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At least 127 records · Page 7Linked to original sources

PubMed2003

Selective inhibition of vascular endothelial growth factor receptor-2 (VEGFR-2) identifies a central role for VEGFR-2 in human aortic endothelial cell responses to VEGF.

Vascular endothelial growth factor receptors (VEGFR) are considered essential for angiogenesis. The VEGFR-family proteins consist of VEGFR-1/Flt-1, VEGFR-2/KDR/Flk-1, and VEGFR-3/Flt-4. Among these, VEGFR-2 is thought to be principally responsible for angiogenesis. However, the precise role of VEGFRs1-3 in endothelial cell biology and angiogenesis remains unclear due in part to the lack of VEGFR-specific inhibitors. We used the newly described, highly selective anilinoquinazoline inhibitor of VEGFR-2 tyrosine kinase, ZM323881 (5-[[7-(benzyloxy) quinazolin-4-yl]amino]-4-fluoro-2-methylphenol), to explore the role of VEGFR-2 in endothelial cell function. Consistent with its reported effects on VEGFR-2 [IC(50) < 2 nM], ZM323881 inhibited activation of VEGFR-2, but not of VEGFR-1, epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), or hepatocyte growth factor (HGF) receptor. We studied the effects of VEGF on human aortic endothelial cells (HAECs), which express VEGFR-1 and VEGFR-2, but not VEGFR-3, in the absence or presence of ZM323881. Inhibition of VEGFR-2 blocked activation of extracellular regulated-kinase, p38, Akt, and endothelial nitric oxide synthetase (eNOS) by VEGF, but did not inhibit p38 activation by the VEGFR-1-specific ligand, placental growth factor (PIGF). Inhibition of VEGFR-2 also perturbed VEGF-induced membrane extension, cell migration, and tube formation by HAECs. Vascular endothelial growth factor receptor-2 inhibition also reversed VEGF-stimulated phosphorylation of CrkII and its Src homology 2 (SH2)-binding protein p130Cas, which are known to play a pivotal role in regulating endothelial cell migration. Inhibition of VEGFR-2 thus blocked all VEGF-induced endothelial cellular responses tested, supporting that the catalytic activity of VEGFR-2 is critical for VEGF signaling and/or that VEGFR-2 may function in a heterodimer with VEGFR-1 in human vascular endothelial cells.

Animals↗

PubMed2003

Hypoxia activates the human placental vascular endothelial growth factor system in vitro and in vivo: up-regulation of vascular endothelial growth factor in clinically relevant hypoxic ischemia in birth asphyxia.

OBJECTIVE: We investigated the influence of acute hypoxia on the placental vascular endothelial growth factor system in vitro and in vivo in acute birth asphyxia compared with pregnancies that were complicated by preeclampsia and with healthy control subjects. STUDY DESIGN: Messenger RNA levels for vascular endothelial growth factor, flt-1, and KDR were measured by TaqMan real-time polymerase chain reaction in human placental choriocarcinoma cells (BeWo) that were exposed to hypoxia (1% oxygen, 5% carbon dioxide, 94% nitrogen) and in placental tissue of neonates with birth asphyxia (n = 20), newborn infants of mothers with preeclampsia (n = 20), and gestational age-matched control subjects. Immunhistologically, placental vascular endothelial growth factor protein expression was compared among the groups. RESULTS: In BeWo cells, vascular endothelial growth factor, flt-1 and KDR messenger RNA increased in a time-dependent manner in response to hypoxia. In vivo, vascular endothelial growth factor/beta-actin and KDR/beta-actin messenger RNA were significantly higher in placental tissue of newborn infants with severe hypoxic-ischemic encephalopathy than with newborn infants with mild or no hypoxic-ischemic encephalopathy and control subjects. In chronic placental hypoxia (preeclampsia), vascular endothelial growth factor and both receptors were found to be up-regulated. Increased placental vascular endothelial growth factor expression was confirmed by immunohistologic examination. CONCLUSION: The vascular endothelial growth factor system is up-regulated in response to placental hypoxia and is assumed to be a potential early indicator of severe birth asphyxia.

