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Endothelial Cell Line (endothelial + cell_line)
Selected AbstractsEstablishment of Rat Lymphatic Endothelial Cell LineMICROCIRCULATION, Issue 2 2003Risuke Mizuno ABSTRACT Objective: The objective of the present study was to establish a rat lymphatic endothelial cell line and then to investigate the morphological and immunohistochemical properties of the cells. Methods: The lymphatic endothelial cells of rat thoracic ducts were isolated enzymatically by trypsin digestion and were cultured in endothelium growth medium (EGM)-2 in an atmosphere of low oxygen (5% O2, 5% CO2, and 90% N2) or high oxygen (21% O2, 5% CO2, and 74% N2). Results: The number of the cells cultured in the low-oxygen atmosphere was significantly larger than that obtained in the high-oxygen atmosphere. The cultured cells in the low-oxygen atmosphere showed a monolayer with uniform cobblestone appearance, suggesting the morphological properties of endothelial cells. Factor VIII-related antigen and cell surface carbohydrates (i.e., D-galactose , and D- N -acetylgalactosamine ,) were found on the lymphatic cultured cells. The phagocytosis of 1,1-diocadecyl1-3,3,3,,3,-tetramethylindocarbocyanine perchlorate-labeled acetylated low-density lipoprotein also was observed in the cultured cells. The cytoskeleton protein F-actin was located on the plasma membrane of the cultured cells as circumferential thin bundles and in the cytoplasma as filamentous bundles. Conclusions: The present study indicates that the choice of EGM-2 as a culture medium and the hypoxic atmosphere (,5%) enabled us to establish rat lymphatic endothelial cell line. [source] Cells meeting our immunophenotypic criteria of endothelial cells are large plateletsCYTOMETRY, Issue 2 2007Michiel H. Strijbos Abstract Background Circulating endothelial cells (CEC) are shed from damaged vasculature, making them a rational choice to serve as surrogate marker for vascular damage. Currently, various techniques and CEC definitions are in use, and their standardization and validation is needed. A flow cytometric single platform assay defining CEC as forward light scatter (FSC)low-to-intermedate, sideward light scatter (SSC)low, CD45,, CD31++ and CD146+ is a promising approach to enumerate CEC because of its simplicity (Mancuso et al., Blood 2001;97:3658,3661). Here, we set out to confirm the endothelial nature of these cells. Methods We isolated cells with a FSClow-to-intermediate, SSClow, CD31++, CD45dim immunophenotype (termed "cells meeting our immunophenotypic criteria for endothelial cells" [CMOIC]) from healthy donors to study the expression of endothelium-associated markers using several techniques. Special attention was paid to reagents identifying the endothelial cell-specific marker CD146. We compared antigen expression patterns of CMOIC with those of the HUVEC endothelial cell line and lymphocytes. Electron microscopy was used to detect the presence of endothelial cell-specific Weibel,Palade bodies in the sorted cells. Results CD146 expression was negative on CMOIC for all tested CD146 mAbs, but positive on HUVEC cells and a minor subset of T lymphocytes. Using flow cytometry, we found no expression of any endothelium-associated marker except for CD31 and CD34. HUVEC cells were positive for all endothelial markers except for CD34. Evaluation of CMOIC morphology showed a homogenous population of cells with a highly irregular nucleus-like structure and positive endothelial immunohistochemistry. CMOIC contained neither nuclei nor DNA. Electron microscopy revealed the absence of a nucleus, the absence of endothelial specific Weibel,Palade bodies, and revealed CMOIC to be large platelets. Conclusion The vast majority of cells with the immunophenotype FSClow-to-intermediate, SSClow, CD45,, CD31++ do not express CD146 and are large platelets rather than endothelial cells. © 2007 Clinical Cytometry Society. [source] Fatty acid incorporation in endothelial cells and effects on endothelial nitric oxide synthaseEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 9 2007S. Couloubaly Abstract Background The nature of fatty acids provided by the diet as well as plasma lipid metabolism can modify the composition and properties of plasma membrane and thus the activity of membrane proteins. In humans, as well as in experimental models, diabetes is associated with both an alteration in serum lipid profile and a documented endothelial dysfunction. This in vitro study investigated on an immortalized human endothelial cell line (EA.hy 926) the specific effects of several free fatty acids (FFAs) on the composition of cellular membranes and the regulation of endothelial nitric oxide synthase (eNOS). Materials and methods 0·1% of lipid deprived serum was added to the incubation medium with 25 mm glucose in order to study the effects of individual fatty acids: myristic acid, palmitic acid, stearic acid, oleic acid or linoleic acid at 100 µm bound with albumin. The effects of the FFAs on the endothelial nitric oxide synthase were investigated on mRNA level by quantitative PCR, on protein level and Ser1177 phosphorylation by Western blot and on enzymatic activity on living cells using radiolabelled arginine. Results Free linoleic acid increased the membrane content in n-6 fatty acids (mainly C18: n-6 and its metabolites) with a decrease in saturated and monounsaturated fatty acids. These conditions decreased the basal eNOS activity and reduced the phosphorylation of eNOS-Ser1177 due to activation by histamine. Free palmitic acid enriched the membranes with 16 : 0 with a slight decrease in monounsaturated fatty acids. These conditions increased eNOS activation without increasing Ser1177 phosphorylation upon histamine activation. The addition of the other FFAs also resulted in modifications of membrane composition, which did not to affect eNOS-Ser1177 phosphorylation. Conclusion Among the fatty acids used, only modification of the membrane composition due to linoleic acid supply disturbed the basal enzymatic activity and Ser1177 phosphorylation of eNOS in a way that limited the role of histamine activation. Linoleic acid might involve the dysfunction of both eNOS basal activity and its phosphorylation status and may then contribute to an impaired vasodilatation in vivo. [source] Cytotoxicity of MTA and Portland cement on human ECV 304 endothelial cellsINTERNATIONAL ENDODONTIC JOURNAL, Issue 9 2005G. De Deus Abstract Aim, To evaluate the cytotoxic effects of two brands of mineral trioxide aggregate (MTA) (Pro-Root MTA® and MTA Angelus®) and Portland cement (PC) on the human ECV 304 endothelial cell line. Methodology, Endothelial ECV 304 cells were incubated at 37 °C in an atmosphere of 95% air, 5% carbon dioxide and 100% humidity for 7 days and grown in F12 medium supplemented with 10% fetal bovine serum with 50 ,g mL,1 of gentamicin sulphate. Effects of the materials on mitochondrial functions were measured by a colorimetric assay. At each experimental time interval (24, 48 and 72 h), a dimethyl-thiazol-diphenyl tetrazolium bromid assay was conducted to measure cell viability. All assays were repeated three times to ensure reproducibility. Results were expressed as average absorbance (A570\,nm) ± SD and the data were analysed statistically by one-way analysis of variance and the Bonferroni post-test. A P -value <0.05 was considered statistically significant. Results, No statistically significant difference was shown between any of the experimental materials (P > 0.05). Conclusions, The two brands of MTA analysed, as well as the PC, initially showed a similar elevated cytotoxic effect that decreased gradually with time allowing the cell culture to become reestablished. [source] Modulation of p-glycoprotein function by caveolin-1 phosphorylationJOURNAL OF NEUROCHEMISTRY, Issue 1 2007Stéphane Barakat Abstract p-glycoprotein (p-gp) is an ATP-binding cassette transporter and its overexpression is responsible for the acquisition of the multidrug resistance phenotype in human tumors. p-gp is localized at the blood,brain barrier and is involved in brain cytoprotection. Our previous work used immunoprecipitation to show that caveolin-1 can interact with p-gp. In this study, we provide evidence that caveolin-1 regulates p-gp transport activity in a rat brain endothelial cell line (RBE4). Down-regulation of caveolin-1 by siRNA reduced the interaction between p-gp and caveolin-1, followed by a decrease in [3H]-Taxol and [3H]-Vinblastine accumulation in RBE4 cells. The latter result showed that down-regulation of caveolin-1 enhanced p-gp transport activity. RBE4 cells were also transfected with Sarcoma in order to modulate caveolin-1 phosphorylation. Overexpression of Sarcoma, a protein tyrosine kinase, stimulated caveolin-1 phosphorylation and increased both [3H]-Taxol and [3H]-Vinblastine accumulation as well as Hoechst 33342 accumulation. Transfection of caveolin-1 inhibits p-gp transport activity. Conversely, transfection of the mutant cavY14F decreased the p-gp/caveolin-1 interaction and reduced accumulation of the two p-gp