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Results for “Endothelialization”

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At least 19 recordsLinked to original sources

PubMed1991

Role of endothelial leukocyte adhesion molecule-1 and platelet-activating factor in neutrophil adherence to IL-1-prestimulated endothelial cells. Endothelial leukocyte adhesion molecule-1-mediated CD18 activation.

Adherence of neutrophils to endothelium is a key event in the sequence of inflammatory leukocyte responses. Double-color FACS analysis was used to determine the extent and kinetics of neutrophil adherence to rIL-1 beta-pretreated endothelial cells (EC). Neutrophils bound very avidly when the EC were prestimulated for 4 to 6 h with rIL-1 beta. Anti-ELAM-1 F(ab)2 fragments inhibited this adherence for more than 80%. On the other hand, anti-CD18 F(ab)2 fragments also inhibited the neutrophil adherence (40 to 50%). Combined use of anti-ELAM-1 and anti-CD18 F(ab)2 fragments completely prevented adherence. Neutrophils became activated as soon as they made contact with the rIL-1 beta-pretreated EC. First, neutrophils depleted of intracellular ATP showed a clearly decreased adherence completely dependent on ELAM-1-mediated binding, i.e., without additional effects of CD18 adhesion proteins. Thus, CD18 is activated during neutrophil adherence and then participates in the binding process. Secondly, the neutrophils responded with a transient rise in [Ca2+]i upon binding to rIL-1 beta-pretreated EC, which was demonstrated to be caused by endothelial cell-associated platelet-activating factor (PAF). However, the extent of neutrophil adherence to rIL-1 beta-pretreated EC was not affected by the use of the PAF-receptor antagonist WEB 2086, or removal of the EC-bound PAF. The only effect was a complete dependency of the neutrophil adherence on ELAM-1-mediated binding, although anti-CD18 mAb still induced 40 to 50% inhibition under these conditions. We therefore conclude that ELAM-1-mediated binding is the major mechanism for CD18 activation during neutrophil adherence to rIL-1 beta-pretreated EC.

Antigens, CD↗

PubMed1992

Electrical method for detection of endothelial cell shape change in real time: assessment of endothelial barrier function.

We have developed an electrical method to study endothelial cell shape changes in real time in order to examine the mechanisms of alterations in the endothelial barrier function. Endothelial shape changes were quantified by using a monolayer of endothelial cells grown on a small (10(-3) cm2) evaporated gold electrode and measuring the changes in electrical impedance. Bovine pulmonary microvessel endothelial cells and bovine pulmonary artery endothelial cells were used to study the effects of alpha-thrombin on cell-shape dynamics by the impedance measurement. alpha-Thrombin produced a dose-dependent decrease in impedance that occurred within 0.5 min in both cell types, indicative of retraction of endothelial cells and widening of interendothelial junctions because of "rounding up" of the cells. The alpha-thrombin-induced decrease in impedance persisted for approximately 2 hr, after which the value recovered to basal levels. Pretreatment of endothelial cells with the protein kinase C inhibitor, calphostin C, or with 8-bromoadenosine 3',5'-cyclic monophosphate prevented the decreased impedance, suggesting that the endothelial cell change is modulated by activation of second-messenger pathways. The alpha-thrombin-induced decrease in impedance was in agreement with the previously observed increases in transendothelial albumin permeability and evidence of formation of intercellular gaps after alpha-thrombin challenge. The impedance measurement may be a valuable in vitro method for the assessment of mechanisms of decreased endothelial barrier function occurring with inflammatory mediators. Since the rapidly occurring changes in endothelial cell shape in response to mediators such as thrombin are mediated activation of second-messenger pathways, the ability to monitor endothelial cell dynamics in real time may provide insights into the signal-transduction events mediating the increased endothelial permeability.

Animals↗

PubMed1978

Control of proliferation of human vascular endothelial cells. Characterization of the response of human umbilical vein endothelial cells to fibroblast growth factor, epidermal growth factor, and thrombin.