Actins↗

PubMedDate not supplied

Simultaneous measurement of endothelial cell damage, elastase release and chemiluminescence response during interaction between polymorphonuclear leukocytes and endothelial cells.

Using cultured human umbilical cord vein endothelial cells and human blood neutrophils, the interaction between neutrophils and endothelial cells, in vitro, was studied. The aim of the study was to examine whether a respiratory burst stimulation by neutrophils would be observed by neutrophil/endothelial cell interaction and whether the respiratory burst stimulation of neutrophils by endothelial cells could be enhanced by lipopolysaccharide stimulation of neutrophils. The second aim was whether such an effect, or secretion of elastase, could cause an endothelial cell damage in vitro. Chemiluminescence as an indicator of oxygen-derived metabolites produced by neutrophils, elastase release by neutrophils, and endothelial cell damage, based on 111 In-oxine release from labelled endothelial cells, were measured simultaneously. The present investigation demonstrates that neutrophils can be directly stimulated by endothelial cells. A further amplification of this process following lipopolysaccharide priming up to 10 ng/ml blood could be demonstrated. A slight endothelial cell damage occurs following neutrophil stimulation, although elastase secretion does not increase during interaction between neutrophils and endothelial cells. These results raise the possibility that oxygen-derived metabolites rather than elastase contribute to an endothelial cell damage which might occur in conditions such as endotoxin-induced adult respiratory distress syndrome.

Cell Communication↗

PubMed1993

Neutrophil adhesion to endothelial cells impairs the effects of catalase and glutathione in preventing endothelial injury.

We studied the effects of the CuZn superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) on endothelial permeability to 125I-albumin after activation of neutrophils (PMN) with phorbol 12-myristate-13-acetate (PMA; 10(-8) M). PMN were either in direct contact with the endothelial cell monolayer grown on a porous gelatin-coated microporous 10-microns-thick polycarbonate filter (upright system) or separated from the endothelium by a similar filter (inverted system). Transendothelial 125I-albumin clearance rates were measured as an index of endothelial permeability. In the absence of antioxidants, activation of PMN increased transendothelial 125I-albumin clearance rates in both systems from 0.041 +/- 0.006 microliters/min (baseline) to 0.262 +/- 0.18 microliters/min (upright system) and from 0.063 +/- 0.02 microliters/min to 0.244 +/- 0.06 microliters/min (inverted system). PMA induced 80-90% of PMN to adhere to either gelatin-coated filters or to endothelial cells, from the basal PMN adhesion value of 5.3 +/- 2.2% and 4.3 +/- 1.1%, respectively. SOD, which dismutases superoxide anion to hydrogen peroxide (H2O2), did not alter the transendothelial 125I-albumin clearance rates in either system at any concentration from 10-300 U/ml. CAT (100-1,000 U/ml) and GSH (0.5-10 mM), which remove the H2O2 generated during PMN activation, did not alter the increase in transendothelial 125I-clearance rates after PMN activation in the upright system, but both agents prevented the increase in transendothelial 125I-clearance rates in the inverted system. We conclude that PMN activation with PMA causes endothelial injury irrespective of PMN contact to the endothelial monolayer. Moreover, H2O2, a release product of PMN activation, is a critical mediator of PMN-dependent endothelial injury. Finally, the results indicate that CAT and GSH prevent endothelial injury only in the absence of direct PMN contact with endothelial cells, suggesting that antioxidants such as GSH and CAT are excluded from sites of PMN-endothelial contact and thus are ineffective antioxidants.

Animals↗

PubMed1991

A comparison of primary cultures of rat cerebral microvascular endothelial cells to rat aortic endothelial cells.