substrates. Thus, our data show that caveolin-1 regulates p-gp function through the phosphorylation state of caveolin-1 in endothelial cells from the blood,brain barrier. [source] Assessment of the role of heparan sulfate in high molecular weight kininogen binding to human umbilical vein endothelial cellsJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 11 2003L.P. Fernando Summary., The assembly and activation of the kinin forming system components on human umbilical vein endothelial cells (HUVEC) have been studied in great detail. Proteins such as gC1qR, cytokeratin-1 and u-PAR have been identified to be responsible for Zn2+ -dependent binding of high molecular weight kininogen (HK) to HUVEC. Heparan sulfate has also been shown to have a major role in Zn2+ -dependent binding of HK to the endothelial cell line, Ea.hy 926. In this study, we have analyzed the possible contribution of heparan sulfate to high molecular weight kininogen binding to HUVEC using multiple approaches. The presence of heparan sulfate on HUVEC was analyzed by staining with an antibody specific for heparan sulfate. Incubation of the cells with bacterial heparinases removed the heparan sulfate from the cell surface to the level seen with a control antibody, however, the Zn2+ -dependent binding of HK was not affected. Further, blocking of heparan sulfate with a specific antibody to heparan sulfate even after digestion with heparinases did not reduce HK binding whereas antibodies to the proteins gC1qR and cytokeratin-1 consistently reduced the binding of HK to the endothelial cells. The binding intensities of FITC-labeled HK were similar in heparinase-treated and -untreated HUVEC. The rate of kallikrein formation by the assembly of factor XII, HK and PK were similar in both heparinase-treated and non-treated HUVEC. All of these data indicate that heparan sulfate does not contribute significantly to HK binding to HUVEC. [source] Establishment of Rat Lymphatic Endothelial Cell LineMICROCIRCULATION, Issue 2 2003Risuke Mizuno ABSTRACT Objective: The objective of the present study was to establish a rat lymphatic endothelial cell line and then to investigate the morphological and immunohistochemical properties of the cells. Methods: The lymphatic endothelial cells of rat thoracic ducts were isolated enzymatically by trypsin digestion and were cultured in endothelium growth medium (EGM)-2 in an atmosphere of low oxygen (5% O2, 5% CO2, and 90% N2) or high oxygen (21% O2, 5% CO2, and 74% N2). Results: The number of the cells cultured in the low-oxygen atmosphere was significantly larger than that obtained in the high-oxygen atmosphere. The cultured cells in the low-oxygen atmosphere showed a monolayer with uniform cobblestone appearance, suggesting the morphological properties of endothelial cells. Factor VIII-related antigen and cell surface carbohydrates (i.e., D-galactose , and D- N -acetylgalactosamine ,) were found on the lymphatic cultured cells. The phagocytosis of 1,1-diocadecyl1-3,3,3,,3,-tetramethylindocarbocyanine perchlorate-labeled acetylated low-density lipoprotein also was observed in the cultured cells. The cytoskeleton protein F-actin was located on the plasma membrane of the cultured cells as circumferential thin bundles and in the cytoplasma as filamentous bundles. Conclusions: The present study indicates that the choice of EGM-2 as a culture medium and the hypoxic atmosphere (,5%) enabled us to establish rat lymphatic endothelial cell line. [source] Absent reduction by HIV protease inhibitors of Candida albicans adhesion to endothelial cellsMYCOSES, Issue 3 2007Barbara Falkensammer Summary Highly active antiretroviral therapy including HIV protease inhibitors has led to a marked reduction of clinically relevant mucosal candidiasis. We have previously shown that HIV protease inhibitors directly inhibit adhesion of Candida albicans to epithelial cells at concentrations that are reached in vivo during antiretroviral therapy. The aim of this study was to establish whether HIV protease inhibitors also inhibit adhesion of Candida to endothelial cells, which play a major role in systemic fungal disease. Three C. albicans strains were incubated with human umbilical vein endothelial cells or an endothelial cell line in the presence of either Ritonavir, Saquinavir or Indinavir. Subsequently, adherence was determined by counting colony-forming units. The results were comparable and revealed that Ritonavir and Saquinavir significantly inhibited adherence to endothelial cells at only very high concentrations