Endothelial Cell Contacts Research Articles

Phosphorylation and disorganization of vascular-endothelial cadherin in interaction between breast cancer and vascular endothelial cells.

Adherens junctions of the endothelium play a key role in the maintenance of endothelial permeability and are composed of the vascular endothelial (VE)-cadherin/catenin adhesion complex. We report that following tumour cell (MDA MB231 cells) adherence to the HUVECs, there was a rapid (within 5 min) redistribution of VE-cadherin, resulting in its transient loss from regions of endothelial cell-cell contact. The molecule gradually reorganised within the endothelial cell contacts after this time. It was further shown that the overall expression of VE-cadherin did not change, however, the amount of alpha- and beta-catenins coprecipitated with VE-cadherin markedly decreased after 5 min of tumour cell adhesion to the HUVECs. Immunoprobing of these samples with anti-phosphotyrosine antibodies demonstrated that the tyrosine phosphorylation of VE-cadherin was significantly increased following 5 min of tumour cell adhesion. Together, these results suggest that the adhesion of tumour cells to HUVEC promotes the redistribution of VE-cadherin from interendothelial adherens junctions, an effect that may be attributed to the increase in tyrosine phosphorylation of members of the VE-cadherin/catenin adhesion complex. This, in turn, may increase vascular endothelial permeability and facilitate the transendothelial migration of tumour cells during extravasation.

Inhibitory effects of malotilate on invasion and metastasis of rat mammary carcinoma cells by modifying the functions of vascular endothelial cells.

Malotilate (diisopropyl,1,3-dithiol-2-ylidenemalonate, MT) is clinically used as a hepatoprotective agent. Because we noticed that MT induced the differentiation of cultured vascular endothelial cells, we have examined its effects on lung metastasis of the highly metastatic rat mammary carcinoma c-SST-2. MT was orally administered to syngeneic SHR rats from 7 days before or after s.c. inoculation of c-SST-2 cells to the end of the experiments. In the MT-treated rats, pulmonary metastasis was markedly suppressed compared with the non-treated rats. In the rats treated with MT for 19 days after i.v. inoculation of c-SST-2 cells, lung metastasis was also significantly suppressed. An in vitro invasion assay using a rat lung endothelial (RLE) cell monolayer revealed that pretreatment of the RLE cells with MT, but not c-SST-2 cells, significantly reduced the invasion of the RLE monolayer by c-SST-2 cells. An in vitro vascular permeability assay demonstrated that MT prevented the increase in permeability of the RLE monolayer by serum starvation. On the other hand, in vivo and in vitro growth, gelatinase production and adhesion to the RLE cell monolayer of c-SST-2 cells were not affected by MT treatment. These findings suggest that MT suppressed tumour metastasis by intensifying the cell-to-cell contact of endothelial cells, thus preventing tumour cells from invading vascular endothelium.

Endothelial cell density and contact lens-induced corneal swelling.

To determine the relationships among endothelial morphometric variables and contact lens-induced corneal swelling in a homogeneous sample of adapted contact-lens wearers. Fifteen male subjects ranging in age from 20 to 40 years, all adapted to daily wear of hydrogel lenses, wore uniform-thickness lenses (Dk/L = 5.78) under unilaterally patched eyes for 4 h. Unpatched fellow eyes served as controls. Central corneal thickness was measured with an optical pachometer. Central endothelial images were obtained with a Topcon SP-1000 Specular Microscope and analyzed by the Topcon IMAGEnet processing system. Thickness and morphometric data were collected on test and control eyes before and after the patching sessions. A strong correlation (r = -0.795; p < 0.001) was found between central corneal swelling and endothelial-cell density. Correlations between swelling and the coefficient of variation in cell area (r = 0.502; p < 0.06) and between swelling and the percentage of six-sided cells (r = -0.200; p < 0.48) were not significant. Correlations among the morphometric variables were not significant. Differences in the morphometric variables between test and control eyes were not significant before or immediately after the patching sessions. Endothelial-cell density is useful in explaining differences in corneal-swelling responses to closed-eye contact-lens wear among adapted wearers, whereas morphometric variables based on cell-size variability and shape are not.