A method to culture rat cerebral microvascular endothelial cells (RCMECs) was developed and adapted to concurrently obtain cultures of rat aortic endothelial cells (RAECs) without subculturing, cloning, or "weeding." The attachment and growth requirements of endothelial cell clusters from isolated brain microvessels were first evaluated. RCMECs required fetal bovine serum to attach efficiently. Attachment and growth also depended on the matrix provided (fibronectin approximately laminin much greater than gelatin greater than poly-D-lysine approximately Matrigel greater than hyaluronic acid approximately plastic) and the presence of endothelial cell growth supplement and heparin in the growth medium. Non-endothelial cells are removed by allowing these cells to attach to a matrix that RCMECs attach to poorly (e.g., poly-D-lysine) and then transferring isolated endothelial cell clusters to fibronectin-coated dishes. These cell cultures, labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarboxyamine perchlorate (DiI-Ac-LDL) and analyzed using flow cytometry, were 97.7 +/- 2.6% (n = 6) pure. By excluding those portions designed to isolate brain microvessels, the method was adapted to obtain RAEC cultures. RAECs do not isolate as clusters and have different morphology in culture, but respond similarly to matrices and growth medium supplements. RCMECs and RAECs have Factor VIII antigen, accumulate DiI-Ac-LDL, contain Weibel-Palade bodies, and have complex junctional structures. The activities of gamma-glutamyl transferase and alkaline phosphatase were measured as a function of time in culture. RCMECs had higher enzymatic activity than RAECs. In both RCMECs and RAECs enzyme activity decreased with time in culture. The function of endothelial cells is specialized depending on its location. This culture method allows comparison of two endothelial cell cultures obtained using very similar culture conditions, and describes their initial characterization. These cultures may provide a model system to study specialized endothelial cell functions and endothelial cell differentiation.

Animals↗

PubMed1987

Separation of luminal and abluminal membrane enriched domains from cultured bovine aortic endothelial cells: monoclonal antibodies specific for endothelial cell plasma membranes.

Two kinds of membrane (luminal and abluminal membrane domains) fractions have been isolated from bovine aortic endothelial cells by fractionation of whole cell homogenate on discontinuous sucrose density gradients. The luminal membrane domain was enriched 12-16-fold for angiotensin-converting enzyme activity and 8-10-fold in alkaline phosphatase activity. The abluminal membrane domain displayed an enrichment of 8-fold in (Na+ + K+)-ATPase activity. Both of the membrane domains were minimally contaminated with mitochondria, microsomes and Golgi bodies, as assessed by their corresponding marker enzyme activities. 125I-labeling of endothelial cell monolayers by the Enzymo-Bead lactoperoxidase-catalyzed iodination procedure, followed by isolation of membranes, revealed that the radioactivity was predominantly associated with membranes enriched in angiotensin-converting enzyme activity, corresponding to the luminal membrane domain. However, when cells were radioiodinated in suspension culture, radioactivity was found equally associated in both the luminal and abluminal membrane fractions. Electron microscopy of freeze-fractured and sectioned material showed both luminal and abluminal membrane domains to be in the form of vesicles varying in size from 100 to 400 nm in diameter. To characterize the separation of endothelial cell membrane domains, we have attempted to prepare monoclonal antibodies specific for endothelial cells. Several clones were obtained, producing antibodies which bound to endothelial cells of arterial, venous and capillary origin. Two antibodies of these clones, XIVC6 and XVD2, were studied in more detail. In the ELISA assay, these antibodies reacted with bovine vascular endothelial cells, but not with human umbilical cord endothelial cells, nor with bovine corneal endothelial cells, smooth muscle cells or fibroblasts. Both of these antibodies are directed against an antigen of approximately 130 kDa, under reducing and non-reducing conditions, as assayed by the immunoprecipitation method. Western blot analysis of luminal and abluminal membrane fractions revealed that only MAb XVD2 reacted with an antigen, indicating that the antibody XIVC6 is directed against an epitope which is denatured by SDS. Moreover, MAb XVD2 preferentially reacted with the luminal membrane compared to the abluminal membrane domain of the endothelial cell. These monoclonal antibodies do not react with platelet membrane proteins, indicating that this 130 kDa membrane antigen is not common to both endothelial cells and platelets.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1987

Prostaglandin synthesis in bovine coronary endothelial cells: comparison with other commonly studied endothelial cells.