which are likely not reached in vivo, and Indinavir did not even inhibit then. Inhibition of adhesion of C. albicans to human cells by HIV protease inhibitors is not a general feature, but strongly cell type-dependent, and clearly not observed for endothelial cells in vitro, which are a main target of systemic candidiasis in vivo. [source] A Novel Three-Dimensional In Vitro System to Study Trophoblast,Endothelium Cell InteractionsAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 2 2007Paulomi B. Aldo Introduction Pregnancy complications have been linked to improper trophoblast migration and failure of spiral artery transformation. Endothelial cells play an essential role in directing trophoblast migration and transformation, although by an unknown mechanism. We describe a novel in vitro model to evaluate endothelial,trophoblast interaction and signaling in a three-dimensional system. Method of study Immortalized human endometrial endothelial cell line and first trimester trophoblast cells were co-cultured. Endothelial transformation into vessel-like structures occurred in MatrigelTM OpenLab Image Analysis software was used to monitor labeled trophoblast migration and endothelium transformation. Cytokine/chemokine production was determined using Multiplex. Results Trophoblast migrates toward endothelial cells in Matrigel, aligns on top of the endothelium within 4,8 hr and achieves complete replacement of the endothelium by 72,96 hr. Lipopolysaccharide treatment damages the endothelium and disrupts endothelium,trophoblast interaction. Conclusion We report a novel three-dimensional in vitro and in vivo system of trophoblast,endothelium cell interaction. Significant changes in endothelial cells' phenotype are observed upon differentiation in Matrigel. These changes may be necessary for endothelium to direct trophoblast migration and transformation. [source] Novel markers of inflammation identified in tumor necrosis factor receptor,associated periodic syndrome (TRAPS) by transcriptomic analysis of effects of TRAPS-associated tumor necrosis factor receptor type I mutations in an endothelial cell lineARTHRITIS & RHEUMATISM, Issue 1 2009Susana L. Rebelo Objective To analyze the effects of tumor necrosis factor receptor,associated periodic syndrome (TRAPS),associated mutant tumor necrosis factor receptor type I (TNFRI) expression in a cell type directly relevant to the inflammation in TRAPS, and to identify novel markers associated with mutant TNFRI expression. Methods Transcriptome analysis on 30,000 human genes was performed on SK-Hep-1 human endothelial cells transfected with either wild-type (WT) or TRAPS-associated mutant TNFRI. Quantitative reverse transcriptase,polymerase chain reaction and protein expression levels measured by enzyme-linked immunosorbent assay verified transcriptional changes for selected genes both in supernatants from cells expressing mutant TNFRI and in patient plasma. Results Cells expressing mutant TNFRI showed up-regulation of multiple proinflammatory genes relative to WT transfectants, including genes for pentraxin 3, granulocyte,macrophage colony-stimulating factor, granulocyte colony-stimulating factor, CCL2, and CCL5, which were also expressed as proteins. In addition, the expression of most of these markers was increased in the plasma and peripheral blood mononuclear cells from TRAPS patients relative to those from healthy controls. The cysteine mutations (C33Y and C52F), which are associated with a more severe clinical phenotype, induced more genes than the low-penetrance mutation R92Q, which is associated with a milder phenotype. The expression of most genes was induced by a death domain (DD),dependent mechanism, since they were not induced by expression of TNFRI mutants with an inactivated DD. Conclusion TRAPS-associated TNFRI mutants induce the expression of multiple genes encoding inflammatory molecules, cellular receptors, transcription factors, and regulators of apoptosis in endothelial cells that require the cytoplasmic signaling properties of the receptor. Different mutants have specific expression profiles, indicating mutation-specific effects. The expression of some of these markers was also elevated in samples from TRAPS patients. [source] Down-Regulation of CD40 Gene Expression and Inhibition of Apoptosis with Danshensu in Endothelial CellsBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 2 2009Guang-De Yang Research shows that it also has immunostimulation properties. The present study evaluates the protective effect of danshensu, an