like structure, which was not observed in control siRNA-treated HRECs. These results suggest that MRP4 is uniquely involved in retinal angiogenesis.

Ionizing radiation activates vascular endothelial growth factor-A transcription in human umbilical vein endothelial cells

Energy Technology Data Exchange (ETDEWEB)

Lee, Hyounji; Kim, Kwang Seok; Jeong, Jae Hoon; Lim, Young Bin [Radiation Cancer Biology Team, Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

2016-12-15

Vascular endothelial growth factor (VEGF) is an essential paracrine factor for developmental and pathological angiogenesis. VEGF also exerts its effects in an autocrine manner in VEGF-producing cells. For instance, autocrine VEGF signaling occurs in tumor cells and contributes to key aspects of tumorigenesis, such as in the function of cancer stem cells and tumor initiation, which are independent of angiogenesis. In addition to tumors cells, non-transformed cells also express VEGF. For example, a VEGF dependent intracellular autocrine mechanism is crucial for the survival of hematopoietic stem cells and hematopoiesis. Stereotactic body radiation therapy (SBRT) is a novel treatment modality for early primary cancer and oligometastatic disease. SBRT delivers high-dose hypofractionated radiation, such as 20-60 Gy, to tumors in a single fraction or 2-5 fractions. As VEGF is a critical regulator of functional integrity and viability of vascular endothelial cells, we examined whether high-dose irradiation alters VEGF signaling by measuring the expression levels of VEGFA transcript. It is generally believed that endothelial cells do not produce VEGF in response to radiation. In present study, however, we provide the first demonstration of transcriptional regulation of VEGFA in human vascular endothelial cells by IR treatment. Irradiation with doses higher than 10 Gy in a single exposure triggers up-regulation of VEGFA transcription within 2 hours in HUVECs, whereas irradiation with 10 Gy does not alter VEGFA levels. Our data have shown that high-dose irradiation triggers immediate transactivation of VEGFA in human vascular endothelial cells.

Changes in insulin-like growth factor-binding protein-3 messenger ribonucleic acid in endothelial cells of the human corpus luteum: a possible role in luteal development and rescue.

Science.gov (United States)

Fraser, H M; Lunn, S F; Kim, H; Duncan, W C; Rodger, F E; Illingworth, P J; Erickson, G F

2000-04-01

In the human menstrual cycle, extensive angiogenesis accompanies luteinization; and the process is physiologically important for corpus luteum (CL) function. During luteolysis, the vasculature collapses, and the endothelial cells die. In a conceptual cycle, the CL persists both functionally and structurally beyond the luteoplacental shift. Although luteal rescue is not associated with increased angiogenesis, endothelial survival is extended. Despite the central role of the luteal vasculature in fertility, the mechanisms regulating its development and demise are poorly understood. There is increasing evidence that insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) may be important effectors of luteal function. Here, we have found that IGFBP-3 messenger RNA is expressed in the endothelium of the human CL and that the levels of message change during luteal development and rescue by human CG. The signal was strong during the early luteal phase, but it showed significant reduction during the mid- and late luteal phases. Interestingly, administration of human CG caused a marked increase in the levels of IGFBP-3 messenger RNA in luteal endothelial cells that was comparable to that observed during the early luteal phase. We conclude that endothelial cell IGFBP-3 expression is a physiological property of the CL of menstruation and pregnancy. These observations raise the intriguing possibility that the regulated expression of endothelial IGFBP-3 may play a role in controlling angiogenesis and cell responses in the human CL by autocrine/paracrine mechanisms.

Protein Phosphotyrosine Phosphatase 1B (PTP1B) in Calpain-dependent Feedback Regulation of Vascular Endothelial Growth Factor Receptor (VEGFR2) in Endothelial Cells

Science.gov (United States)

Zhang, Yixuan; Li, Qiang; Youn, Ji Youn; Cai, Hua

2017-01-01

The VEGF/VEGFR2/Akt/eNOS/NO pathway is essential to VEGF-induced angiogenesis. We have previously discovered a novel role of calpain in mediating VEGF-induced PI3K/AMPK/Akt/eNOS activation through Ezrin. Here, we sought to identify possible feedback regulation of VEGFR2 by calpain via its substrate protein phosphotyrosine phosphatase 1B (PTP1B), and the relevance of this pathway to VEGF-induced angiogenesis, especially in diabetic wound healing. Overexpression of PTP1B inhibited VEGF-induced VEGFR2 and Akt phosphorylation in bovine aortic endothelial cells, while PTP1B siRNA increased both, implicating negative regulation of VEGFR2 by PTP1B. Calpain inhibitor ALLN induced VEGFR2 activation, which can be completely blocked by PTP1B overexpression. Calpain activation induced by overexpression or Ca/A23187 resulted in PTP1B cleavage, which can be blocked by ALLN. Moreover, calpain activation inhibited VEGF-induced VEGFR2 phosphorylation, which can be restored by PTP1B siRNA. These data implicate calpain/PTP1B negative feedback regulation of VEGFR2, in addition to the primary signaling pathway of VEGF/VEGFR2/calpain/PI3K/AMPK/Akt/eNOS. We next examined a potential role of PTP1B in VEGF-induced angiogenesis. Endothelial cells transfected with PTP1B siRNA showed faster wound closure in response to VEGF. Aortic discs isolated from PTP1B siRNA-transfected mice also had augmented endothelial outgrowth. Importantly, PTP1B inhibition and/or calpain overexpression significantly accelerated wound healing in STZ-induced diabetic mice. In conclusion, our data for the first time demonstrate a calpain/PTP1B/VEGFR2 negative feedback loop in the regulation of VEGF-induced angiogenesis. Modulation of local PTP1B and/or calpain activities may prove beneficial in the treatment of impaired wound healing in diabetes. PMID:27872190

Lipoprotein receptors in cultured bovine endothelial cells

International Nuclear Information System (INIS)

Struempfer, A.E.M.