Comparison of arachidonic acid metabolism by bovine coronary artery endothelial cells, bovine aortic endothelial cells and human umbilical endothelial cells indicated potentially important differences in relative amounts of the different prostaglandins produced. Bovine coronary endothelial cells converted 14C-arachidonic acid to radioactive 6-keto PGF1 alpha (the stable metabolite of PGI2) and to a lesser extent PGE2. Bovine aortic cells synthesized 6-keto PGF1 alpha and 6,15-diketo PGF1 alpha as the major products. PGE2, 6-keto PGE1, PGF2 alpha and PGD2 were minor metabolites. By comparison, endothelial cells isolated from human umbilical artery or vein formed mainly 6-keto PGF1 alpha and substantial amounts of PGF2 alpha, PGE2 and PGD2. Basal concentrations of 6-keto PGF1 alpha were two-fold higher in bovine coronary cells than in bovine aortic endothelial cells, but seven-fold less than in endothelial cells cultured from human umbilical vessels. Histamine, bradykinin and thrombin stimulated PGI2 synthesis in both coronary endothelial cells and human umbilical cells, but only bradykinin stimulated PGI2 synthesis in bovine aortic cells. This comparative study indicates that endothelial cells vary in the metabolites of arachidonic acid that they produce depending upon the vascular origin of the cells. Also, endothelial cells from different vascular beds respond differently to specific vasoactive agents.

Animals↗

PubMed1987

Iridocorneal endothelial syndrome with contralateral guttate endothelial dystrophy. A light and electron microscopic study.

Both eyes from a 75-year-old woman who had unilateral closed-angle glaucoma, characteristic endothelial specular microscopic findings, and slowly progressive tractional iris abnormalities consistent with essential iris atrophy were obtained postmortem and studied by light, scanning, and transmission electron microscopy. Clinically, the nonglaucomatous right eye had an open-angle, normal iris, and severe endothelial dystrophy with three-plus corneal guttae. In this eye, results of histopathologic examination disclosed marked endothelial degeneration and typical guttate excrescences on Descemet's membrane. In contrast, the endothelium in the fellow eye with the essential iris atrophy variant of the iridocorneal endothelial (ICE) syndrome was strikingly different. The endothelial cells were relatively well preserved and exhibited marked pleomorphism, anisocytosis, and flat, occasionally paired oval nuclei. Binucleate cells also were observed. The endothelial monolayer had secreted a new layer of fibrillar extracellular matrix material on the posterior surface of Descemet's membrane. Extracellular matrix material was found interposed between the iris and cornea in zones of synechial closure, and an endothelial membrane was present on the anterior surface of the iris. These observations support the hypothesis that the iris abnormalities in essential iris atrophy are secondary to endothelial proliferation. The endothelial cells displayed neither tonofilaments nor abundant apical microvilli. The latter observation suggests that increased numbers of endothelial microvilli are not responsible for the characteristic specular microscopic picture seen in the ICE syndrome.

Aged↗

PubMed2001

[Regression analysis of idiopathic endothelial cell loss after perforating normal risk keratoplasty: basic principles for long-term analysis of endothelial risk factors in a retrospective clinical study].