active water-extractable component isolated from danshen, on an endothelial cell line (CRL-1730) treated with hydrogen peroxide (H2O2). Danshensu significantly inhibited endothelial cell viability induced by H2O2. The treatment of endothelial cells with danshensu resulted in most cells being arrested in the S and G2/M phases of the cell cycle. The fraction of cells in G0/G1 phase was markedly decreased by danshensu treatment compared to the control groups. The apoptosis was also markedly decreased after danshensu treatment. Additionally, danshensu restrains decreased nitric oxide level, increased the release of lactate dehydrogenase and expression of cluster of differentiation 40 (CD40) significantly. These results suggest that danshensu protects endothelial cells from the damage induced by H2O2 through its CD40 anti-inflammatory approach and cell apoptosis inhibition. [source] Vascular and Biology 05BRITISH JOURNAL OF SURGERY (NOW INCLUDES EUROPEAN JOURNAL OF SURGERY), Issue S1 2002L. Yea Background: Tight junctions govern the permeability of endothelial and epithelial cells. Changes in tight junction function are thus an early and key event in cancer metastasis and tissue permeability. This study sought to determine the role of oestrogen in the regulation of tight junctions and expression of occludin in endothelial cells. Methods: Human vascular endothelial cell line was incubated with 17-,-oestradiol at different concentrations (from 10,11m to 10,7m) over 1,24 h. Expression of occludin mRNA was determined using RT-PCR, and change of occludin protein using Western blotting. Transendothelial resistance (TER) was measured with an EVOM, and transendothelial cell permeability was determined using fluorescence labelled dextran (FITC-dextran 10) with a multichannel fluorescence reader. Results: 17-,-oestradiol reduced the expression of occludin mRNA in a time and concentration dependent manner with an obvious effect starting from 4 h. Reduced level of occludin protein was similarly seen after treatment with 17-,-oestradiol. Incubation of HECV with 17-,-oestradiol resulted in an increase in paracellular permeability by 9.9 ± 8.9 per cent at 10,10m (P > 0.05 versus control), 42.1 ± 15.2 per cent at 10,9m (P < 0.05 versus control), and 40.1 ± 22.4 per cent at 10,8m (P < 0.05, versus control). A decrease in the transendothelial cell resistance (TER) was seen with oestradiol (a reduction by 18.6 ± 16.6 per cent (P > 0.05 versus control), 31.5 ± 10.6 per cent (P < 0.01), or 44.4 ± 18.4 per cent (P < 0.01), at concentration 10,10, 10,9, 10,8m, respectively. Conclusions: This study shows a perturbation of tight junction functions in endothelial cells by oestrogen, which may have implications in the aetiology of mastalgia and vascular spread of breast cancer. [source] Inhibitory effect of c-Met mutants on the formation of branching tubules by a porcine aortic endothelial cell lineCANCER SCIENCE, Issue 12 2006Marino Maemura The association of hepatocyte growth factor (HGF) with its high-affinity receptor (c-Met) has been shown to induce mitogenesis, motogenesis and morphogenesis in a variety of cell types. Various point mutations in c-Met have been identified in hereditary and sporadic papillary renal carcinomas as well as in other carcinomas. In the present study, we examined the effects of c-Met point mutations on the morphology of a porcine aortic endothelial (PAE) cell line. When cultured in three-dimensional collagen gel, PAE cells formed branching tubule structures, and HGF treatment caused breakdown of the structures and induced a scattered morphology. The exogenous expression of c-Met point mutants inhibited the formation of tubules. HGF treatment induced the formation of tubules by PAE cells expressing some c-Met mutants, but it induced the scattering of PAE cells expressing other c-Met mutants. The presence of a low concentration of a mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) inhibitor cancelled the inhibitory effect of the c-Met point mutations on the formation of tubules. These results suggest that c-Met point mutations affect the extracellular signal-regulated kinase (ERK) signaling required for the formation of tubules by PAE cells, and HGF binding changes the conformation of c-Met mutants, leading to the different signals required for formation of tubules and cell scattering. (Cancer Sci 2006; 97: 1343,1350) [source] Interaction between flavonoids and the blood,brain barrier: in vitro studiesJOURNAL OF NEUROCHEMISTRY, Issue 1 2003Kuresh A. Youdim Abstract There is considerable