Because the response of human endothelial cells to growth factors and conditioning agents has broad implications for our understanding of wound healing angiogenesis, and human atherogenesis, we have investigated the responses of these cells to the fibroblast (FGF) and epidermal growth factors (EGF), as well as to the protease thrombin, which has been previously shown to potentiate the growth response of other cell types of FGF and EGF. Because the vascular endothelial cells that form the inner lining of blood vessels may be expected to be exposed to high thrombin concentrations after trauma or in pathological states associated with thrombosis, they are of particular interest with respect to the physiological role of this protease in potentiating cell proliferation. Our results indicate that human vascular endothelial cells respond poorly to either FGF or thrombin alone. In contrast, when cells are maintained in the presence of thrombin, their proliferative response to FGF is greatly increased even in cultures seeded at a density as low as 3 cells/mm2. Human vascular endothelial cells also respond to EGF and thrombin, although their rate of proliferation is much slower than when maintained with FGF and thrombin. In contrast, bovine vascular endothelial cells derived from vascular territories as diverse as the bovine heart, aortic arch, and umbilical vein respond maximally to FGF alone and neither respond to nor bind EGF. Furthermore, the response of bovine vascular endothelial cells to FGF was not potentiated by thrombin, indicating that the set of factors controlling the proliferation of vascular endothelial cells could be species-dependent. The requirement of cultured human vascular endothelial cells for thrombin could explain why the human cells, in contrast to bovine endothelial cells, are so difficult to maintain in tissue culture. Our results demonstrate that by using FGF and thrombin one can develop cultures of human vascular endothelial cells capable of being passage repeatedly while maintaining a high mitotic index. The stock cultures used for these studies have been passed weekly with a split ratio of 1 to 10 and are currently in their 30th passage. These cultures are indistinguishable from earlier passages when examined for the presence of Weibel-Palade bodies or Factor VIII antigen. We conclude that the use of FGF and thrombin can prevent the precocious senescence observed in most human endothelial cells cultures previously described.

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↗

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↗

PubMed1992

Endothelial cell senescence inhibits unidirectional endothelialization in vitro.

We investigated the effects of cellular senescence on unidirectional endothelialization in vitro, simulating the anastomotic endothelialization of vascular prosthesis. The experiments were carried out with three different cumulative population-doubling levels (CPDLs) of bovine aortic endothelial cells (ECs), which have finite life span. Young ECs with 22 CPDL, middle aged with 46, and senescent with 70 at the time of inoculation were used. The effect of aging on unidirectional endothelialization, as well as cellular morphology and proliferative and migratory potentials of isolated cells, were qualitatively and quantitatively analyzed. The unidirectional endothelialization rate was determined by our newly designed method to prepare the square monolayer sheet with linear margins between cell-adhesion and noncell-adhesion regions. The results showed that endothelial cell senescence retarded not only proliferation and migration but also unidirectional endothelialization. Time-lapsed videomicroscopic study of unidirectional endothelialization process revealed that ECs at several rows back from the leading edge represented much slower rate of migration than did the ECs at the leading edge. These findings suggest that high cellular mobility observed for the ECs at the leading edge may result in localized excessive cell replication. Thus, atherosclerotic vessels containing senescent or injured ECs may have limited capability of anastomotic endothelialization.

Animals↗

PubMed1975

Endothelial regeneration in the rat carotid artery and the significance of endothelial denudation in the pathogenesis of myointimal thickening.