BACKGROUND: For the characterisation of influencing factors on chronic endothelial cell loss after penetrating keratoplasty by means of multivariate statistics, a mathematical description of the course of the individual postoperative endothelial cell density is a prerequisite. This mathematical description should result in a standardized index value describing course and amount of the postoperative endothelial cell loss over time in a canonical way. The slopes of the linear regression lines for each individual scatter plot of a) the endothelial cell density values plotted against the respective postoperative time directly (linear regression), and b) after logarithmic transformation (exponential regression) are evaluated, respectively. PATIENTS AND METHODS: 58 patients after normal-risk keratoplasty (26x keratokonus, 22x Fuchs-dystrophy and 10 cases of corneal decompensation after cataract surgery) with 5 or more postoperatively acquired endothelial density values and without any episodes of graft rejection were included in this study. Mean follow up was 2.9 +/- 1.1 years. The postoperative endothelial cell density values were plotted against the respective time for each patient individually. The coefficients of variation (R2) derived from the linear and the exponential regression models were calculated for each of these scatter plots. The pairs of R2 values (linear vs. exponential) were compared statistically. A dependence of the difference of linear and exponential R2-values on the ophthalmologic diagnosis was tested as well. RESULTS: The linear model is able to declare 83% the total variance of the course of the endothelial cell density. The exponential model even declares 86%. This small difference was statistically significant. Since both methods of regression describe the course of the cell density well, intra/and extrapolation of missing endothelial values is possible with both models. No dependence of the difference of linear and exponential R2-values on the ophthalmologic diagnosis could be demonstrated. CONCLUSIONS: Both, the intuitively understandable slope of the linear regression line and the constant of decay of the exponential regression curve, are suitable for describing the amount of the postoperative loss of endothelial cells after normal-risk keratoplasty independent of the ophthalmologic diagnosis. Both can thus be used as target variable in forthcoming statistical analyses for chronic endothelial cell loss.

Adult↗

PubMed2005

Effect of shear stress on attachment of corneal endothelial cells in association with corneal endothelial cell loss after laser iridotomy.

PURPOSE: Laser iridotomy often causes bullous keratopathy; however, the mechanism is unclear. We investigated whether changes in aqueous humor hydrodynamics after laser iridotomy have any role in corneal endothelial cell loss. MATERIALS AND METHODS: Porcine corneal endothelial cells were plated onto glass slides. Following 1 or 3 hours for adhesion, the endothelial cells were exposed to shear stresses (0.1-10 dyne/cm) for 15 minutes, and the number of detached cells was counted. In addition, the pressure and shear stress on corneal endothelial layer were calculated in a virtual model of laser iridotomy. RESULTS: The number of detached corneal endothelial cells increased with shear stresses in a dose-dependent manner. Significant increase of rate of detached corneal endothelial cells was observed at >0.3 dyne/cm after 1-hour attachment and at 1 dyne/cm after 3-hour attachment. The maximum pressure on corneal endothelial layer was 0.007 mm Hg, which is negligible compared with intraocular pressure. However, the maximum shear stress on the corneal endothelial layer could be> dyne/cm in some conditions of laser iridotomy. CONCLUSIONS: The resistance of corneal endothelial cell loss to shear stress is time dependent. Shear stress could be a cause of corneal endothelial cell loss in some conditions of laser iridotomy.

Animals↗

PubMed1992

Secretion of SPARC by endothelial cells transformed by polyoma middle T oncogene inhibits the growth of normal endothelial cells in vitro.

Endothelioma cells expressing the polyoma virus middle T oncogene induced hemangiomas in mice by the recruitment of nonproliferating endothelial cells from host blood vessels (Williams et al. 1989). I now report that SPARC, a Ca(2+)-binding glycoprotein that perturbs cell-matrix interactions and inhibits the endothelial cell cycle, is produced by endothelioma cells and is in part responsible for the alterations in the morphology and growth that occur when nontransformed bovine aortic endothelial cells are cocultured with endothelioma cells. Normal endothelial cells cocultured with two different middle T-positive endothelial cell lines, termed End cells, exhibited changes in shape that were accompanied by the formation of cell clusters. Media conditioned by End cells repressed proliferation of normal endothelial cells, but enhanced that of an established line of murine capillary endothelium. Radiolabeling studies revealed no apparent differences in the profile of proteins secreted by aortic or capillary cells cultured in End cell conditioned media. Characterization of proteins produced by End cells led to the identification of type IV collagen, laminin, entactin, and SPARC as major secreted products. Although SPARC did not affect the morphology of End or capillary cells, it was associated with overt changes in the shape of aortic endothelial cells. Moreover, SPARC and a synthetic peptide from SPARC domain II inhibited the incorporation of [3H]thymidine by aortic cells, but had minimal to no effect on the capillary endothelial cell line. The inhibition of growth exhibited by aortic endothelial cells cultured in End cell conditioned media could be partially reversed by antibodies specific for SPARC and SPARC peptides. These studies indicate a potential role for SPARC in the generation of hemangiomas by End cells in vivo, a process that requires normal (host) endothelial cells to disengage from the extracellular matrix, withdraw from the cell cycle, migrate, and reassociate into the disorganized cellular networks that comprise cavernous and capillary hemangiomas.