current interest in the neuroprotective effects of flavonoids. This study focuses on the potential for dietary flavonoids, and their known physiologically relevant metabolites, to enter the brain endothelium and cross the blood,brain barrier (BBB) using well-established in vitro models (brain endothelial cell lines and ECV304 monolayers co-cultured with C6 glioma cells). We report that the citrus flavonoids, hesperetin, naringenin and their relevant in vivo metabolites, as well as the dietary anthocyanins and in vivo forms, cyanidin-3-rutinoside and pelargonidin-3-glucoside, are taken up by two brain endothelial cell lines from mouse (b.END5) and rat (RBE4). In both cell types, uptake of hesperetin and naringenin was greatest, increasing significantly with time and as a function of concentration. In support of these observations we report for the first time high apparent permeability (Papp) of the citrus flavonoids, hesperetin and naringenin, across the in vitro BBB model (apical to basolateral) relative to their more polar glucuronidated conjugates, as well as those of epicatechin and its in vivo metabolites, the dietary anthocyanins and to specific phenolic acids derived from colonic biotransformation of flavonoids. The results demonstrate that flavonoids and some metabolites are able to traverse the BBB, and that the potential for permeation is consistent with compound lipophilicity. [source] Progress and limitations in the use of in vitro cell cultures to serve as a permeability screen for the blood-brain barrierJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 11 2001Mark Gumbleton Abstract A relatively simple, widely applicable, and robust in vitro method of predicting blood-brain barrier (BBB) permeability to central nervous system-acting drugs is an increasing need. A cell-based model offers the potential to account for transcellular and paracellular drug diffusional processes, metabolism, and active transport processes, as well as nondefined interactions between a drug and cellular material that may impact upon a membrane's overall permeability profile. Any in vitro BBB cell model to be utilized for the transendothelial BBB permeability screening of potential central nervous system drugs must display reproducible solute permeability, and a number of other general criteria including: a restrictive paracellular barrier; a physiologically realistic cell architecture; the functional expression of key transporter mechanisms; and allow ease of culture to meet the technical and time constraints of a screening program. This article reviews the range of in vitro cell-based BBB models available, including the primary/low passage bovine and porcine brain endothelial cultures as well as the spectrum of immortalized brain endothelial cell lines that have been established. The article further discusses the benefits and limitations of exploiting such systems as in vitro BBB permeability screens. © 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:1681,1698, 2001 [source] Antiangiogenic and Immunomodulatory Effects of Rapamycin on Islet Endothelium: Relevance for Islet TransplantationAMERICAN JOURNAL OF TRANSPLANTATION, Issue 11 2006V. Cantaluppi Donor intra-islet endothelial cells contribute to neovascularization after transplantation. Several factors may interfere with this process and ultimately influence islet engraftment. Rapamycin, a central immunosuppressant in islet transplantation, is an mTOR inhibitor that has been shown to inhibit cancer angiogenesis. The aim of this study was to evaluate the effects of rapamycin on islet endothelium. Rapamycin inhibited the outgrowth of endothelial cells from freshly purified human islets and the formation of capillary-like structures in vitro and in vivo after subcutaneous injection within Matrigel plugs into SCID mice. Rapamycin decreased migration, proliferation and angiogenic properties of human and mouse islet-derived endothelial cell lines with appearance of apoptosis. The expression of angionesis-related factors VEGF, ,V,3 integrin and thrombospondin-1 on islet endothelium was altered in the presence of rapamycin. On the other hand, rapamycin decreased the surface expression of molecules involved in immune processes such as ICAM-1 and CD40 and reduced the adhesion of T cells to islet endothelium. Our results suggest that rapamycin exerts dual effects on islet endothelium inducing a simultaneous inhibition of angiogenesis and a down-regulation of receptors involved in lymphocyte adhesion and activation. [source] | |||||||||||||