A new model was developed to study endothelial regeneration and the effects of endothelial denudation in large arteries. Complete endothelial loss was produced in a sharply defined, unbranched segment of the rat common carotid artery by brief drying with a gentle stream of air along the lumen of the vessel. Platelets became attached to the denuded surface, but no polymorphonuclear or mononuclear leukocytic adherence or infiltration was observed. Regeneration occurred by the ingrowth, from each end of the denuded segment, of sheets of rapidly dividing endothelial cells. Endothelial replacement was complete by 7 to 10 days after drying. It was also noted that, by 14 days after drying, a striking myointimal thickening developed in the central region of the denuded segment. This central region was the last area to be covered with new endothelium; while still denuded, the region showed edema and signs of cellular proliferation in the innermost medial layer. The myointimal thickening consisted of smooth muscle-like cells in a fibroelastic stroma in the deeper zones, and a layer of rounded, relatively undifferentiated cells in the superficial zones. Progressive regression and condensation of the thickening was observed between 14 days and 3 months. In this model, the clear demarcation and completeness of endothelial denudation in the arterial segment allow study of the over-all process of endothelial replacement, avoiding the confusion imposed by islands of residual endothelium or contributions from branch vessels. The results suggest that the new endothelium in this situation is derived from each end of the segment. In addition, because the method of inducing denudation does not cause significant damage to the underlying media (as shown by the absence of inflammatory cell attachment or infiltration), the model offers a unique opportunity to study the relationship between endothelial loss and changes in the vessel wall. The correlation between the duration of endothelial denudation and the extent of myointimal thickening suggests that sustained insudation of certain, as yet undefined, factors from the lumen may provoke cellular proliferation in the arterial wall. Further investigation of this model should provide information relevant to the pathogenesis of the fibromuscular thickenings of human atherosclerosis.

Animals↗

PubMed1992

Human dermal microvascular endothelial but not human umbilical vein endothelial cells express CD36 in vivo and in vitro.

CD36 is an 88-kDa glycoprotein that has been identified on platelets, monocytes, and some endothelial cells. Experimental evidence suggests that CD36 mediates the binding of Plasmodium falciparum-infected RBC to a variety of cells, and therefore may play a role in the vascular complications associated with malaria. Additionally, CD36 may also bind the extracellular matrix proteins thrombospondin and collagen. Human umbilical vein endothelial cells have been used in in vitro models examining the binding of P. falciparum RBC to endothelial cells, but they do not consistently express cell surface CD36. Inasmuch as human dermal microvascular endothelial cells (HDMEC) differ in a variety of ways from large vessel endothelial cells, we have examined HDMEC for cell surface expression of CD36 in vivo and in vitro. Direct immunofluorescence of skin showed bright staining of HDMEC with antibody recognizing CD36 and flow cytometric analysis of cultured HDMEC revealed cell surface expression. In contrast, large vessel endothelial cells were not stained with antibody recognizing CD36 in vivo and cultured cells derived from umbilical vein failed to express cell surface CD36 in vitro. Western immunoblots of lysates of HDMEC but not human umbilical vein endothelial cells demonstrated an 88-kDa protein that comigrated with CD36 from platelets. Functional studies demonstrated that adherence of PRBC to HDMEC was inhibited up to 66% by mAb recognizing CD36. Furthermore, the expression of CD36 on HDMEC was increased in a dose- and time-dependent manner by IFN-gamma, and was decreased by protein kinase C agonists. These data demonstrate that HDMEC express functionally active CD36 and this expression can be positively and negatively regulated by soluble factors. This study demonstrates that HDMEC are useful in the study of CD36-mediated binding of PRBC to endothelial cells in vitro and provides further evidence of distinct phenotypic differences between HDMEC and large vessel endothelial cells.

Antigens, CD↗

PubMed1992

Expression of vascular endothelial growth factor during embryonic angiogenesis and endothelial cell differentiation.