Occludin as a possible determinant of tight junction permeability in endothelial cells

Endothelial cells provide a crucial interface between blood and tissue environments. Free diffusion of substances across endothelia is prevented by the endothelial tight junction, the permeability of which varies enormously depending on tissue. Endothelial cells of the blood-brain barrier possess tight junctions of severely limited permeability, whereas those of non-neural tissue are considerably leakier, but the molecular basis for this difference is not clear. Occludin is a major transmembrane protein localizing at the tight junction. In this study, we show, by immunocytochemistry, that occludin is present at high levels and is distributed continuously at cell-cell contacts in brain endothelial cells. In contrast, endothelial cells of non-neural tissue have a much lower expression of occludin, which is distributed in a discontinuous fashion at cell-cell contacts. The apparent differences in occludin expression levels were directly confirmed by immunoblotting. The differences in occludin protein were reflected at the message level, suggesting transcriptional regulation of expression. We also show that occludin expression is developmentally regulated, being low in rat brain endothelial cells at postnatal day 8 but clearly detectable at post-natal day 70. Our data indicate that regulation of occludin expression may be a crucial determinant of the tight junction permeability properties of endothelial cells in different tissues.

Aortic endothelial cells regulate proliferation of human monocytes in vitro via a mechanism synergistic with macrophage colony-stimulating factor. Convergence at the cyclin E/p27(Kip1) regulatory checkpoint.

Monocyte-derived macrophages (Mphis) are pivotal participants in the pathogenesis of atherosclerosis. Evidence from both animal and human plaques indicates that local proliferation may contribute to accumulation of lesion Mphis, and the major Mphi growth factor, macrophage colony stimulating factor (MCSF), is present in atherosclerotic plaques. However, most in vitro studies have failed to demonstrate that human monocytes/Mphis possess significant proliferative capacity. We now report that, although human monocytes cultured in isolation showed only limited MCSF-induced proliferation, monocytes cocultured with aortic endothelial cells at identical MCSF concentrations underwent enhanced (up to 40-fold) and prolonged (21 d) proliferation. In contrast with monocytes in isolation, this was optimal at low seeding densities, required endothelial cell contact, and could not be reproduced by coculture with smooth muscle cells. Intimal Mphi isolated from human aortas likewise showed endothelial cell contact-dependent, MCSF-induced proliferation. Consistent with a two-signal mechanism governing Mphi proliferation, the cell cycle regulatory protein, cyclin E, was rapidly upregulated by endothelial cell contact in an MCSFindependent fashion, but MCSF was required for successful downregulation of the cell cycle inhibitory protein p27(Kip1) before cell cycling. Thus endothelial cells and MCSF differentially and synergistically regulate two Mphi genes critical for progression through the cell cycle.

VE-cadherin antibody accelerates neutrophil recruitment in vivo.

Neutrophils enter sites of inflammation by crossing the endothelial lining of the blood vessel wall. VE-cadherin is an endothelial specific, homophilic adhesion molecule located at the lateral cell surface. We have generated a monoclonal antibody against mouse VE-cadherin which inhibits electrical resistance of endothelial cell monolayers in vitro as well as aggregation of VE-cadherin transfected cells. In vivo, this antibody was found to increase vascular permeability and to accelerate the entry of neutrophils into chemically inflamed mouse peritoneum. Thus, VE-cadherin is essential for the integrity of the endothelial barrier in vivo. Our data suggest that opening of VE-cadherin mediated endothelial cell contacts may be a relevant step during neutrophil extravasation.

Matrix metalloprotease inhibitory effect of endothelial cell contact during exocytosis of adherent neutrophils

Basal membrane turnover implies the regulation of specific metalloproteases (MMP) with cleavage abilities for collage IV. We have set up a model of neutrophil (PMN)-endothelial interactionin vitro which allowed to observe a net gelatinase exocytosis when PMN adhered to plastic substrates. When PMN adhered to endothelial monolayers, this activity was significantly diminished (−30%;p<0.05) and furthermore, RNA transcripts for MMP were specifically down-regulated. Our study points to an endothelial inhibitory regulation of matrix remodeling during PMN-endothelial interaction.

Activation-dependent changes in human platelet PECAM-1: phosphorylation, cytoskeletal association, and surface membrane redistribution.