Animals↗

PubMed2002

Endothelial dysfunction in ischemic acute renal failure: rescue by transplanted endothelial cells.

There is accumulating circumstantial evidence suggesting that endothelial cell dysfunction contributes to the "no-reflow" phenomenon in postischemic kidneys. Here, we demonstrated the vulnerability of in vitro, ex vivo, and in vivo endothelial cells exposed to pathophysiologically relevant insults, such as oxidative and nitrosative stress or ischemia. All of these stimuli compromised the integrity of the endothelial lining. Next, we performed minimally invasive intravital microscopy of blood flow in peritubular capillaries, which provided direct evidence of the existence of the no-reflow phenomenon, attributable, at least in part, to endothelial injury. In an attempt to ameliorate the hemodynamic consequences of lost endothelial integrity, we transplanted endothelial cells or surrogate cells expressing endothelial nitric oxide synthase into rats subjected to renal artery clamping. Implantation of endothelial cells or their surrogates expressing functional endothelial nitric oxide synthase in the renal microvasculature resulted in a dramatic functional protection of ischemic kidneys. These observations strongly suggest that endothelial cell dysfunction is the primary cause of the no-reflow phenomenon, which, when ameliorated, results in prevention of renal injury seen in acute renal failure.

Acute Kidney Injury↗

PubMed2004

Murine endothelial cell lines as models of tumor endothelial cells.

Identification of appropriate models for in vivo and in vitro preclinical testing of inhibitors of tumor angiogenesis and progression is vital to the successful development of anticancer therapeutics. Although the focus is on human molecular targets, most preclinical in vivo efficacy testing occurs in mice. The goal of the current studies was to identify a murine endothelial cell line to model tumor endothelium for studying the antiangiogenic activity of therapeutic compounds in vitro. In situ hybridization was performed on three s.c. grown syngeneic murine tumors (B16 melanoma, Lewis lung carcinoma, and CT26 colon carcinoma) to assess expression of murine homologs of human tumor endothelial cell markers in the vasculature of these tumor models. Seven murine endothelial cell lines were characterized for expression of the murine homologs of recognized endothelial cell surface markers as well as for tumor endothelial cell surface markers. The seven murine endothelial cell lines had similar generation times and five of the seven lines were able to form tubes on Matrigel. Real-time-PCR and flow cytometry analysis were used to evaluate relative mRNA and protein expression of murine homologs of several recognized endothelial cell surface markers in the seven cell lines. The expression of the mRNA for the murine homologs of five tumor endothelial cell surface markers was also evaluated. The 2H11 cell line expressed all five of the tumor endothelial cell surface markers as well as several well-recognized endothelial cells markers. The 2H11 cell line responds to known and novel antiangiogenic agents by inhibition of proliferation and tube formation. These cells can be used in in vitro angiogenesis assays for evaluating the potential antiangiogenic properties and interspecies cross-reactivity of novel compounds.

Animals↗

PubMed2003

Endothelial progenitor cells are decreased in blood of cardiac allograft patients with vasculopathy and endothelial cells of noncardiac origin are enriched in transplant atherosclerosis.