Vascular endothelial growth factor (VEGF) is a secreted angiogenic mitogen whose target cell specificity appears to be restricted to vascular endothelial cells. Such factors are likely candidates for regulatory molecules involved in endothelial growth control. We have characterized the murine VEGF gene and have analysed its expression pattern in embryogenesis, particularly during brain angiogenesis. Analysis of cDNA clones predicted the existence of three molecular forms of VEGF which differ in size due to heterogeneity at the carboxy terminus of the protein. The predicted mature proteins consist of 120, 164 or 188 amino acid residues. Homodimers of the two lower molecular weight forms, but not of the higher molecular weight form, were secreted by COS cells transfected with the corresponding cDNAs and were equally potent in stimulating the growth of endothelial cells. During brain development, VEGF transcript levels were abundant in the ventricular neuroectoderm of embryonic and postnatal brain when endothelial cells proliferate rapidly but were reduced in the adult when endothelial cell proliferation has ceased. The temporal and spatial expression of VEGF is consistent with the hypothesis that VEGF is synthesized and released by the ventricular neuroectoderm and may induce the ingrowth of capillaries from the perineural vascular plexus. In addition to the transient expression during brain development, a persistent expression of VEGF was observed in epithelial cells adjacent to fenestrated endothelium, e.g. in choroid plexus and in kidney glomeruli. The data are consistent with a role of VEGF as a multifunctional regulator of endothelial cell growth and differentiation.

Amino Acid Sequence↗

PubMed1991

Direct interaction with primed CD4+ CD45R0+ memory T lymphocytes induces expression of endothelial leukocyte adhesion molecule-1 and vascular cell adhesion molecule-1 on the surface of vascular endothelial cells.

The process of recruitment of leukocytes at sites of inflammation involves direct cell-to-cell interactions between leukocytes and vascular endothelial cells (EC) mediated by various adhesion receptors on leukocytes and their inducible endothelial ligands. In this study we have examined the induction on EC of endothelial leukocyte adhesion molecule-1 (ELAM-1), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) upon their interaction with subpopulations of human T cells. When co-cultured with EC both resting CD4+ T and CD8+ T cells caused a modest increase in the expression of endothelial ICAM-1. Moreover, resting CD4+ but not CD8+ T cells induced expression of ELAM-1 and VCAM-1 on a small fraction of unstimulated EC. Prior activation with phorbol 12-myristate 13-acetate (PMA) significantly increased the ability of T cells to up-regulate endothelial ICAM-1 and also induced the expression of both ELAM-1 and VCAM-1. PMA-primed CD4+ T cells induced both VCAM-1 and ELAM-1 on EC more efficiently than CD8+ T cells. Furthermore, the ability to induce the expression of ELAM-1 and VCAM-1 was confined to the CD4+ CD45R0+ memory/primed subpopulation of T cells. This induction of various endothelial adhesion ligands could also be mediated by antigen-primed CD4+ T cell lines. The CD4+ T cell-mediated induction of adhesion ligands required direct intercellular contact with EC because neither cultures of EC and PMA-primed CD4+ T cells separated by a microporous membrane insert nor the conditioned medium of PMA-primed T cells induced expression of ELAM-1 and VCAM-1 on EC. Cyclosporin A significantly inhibited the activation of T cells with PMA but had no effect on the ability of PMA-primed T cells to up-regulate endothelial CAM. Thus, CD4+CD45R0+ T cells via as yet unknown mechanism can significantly enhance the expression of each of the three endothelial adhesion ligands and, thereby, may facilitate the process of recruitment of additional leukocytes to exacerbate inflammation.

Antigens, CD↗

PubMed1992

Stimulation of endothelial cell proliferation by vanadate is specific for microvascular endothelial cells.

Micromolar concentrations of sodium orthovanadate stimulated the proliferation of bovine capillary endothelial cells, but not bovine aortic endothelial cells. Vanadate was equally potent at inducing protein tyrosine phosphorylation and changes in morphology in both types of cells. However, vanadate treatment lead to an inhibition of protein tyrosine kinase activity in the aortic endothelial cells, but not the capillary endothelial cells. In capillary endothelial cells, the effect of vanadate was additive with basic FGF (bFGF) at low concentrations of bFGF. There was no interaction between bFGF and vanadate in aortic endothelial cells. TGF-beta, which inhibits the induction of endothelial cell proliferation by bFGF, appeared to shift the dose response curve to vanadate in capillary endothelial cells, increasing the proliferative effect of vanadate at low vanadate concentrations, but decreasing the proliferative effect at higher vanadate concentrations.