PECAM-1 is a recently described member of the immunoglobulin gene (Ig) superfamily that is expressed on the surface on platelets, several leukocyte subsets, and at the endothelial cell intracellular junction. Recent studies have shown that the extracellular domain of PECAM-1, which is comprised of 6 Ig-like homology units, participates in mediating cell-cell adhesion, plays a role in initiating endothelial cell contact, and may later serve to stabilize the endothelial cell monolayer. PECAM-1 also has a relatively large 108 amino acid cytoplasmic domain, with potential sites for phosphorylation, lipid modification, and other posttranslational events that could potentially modulate its adhesive function or regulate its subcellular distribution. Virtually nothing is known about the contribution of the intracellular region of the PECAM-1 molecule to either of these cellular processes. Using human platelets as a model, we now demonstrate that PECAM-1 becomes highly phosphorylated in response to cellular activation, and coincident with phosphorylation associates with the cytoskeleton of activated, but not resting, platelets. The engagement of PECAM-1 with the platelet cytoskeleton enables it to move large distances within the plane of the membrane of fully-spread, adherent platelets. This redistribution may similarly account for the ability of PECAM-1 to localize to the intracellular borders of endothelial cells once cell-cell contact has been achieved.

Cultured microvascular endothelial cells derived from the bovine corpus luteum possess NCAM-140

Previously, five phenotypically different, stable types of microvascular endothelial cells (MVE) were isolated from the bovine corpus and cultured successfully. We found that three out of these five types of MVE express the neural cell adhesion molecule (NCAM). As shown by immunocytochemistry, weak NCAM immunoreactivity occurred mainly in the perinuclear area of cell type 1. Monolayers of types 2 and 5 revealed heavy NCAM immunoreactivity, which was localized predominantly at the lateral cell surface outlining the contact zones of adjacent cells. In contrast, cell types 3 and 4 were not NCAM immunoreactive. Western blot analyses substantiated these results: While cell type 1 showed a weak immunoreactive band, cell types 2 and 5 displayed strong NCAM-immunoreactive bands of a molecular weight of approximately 140 kDa (NCAM-140), which was absent in cell types 3 and 4. These results reveal for the first time that NCAM can be expressed by cultured MVE and may serve in mediating endothelial cell contacts. Since luteal cells also express NCAM-140, this adhesion molecule could in addition be involved in the interactions of luteal cells with MVE.

Mechanisms of endothelial cell killing by H 2O 2 or products of activated neutrophils

Interactions between rat pulmonary artery endothelial cells and hydrogen peroxide or toxic oxygen products from phorbol ester-activated human neutrophils result in endothelial cell killing defined by 51Cr release. It has been shown that this cytotoxic reaction can be blocked by the presence of catalase, iron chelators, or scavengers of the hydroxyl radical. Evidence shows that products from xanthine oxidase of endothelial cells are necessary for the toxic effects of hydrogen peroxide or phorbol ester-activated neutrophils. Addition of xanthine oxidase inhibitors protects against phorbol ester-mediated injury of endothelial cells. Preloading of endothelial cells with superoxide dismutase attenuates injury caused either by hydrogen peroxide or phorbol ester-activated neutrophils. Conversion of xanthine dehydrogenase to xanthine oxidase in endothelial cells occurs during contact of endothelial cells by activated neutrophils. This conversion is not related to oxygen products of neutrophils. Conversion of xanthine dehydrogenase to xanthine oxidase in endothelial cells is also induced by endothelial cell contact with C5a, N′-formyl-methionyl-leucyl-phenylalanine (fMLP), or tumor necrosis factor alpha (TNFα). Interaction of hydrogen peroxide with endothelial cells rapidly depletes adenosine triphosphate (ATP) and causes the extracellular appearance of xanthine and hypoxanthine. Agents that protect endothelial cells from the toxic effects of hydrogen peroxide do not prevent falls in cellular ATP caused by hydrogen peroxide, indicating that ATP levels do not necessarily correlate with cytotoxic events. A synergy between hydrogen peroxide and proteases in endothelial cell killing has been demonstrated. TNFα causes alterations in endothelial cells, the result of which is increased susceptibility to killing by PMA-activated neutrophils.