1983-07-01

In this study, receptors that may be involved in the uptake of low density lipoproteins (LDL) and low density lipoproteins which have been modified by acetylation (AcLDL), were characterized. Aortic epithelial cells were used and a cell culture system which closely resembled the in vivo monolayer was established. Endothelial cell and lipoprotein interactions were examined by incubating the cells with 125 l-labelled lipoproteins under various conditions. The receptor affinity of bovine aortic endothelial cells was higher for AcLDL than that for LDL. Competition studies demonstrated that there were two distinct receptors for LDL and AcLDL on the endothelial cells. AcLDL did not compete with LDL for the LDL receptor, and conversely LDL did not compete with AcLDL for the AcLDL receptor. The receptor activities for LDL and AcLDL were examined as a function of culture age. Whereas the LDL receptor could be regulated, the AcLDL receptor was not as susceptible to regulation. Upon exposing endothelial cells for 72 h to either LDL or AcLDL, it was found that the total amount of cellular cholesterol increased by about 50%. However, the increase of total cholesterol was largely in the form of free cholesterol. This is in contrast to macrophages, where the increase in total cholesterol upon exposure to AcLDL is largely in the form cholesteryl esters

Culture and Characterization of Circulating Endothelial Progenitor Cells in Patients with Renal Cell Carcinoma.

Science.gov (United States)

Gu, Wenyu; Sun, Wei; Guo, Changcheng; Yan, Yang; Liu, Min; Yao, Xudong; Yang, Bin; Zheng, Junhua

2015-07-01

Although emerging evidence demonstrates increased circulating endothelial progenitor cells in patients with solid tumors, to our knowledge it is still unknown whether such cells can be cultured from patients with highly angiogenic renal cell carcinoma. We cultured and characterized circulating endothelial progenitor cells from patients with renal cell carcinoma. The circulating endothelial progenitor cell level (percent of CD45(-)CD34(+) VEGF-R2(+) cells in total peripheral blood mononuclear cells) was quantified in 47 patients with renal cell carcinoma and 40 healthy controls. Peripheral blood mononuclear cells were then isolated from 33 patients with renal cell carcinoma and 30 healthy controls to culture and characterize circulating endothelial progenitor cells. The circulating endothelial progenitor cell level was significantly higher in patients with renal cell carcinoma than in healthy controls (0.276% vs 0.086%, p cells first emerged significantly earlier in patient than in control preparations (6.72 vs 14.67 days, p culture success rate (87.8% vs 40.0% of participants) and the number of colonies (10.06 vs 1.83) were significantly greater for patients than for controls (each p cell level correlated positively with the number of patient colonies (r = 0.762, p Cells cultured from patients and controls showed a similar growth pattern, immunophenotype, ability to uptake Ac-LDL and bind lectin, and form capillary tubes in vitro. However, significantly more VEGF-R2(+) circulating endothelial progenitor cells were found in preparations from patients with renal cell carcinoma than from healthy controls (21.1% vs 13.4%, p cell colonies, a higher cell culture success rate and more colonies were found for patients with renal cell carcinoma than for healthy controls. Results indicate the important significance of VEGF-R2(+) circulating endothelial progenitors in patients with renal cell carcinoma. Copyright © 2015 American Urological Association Education and Research

Endothelial Induced EMT in Breast Epithelial Cells with Stem Cell Properties

OpenAIRE

Sigurdsson, Valgardur; Hilmarsdottir, Bylgja; Sigmundsdottir, Hekla; Fridriksdottir, Agla J. R.; Ringnér, Markus; Villadsen, Rene; Borg, Ake; Agnarsson, Bjarni A.; Petersen, Ole William; Magnusson, Magnus K.; Gudjonsson, Thorarinn

2011-01-01

Epithelial to mesenchymal transition (EMT) is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell proper...

Tumor-derived circulating endothelial cell clusters in colorectal cancer.

KAUST Repository

Cima, Igor; Kong, Say Li; Sengupta, Debarka; Tan, Iain B; Phyo, Wai Min; Lee, Daniel; Hu, Min; Iliescu, Ciprian; Alexander, Irina; Goh, Wei Lin; Rahmani, Mehran; Suhaimi, Nur-Afidah Mohamed; Vo, Jess H; Tai, Joyce A; Tan, Joanna H; Chua, Clarinda; Ten, Rachel; Lim, Wan Jun; Chew, Min Hoe; Hauser, Charlotte; van Dam, Rob M; Lim, Wei-Yen; Prabhakar, Shyam; Lim, Bing; Koh, Poh Koon; Robson, Paul; Ying, Jackie Y; Hillmer, Axel M; Tan, Min-Han

2016-01-01

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.

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.

Targeted transfection increases siRNA uptake and gene silencing of primary endothelial cells in vitro--a quantitative study.

Science.gov (United States)

Asgeirsdóttir, Sigridur A; Talman, Eduard G; de Graaf, Inge A; Kamps, Jan A A M; Satchell, Simon C; Mathieson, Peter W; Ruiters, Marcel H J; Molema, Grietje