Animals↗

PubMed1992

A mechanism for the antiinflammatory effects of corticosteroids: the glucocorticoid receptor regulates leukocyte adhesion to endothelial cells and expression of endothelial-leukocyte adhesion molecule 1 and intercellular adhesion molecule 1.

Corticosteroids are the preeminent antiinflammatory agents although the molecular mechanisms that impart their efficacy have not been defined. The endothelium plays a critical role in inflammation by directing circulating leukocytes into extravascular tissues by expressing adhesive molecules for leukocytes [e.g., endothelial-leukocyte adhesion molecule 1 (ELAM-1) and intercellular adhesion molecule 1 (ICAM-1)]. We therefore determined whether corticosteroids suppress inflammation by inhibiting endothelial expression of adhesion molecules for neutrophils (polymorphonuclear leukocytes). Preincubation of endothelial cells with endotoxin [lipopolysaccharide (LPS), 1 microgram/ml] led to a 4-fold increase in subsequent adherence of polymorphonuclear leukocytes (P < 0.0001, n = 10) to endothelial cells, an increase that was markedly attenuated when endothelial cells were treated with dexamethasone (IC50 < 1 nM, P < 0.0001, n = 6 or 7) during preincubation with LPS. Moreover, the steroid receptor agonist cortisol (10 microM), but not its inactive metabolite tetrahydrocortisol (10 microM), diminished LPS-induced endothelial cell adhesiveness. Further evidence that the action of dexamethasone was mediated through ligation of corticosteroid receptors [human glucocorticoid receptors (hGRs)] was provided by experiments utilizing the steroid antagonist RU-486. RU-486 (10 microM), which prevents translocation of ligated hGR to the nucleus by inhibiting dissociation of hGR from heat shock protein 90, completely aborted the effect of dexamethasone on adhesiveness of endothelial cells (P < 0.0005, n = 3). Treatment of endothelial cells with LPS (1 microgram/ml) stimulated transcription of ELAM-1, as shown by Northern blot analysis, and expression of membrane-associated ELAM-1 and ICAM-1, as shown by quantitative immunofluorescence (both P < 0.001, n = 9). Dexamethasone markedly inhibited LPS-stimulated accumulation of mRNA for ELAM-1 and expression of ELAM-1 and ICAM-1 (IC50 < 10 nM, both P < 0.001, n = 4-9); inhibition of expression by dexamethasone was reversed by RU-486 (both P < 0.005, n = 4-6). As in the adhesion studies, cortisol but not tetrahydrocortisol inhibited expression of ELAM-1 and ICAM-1 (both P < 0.005, n = 3 or 4). In contrast, sodium salicylate (1 mM) inhibited neither adhesion nor expression of these adhesion molecules. These studies suggest that antagonism by dexamethasone of endotoxin-induced inflammation is a specific instance of the general biological principle that the glucocorticoid receptor is a hormone-dependent regulator of transcription.

Adrenal Cortex Hormones↗

PubMed1992

Production of neutrophil-specific lipid chemoattractant activity by cultured endothelial cells: heterogeneity dependent on species, ligand, or endothelial cell site of origin.

Previous studies have demonstrated that the interaction of cultured bovine aortic and pulmonary arterial endothelial cells and the proinflammatory vasoactive amines histamine, serotonin, and angiotensin II, causes production of three novel lipid neutrophil-specific chemoattractants that are distinct from other phospholipid or lipid neutrophil chemoattractants. In this study, we investigated the species and site specificity of this inflammatory response by incubating human aortic and pulmonary arterial endothelial cells with histamine, serotonin, and angiotensin II and assaying the supernatants for their effect on neutrophil migration. Each of these vasoactive amines caused production of neutrophil chemoattractant activity in a concentration dependent manner in both cell types. For each amine, production was blocked by a specific antagonist: cimetidine for histamine, methiothepin for serotonin-stimulated aortas, ketanserin for serotonin-stimulated pulmonary arteries, and saralasin for angiotensin II. In each case, all chemoattractant activity partitioned into the organic phase and resolution by HPLC yielded two chemotactic lipids. As with the lipid chemoattractants produced by bovine endothelial cells, these lipids did not coelute with PAF, LTB4, 5-HETE, or 15-HETE, nor did they increase lymphocyte or monocyte migration. The pattern of chemotactic activity following resolution by HPLC was similar in both human aortic and pulmonary arterial endothelial cells, but was different from that of bovine aortic and pulmonary arterial endothelial cells in that only two chemoattractant lipids appeared; the third chemotactic lipid was never produced. These studies demonstrate that human endothelial cells may actively participate in neutrophil enriched local inflammatory responses by production of neutrophil-specific chemotactic factors. They also suggest this response may be dissimilar depending on the site and species from which the endothelial cells originate.