Expression of the CD34 Gene in Vascular Endothelial Cells

All seven of a set of CD34 monoclonal antibodies that recognize epitopes on an approximately 110 Kd glycoprotein on human hemopoietic progenitor cells also bind to vascular endothelium. Capillaries of most tissues are CD34 positive, as are umbilical artery and, to a lesser extent, vein, but the endothelium of most large vessels and the endothelium of placental sinuses are not. Angioblastoma cells and parafollicular mesenchymal cells in fetal skin are also CD34 positive, as are some stromal elements. An approximately 110 Kd protein can be identified by Western blot analysis with CD34 antibodies in detergent extracts of freshly isolated umbilical vessel endothelial cells, and CD34 mRNA is present in cultured umbilical vein cells as well as other tissues rich in vascular endothelium (breast, placenta). These data indicate that the binding of CD34 antibodies to vascular endothelium is to the CD34 gene product, and not to crossreactive epitopes. Despite the presence of CD34 mRNA in cultured, proliferating endothelial cells, the latter do not bind CD34 antibodies. In addition, CD34 antigen cannot be upregulated by growth factors. We conclude that under these conditions, CD34 protein is downregulated or processed into another form that is unreactive with CD34 antibodies. Electron microscopy of umbilical artery, breast, and kidney capillary vessels reveals that in all three sites, CD34 molecules are concentrated on membrane processes, many of which interdigitate between adjacent endothelial cells. However, well-established endothelial cell contacts with tight junctions are CD34 negative. CD34 may function as an adhesion molecule on both endothelial cells and hematopoietic progenitors.

Expression of the CD34 gene in vascular endothelial cells

All seven of a set of CD34 monoclonal antibodies that recognize epitopes on an approximately 110 Kd glycoprotein on human hemopoietic progenitor cells also bind to vascular endothelium. Capillaries of most tissues are CD34 positive, as are umbilical artery and, to a lesser extent, vein, but the endothelium of most large vessels and the endothelium of placental sinuses are not. Angioblastoma cells and parafollicular mesenchymal cells in fetal skin are also CD34 positive, as are some stromal elements. An approximately 110 Kd protein can be identified by Western blot analysis with CD34 antibodies in detergent extracts of freshly isolated umbilical vessel endothelial cells, and CD34 mRNA is present in cultured umbilical vein cells as well as other tissues rich in vascular endothelium (breast, placenta). These data indicate that the binding of CD34 antibodies to vascular endothelium is to the CD34 gene product, and not to crossreactive epitopes. Despite the presence of CD34 mRNA in cultured, proliferating endothelial cells, the latter do not bind CD34 antibodies. In addition, CD34 antigen cannot be upregulated by growth factors. We conclude that under these conditions, CD34 protein is downregulated or processed into another form that is unreactive with CD34 antibodies. Electron microscopy of umbilical artery, breast, and kidney capillary vessels reveals that in all three sites, CD34 molecules are concentrated on membrane processes, many of which interdigitate between adjacent endothelial cells. However, well-established endothelial cell contacts with tight junctions are CD34 negative. CD34 may function as an adhesion molecule on both endothelial cells and hematopoietic progenitors.

Organization of the cytoskeleton and the focal contacts of bovine aortic endothelial cells cultured on type I and III collagen.

We investigated the organization of the cytoskeleton and the focal contacts of bovine aortic endothelial cells cultured on type I and III collagen. The influence of these collagens on cell morphology and the distribution pattern of actin, vimentin, talin, and vinculin was analyzed by light microscopy, conventional electron microscopy, immunofluorescence, and immunogold labeling after lysis-squirting. Whereas the morphology of the endothelial cells is not markedly influenced, the structure of the cytoskeleton and the focal contacts of the cells are altered by the different collagen types. Stress fibers are more distinct in cells grown on type I collagen; cells on type III collagen show a more diffuse distribution of actin molecules. Intermediate filaments seem not to be affected by the collagens. The areas of focal contacts are larger in cells on type I collagen. Additionally, the labeling pattern of talin and vinculin is denser in focal contacts of cells grown on type I collagen. These results suggest an important role of the type of collagen in mediation of the organization of the microfilament system and the adhesion structures of bovine aortic endothelial cells in culture.