BACKGROUND: Recent studies in animals suggest that circulating recipient endothelial precursors may participate in the biology of transplant vasculopathy. It is currently unknown whether a similar interaction between recipient endothelial cells and the vessel wall occurs in human subjects undergoing allogeneic cardiac transplantation. METHODS AND RESULTS: Circulating endothelial cells and endothelial progenitor cells (EPCs) were quantified in 15 cardiac transplantation subjects with and without angiographic evidence of vasculopathy. In a separate series of experiments, the origin (donor or recipient) of transplant plaque endothelial cells was assessed in subjects who had undergone a gender-mismatched cardiac transplantation and had histological evidence of severe vasculopathy at the time of heart explantation. Circulating EPC outgrowth colonies in peripheral blood were significantly reduced in subjects with transplant vasculopathy compared with those without angiographic evidence of disease (EPC colony-forming units [CFUEPC]: 4.5+/-1.9 versus 15.1+/-3.7, P<0.05). There was no significant difference in circulating endothelial cell numbers as defined by day 4 culture acetylated LDL/lectin assay in either of these patient groups. In a separate group of 5 subjects who underwent gender-mismatched cardiac transplantation, there was a significant seeding of recipient endothelial cells (range: 1% to 24% of all luminal endothelial cells) in large-vessel lumen and adventitial microvessel lumen of arteriopathic vessels. No opposite-sex chimeric cells were observed in control gender-matched transplantation scenarios. CONCLUSIONS: These data suggest that the human cardiac transplant arteriopathy is associated with reduction in circulating endothelial precursors and with seeding of recipient-derived endothelial cells at the site of plaque development.

Adult↗

PubMed2003

Circulating endothelial cells are a novel marker of cyclosporine-induced endothelial damage.

Microvascular endothelial cells play a key role in transplant immunology. They are also important targets for calcineurin inhibitors. We recently demonstrated elevated numbers of circulating endothelial cells in renal transplant recipients with and without rejection in comparison with healthy controls. Because these patients received either cyclosporine or tacrolimus, we speculated that endothelial damage from calcineurin inhibitors might be responsible for these findings. In the present study, we tested the hypothesis that treatment with calcineurin inhibitors leads to an increase in circulating endothelial cells. We studied 57 renal transplant recipients: 19 on a calcineurin inhibitor-free immunosuppressive regimen and 38 patients on a standard immunosuppressive regimen, including cyclosporine, and matched them for age and serum creatinine. Endothelial cells were isolated from peripheral blood with anti-CD-146-coated immunomagnetic Dynabeads and were counted by fluorescence microscopy. Patients with cyclosporine therapy had elevated numbers of circulating endothelial cells (median 26, range 12 to 82 cells/mL) compared with healthy controls (median 6, range 0 to 82 cells/mL; P<0.001). Patients without calcineurin inhibitor treatment had significantly lower cell numbers (median 12, range 0 to 32 cells/mL; P<0.003) and were not significantly different from normal, untreated controls. In conclusion, renal transplant recipients who do not receive calcineurin inhibitors have significantly lower numbers of circulating endothelial cells than their age- and creatinine-matched counterparts who receive these drugs. We suggest that elevated numbers of circulating endothelial cells indicate damage from calcineurin inhibitors in renal transplant recipients and that circulating endothelial cells are a novel marker of endothelial damage.

Adult↗

PubMed2003

Human endothelial cells derived from circulating progenitors display specific functional properties compared with mature vessel wall endothelial cells.