Animals↗

PubMed1979

Appearance in confluent vascular endothelial cell monolayers of a specific cell surface protein (CSP-60) not detected in actively growing endothelial cells or in cell types growing in multiple layers.

The formation of a highly organized vascular and corneal endothelial cell monolayer is associated with the appearance of a 60,000-dalton cell surface protein (CSP-60) (30,000 daltons after reduction with dithiothreitol) which is not detectable in rapidly growing endothelial cells and in subconfluent cultures that do not yet exhibit the strict morphology of a confluent monolayer. It is also absent from vascular smooth muscle cells and from endothelial cultures that are maintained in the absence of fibroblast growth factor and grow on top of each other at confluence. After disorganization of cells in a confluent endothelial monolayer by urea, EDTA, or trypsin, CPS-60 is no longer exposed on the cell surface, but it reappears as soon as the cells readopt their characteristic two-dimensional configuration. This reorganization can be achieved in the presence of cycloheximide and despite removal of fibronectin by urea, EDTA, or trypsin. Maximal amounts of fibronectin and no CSP-60 are detected in subconfluent, but not yet organized, endothelial cultures or in endothelial cells that no longer form a monolayer of nonoverlapping cells at confluence. Likewise, cultures of vascular smooth muscle cells contain fibronectin but no CSP-60. These results suggest that CSP-60, rather than fibronectin, could be involved in the adoption of a monolayer configuration by confluent endothelial cells.

Aorta↗

PubMed1991

Inhibition of platelet function by an aspirin-insensitive endothelial cell ADPase. Thromboregulation by endothelial cells.

We previously reported that platelets become unresponsive to agonists when stimulated in combined suspension with aspirin-treated human umbilical vein endothelial cells. Inhibition occurred concomitant with metabolism of platelet-derived endoperoxides to prostacyclin by endothelial cells. We now demonstrate that if aspirin-treated platelets which fully respond to appropriate doses of agonists are exposed to aspirin-treated endothelial cells, they remain unresponsive despite absence of prostacyclin. Platelet inhibition is due in large part to ecto-ADPase activity on the endothelial cells. This was established by incubating aspirin-treated endothelial cells with 14C-ADP. Radio-thin layer chromatography and aggregometry demonstrated that 14C-ADP and induction of platelet activation decreased rapidly and concurrently. AMP accumulated transiently, was further metabolized to adenosine, and deaminated to inosine. The apparent Km of the endothelial cell ADPase was 33-42 microM and the Vmax 17-43 nmol/min per 10(6) cells, values in the range of antithrombotic potential. Thus, at least three complementary systems in human endothelial cells control platelet responsiveness: a cell-associated, aspirin-insensitive ADPase which functions in parallel with fluid phase autacoids such as the aspirin-inhibitable eicosanoids, and the aspirin-insensitive endothelium-derived relaxing factor.

Adenosine Diphosphate↗

PubMed1992

Antibodies defining rat endothelial cells: RECA-1, a pan-endothelial cell-specific monoclonal antibody.

We have been searching for antibodies reactive with rat endothelial cells. Two monoclonal antibodies (mAb), named RECA-1 and RECA-2 were produced and tested in immunoperoxidase staining on frozen sections of various rat tissues. Staining patterns were compared to those obtained with the mAbs OX-2, OX-26, OX-43, and the polyclonal antibody to von Willebrand Factor (vWF), which all have been described to react with rat endothelial cells. The RECA-2 mAb showed staining patterns similar to those obtained with OX-2. RECA-1 showed to be the only antibody reactive with all vascular endothelium in the tested tissues. In addition, RECA-1 was endothelial cell-specific whereas all other antibodies crossreacted with one or more other cell types. No reactivity of RECA-1 was found in various tested species other than rat. The RECA-1 antibody was successfully applied in staining of paraformaldehyde fixed, plastic embedded tissue material. Immunofluorescence staining of viable endothelial cells demonstrated that RECA-1 recognizes a cell surface antigen. This was supported by intravenous injection of RECA-1, which showed the antibody to localize along the endothelium lining the vasculature in various organs tested. No reactivity of the antibody was seen when applied in immunoblotting of PAGE-run lysates from endothelial cell cultures and stromal cell preparations. We believe RECA-1 to be a promising antibody for rat endothelial cell studies, and in particular for further defining nature and function of endothelial cell-specific antigens.

Animals↗

PubMed1991

Preservation of natural endothelial cytopathogenicity of cytomegalovirus by propagation in endothelial cells.

Cytomegalovirus (CMV) is a source of major complications in immunosuppressed individuals, and endothelial involvement in CMV infection is well documented. Traditionally the virus has been propagated in fibroblasts, however this process may alter CMV's characteristics, thereby limiting the fibroblast model's utility as a research tool. In our efforts to develop a more accurate in vitro model of CMV/endothelial cell interaction, we have propagated a recent isolate (CMV VHL) through multiple passages in human umbilical vein endothelial cells (HUVE) and, collaterally in neonatal human dermal fibroblasts (NHDF). Infection of HUVE inoculated with either sub-strain of the virus was confirmed by CMV-specific in situ hybridization and by immunocytochemical staining for CMV antigens. Whereas infection of HUVE by substrain VHL/E (endothelial-raised) was accompanied by dramatic cytopathology resembling that observed clinically, the endothelial cytopathic potential of VHL/F (fibroblast-raised) was lost by its 20th passage in NHDF. Similarly, the ability of VHL/F to initiate sustained productive infection in HUVE was severely attenuated; plaque assay of culture supernatants and infected cell fractions, as well as virus-specific DNA polymerase assay of cell lysates, demonstrated progressive viral reproductive activity in VHL/E-inoculated HUVE, whereas VHL/F reproduction was barely detectable. Since properties of VHL/F bear strong resemblance to those of the fibroblast-raised AD169, these studies suggest that while the fibroblast adaptation process commonly employed in the propagation of CMV restricts the host range of the virus and attenuates its spectrum of cytopathic potential, endothelial-based propagation preserves the natural endothelial cytopathogenicity of the original isolate.

Antigens, Viral↗

PubMed1991

Tumor cell adhesion to endothelial cells: endothelial leukocyte adhesion molecule-1 as an inducible adhesive receptor specific for colon carcinoma cells.

Activation of endothelial cells by the two inflammatory mediators interleukin-1 (IL-1) and tumor necrosis factor strongly increases tumor cell adhesion. We describe antibody inhibition studies showing that the endothelial leukocyte adhesion molecule-1 (ELAM-1), a cell-surface glycoprotein selectively expressed by cytokine-activated endothelial cells and responsible for neutrophil adhesion, is the major, if not the only, mediator of colon carcinoma cell adhesion to activated endothelial cells. Among the different tumor cell lines tested, seven colon carcinoma cell lines were sensitive to ELAM-1 antibodies. Adhesion of melanoma, osteosarcoma, and lung, cervix, or kidney carcinoma cell lines to IL-1-treated endothelial cells was not affected by the ELAM-1 antibody. This result suggests that ELAM-1 is selectively recognized by colon carcinoma cells and that adhesion of tumor cells to activated endothelial cells could be mediated by different and specific mechanisms.

Antibodies, Monoclonal↗