Endothelial progenitor cells (EPCs) were shown to be present in systemic circulation and cord blood. We investigated whether EPCs display specific properties compared with mature endothelial cells. Human cord blood CD34+ cells were isolated and adherent cells were amplified under endothelial conditions. Expression of specific markers identified them as endothelial cells, also called endothelial progenitor-derived cells (EPDCs). When compared to mature endothelial cells, human umbilical vein endothelial cells (HUVECs) and human bone marrow endothelial cells (HBMECs), endothelial markers, were expressed to the same extent except for KDR, which is expressed more in EPDCs. They display a higher proliferation potential. Functional studies demonstrated that EPDCs were more sensitive to angiogenic factors, which afford these cells greater protection against cell death compared with HUVECs. Moreover, EPDCs exhibit more hematopoietic supportive activity than HUVECs. Finally, studies in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice demonstrated that human circulating EPCs are able to colonize a Matrigel plug. EPDCs display the morphology and phenotype of endothelial cells. Their functional features indicate, however, that although these cells have undergone some differentiation steps, they still have the properties of immature cells, suggesting greater tissue repair capabilities. Future use of in vitro amplified peripheral blood EPDCs may constitute a challenging strategy for cell therapy.

Animals↗

PubMed1996

The synergistic effect of elastase and hydrogen peroxide on vascular endothelial cell injury is due to the production of hydroxylradical in the endothelial cells.

Protease inhibitors such as aprotinin and urinastatin inhibited vascular endothelial cell injury induced by PMA-stimulated leukocytes, although their inhibitors did not suppress the production of active oxygen species released from leukocytes. On the other hand, in the presence of pancreas elastase (10 micrograms/ml), hydrogen peroxide (50 microM) caused severe injury of endothelial cells isolated from the bovine carotid artery (% specific 51Cr release, % SR = 42.9 +/- 3.3%), although the % SR elicited by elastase or hydrogen peroxide alone, respectively, was below 1%. Elastase and hydrogen peroxide acted synergistically on the injury of endothelial cells from the bovine carotid artery similarly to that in the endothelial cells isolated from the bovine coronary artery and human umbilical vein. Furthermore, elastase derived from both pancreas and leukocyte induced this synergistic action on endothelial cell injury. To clarify the mechanism of vascular endothelial cell injury induced by the combination of elastase and hydrogen peroxide, we examined the effects of various radical scavengers and protease inhibitors. Deferoxamine mesylate completely inhibited the endothelial cell injury, while protease inhibitors such as antitrypsin and macroglobulin had a protective effect. Pretreatment of endothelial cells with deferoxamine mesylate also protected against this cytotoxicity. These findings suggested that the synergistic effect of elastase and hydrogen peroxide on the endothelial cell injury is due to the production of hydroxylradical in the endothelium and that this synergistic action might be partially involved in the endothelial cell injury induced by activated leukocytes.

Animals↗

PubMedDate not supplied

Relationship between anti-phospholipid and anti-endothelial cell antibodies III: beta 2 glycoprotein I mediates the antibody binding to endothelial membranes and induces the expression of adhesion molecules.

OBJECTIVE: To investigate the role of antibodies reacting with beta 2 glycoprotein I (beta 2GPI) in the antiendothelial cell binding activity present in sera from patients with the anti-phospholipid syndrome. METHODS: Sera positive for anti-phospholipid, anti-endothelial and anti-beta 2 GPI antibodies were studied for their binding activity on endothelial monolayers cultured in the presence or absence of media containing bovine serum as a source of beta 2GPI. Anti-endothelial activity was also evaluated on endothelial cells cultured without serum and supplemented with exogenous human purified beta 2GPI. Affinity purified anti-beta 2 GPI antibodies were investigated under the same experimental conditions. Finally, the effect of the incubation of these affinity purified fractions on the expression of adhesion molecules (ELAM-1) was studied. RESULTS: The reactivity of the sera decreased on endothelial cells incubated in serum-free medium, while endothelial cell binding was restored in a dose dependent manner after the addition of exogenous purified human beta 2 GPI. Affinity purified anti-beta 2 GPI antibodies obtained from the same sera retained their endothelial cell binding and were able to activate endothelial cells by inducing the ex novo surface expression of adhesion molecules (ELAM-1). CONCLUSIONS: These findings indicate that the close association between anti-endothelial and anti-phospholipid antibodies is sustained by antibodies which recognize beta 2 GPI adhering to the endothelial cells, and can promote their activation.

Antibodies, Antiphospholipid↗