Topographical specificity in isolated retinal capillary basement membranes: A high-resolution scanning electron microscope analysis

Numerous investigations have demonstrated that basement membranes (BMs) are composed of type IV collagen, laminin, heparan sulfate proteoglycan, nidogen, and possibly fibronectin. The precise proportion and supramolecular organization of these molecules within BMs is unclear, but is believed to be tissue-specific. In an effort to provide morphological evidence for BM specificity, we studied isolated bovine retinal capillary BMs by high-resolution SEM. Cryofractured specimens demonstrated that surfaces of BM leaflets and pericytic matrix (PCM) within the retinal capillary BM complex are composed of 20- to 100-nm granules and beaded fibrils arranged in patterns which are specific for each cell type. Subendothelial BMs and the subjacent PCM are composed of 20- to 30-nm granules loosely arranged and marked by numerous pits, features that are consistent with their TEM morphology and known susceptibility to proteolytic attack. These BMs also frequently exhibit large openings or fenestrations. These compare favorably with their fragmented image by TEM and probably represent BM discontinuities necessary for direct contact of pericytes and endothelial cells. Muller cell BMs are also composed of granules though they are much larger (40–100 nm) and more densely packed then those of subendothelial BMs. Moreover, they frequently contain interstitial collagen fibrils which could account for the tube-like structural rigidity exhibited by acellular retinal vessel BMs in SEM views. Data in the current study provide morphological evidence for direct contact of pericytes and endothelial cells in vivo and support the view that tissue specificity of BMs may be more exquisite than previously believed, extending even to surface topography of BM leaflets within capillary BM complexes.

Interactions Between Bladder Tumor Cells as Tumor Spheroids from the Cell Line J82 and Human Endothelial Cells in Vitro

Multicellular tumor spheroids (MCTS) are a reliable model of nonvascularized tumor cell aggregates showing a well defined three-dimensional growth pattern and are comparable with small metastatic cancer cell complexes in blood circulation.In the present study we have established a co-culture system of multicellular bladder tumor spheroids with human endothelial cells on extracellular matrix (ECM) in order to investigate morphological and proliferative changes of endothelial and tumor cells within a defined time of cell-cell interaction.The MCTS—endothelial cell—extracellular matrix complex was observed within coculture periods from ½ to seven days. Morphological changes (light microscopy, scanning and electron microscopy) indicated that MCTS are not influenced by cocultured endothelial cells. The tumor cells invaded into the ECM after degradation of endothelial cells in the center of the contact zone.Endothelial cells, however, showed degenerative changes as well as a complex reaction in their proliferation activities. We could recognize an initial increase of proliferation of endothelial cells next to the MCTS. Later on, endothelial cells next to invading tumor cells showed changes in morphological polarity.The model system used has the advantage of using human tumor tissue. It distinguishes between basic cellular mechanisms like adherence, migration, DNA synthesis and proliferation in the study of the contact of tumor cells and vascular endothelial cells as an important event in hematogenous tumor spread.

Use of an endothelial monolayer on a vascular graft prior to implantation. Temporal dynamics and compatibility with the operating room.

The temporal sequence of events was examined from initial contact of endothelial cells (ECs) to Dacron until the establishment of a monolayer. Cultured human adult ECs were radiolabeled, seeded onto Dacron, and adherence was quantified after vigorous washing. Firm adherence of 70% of the seeded ECs was seen by 2 hours to untreated Dacron, by 30 minutes to Dacron pretreated with a combination of interstitial type I/III collagen and an amnion-derived basement membrane (Type IV) collagen surface, and by 10 minutes to plasma-coated Dacron. Parallel samples were examined morphologically by scanning electron microscopy (SEM) to evaluate the adherence of ECs to surfaces. ECs seeded onto plain Dacron exhibited limited adherence, while cells on plasma-treated Dacron exhibited limited cell-cell associations. On basement membrane-treated Dacron, by 30 minutes the ECs exhibited a flat attenuated morphology, completely covering the graft surface. This time-frame is compatible with most vascular procedures, making an immediately endothelialized graft feasible.

Platelet Thrombosis and Non-traumatic Intimal Injury in Mouse Aorta

SummaryAortas of mice, not exposed to any experimental injury, were examined by light and electron microscopy for the initial stages of mural thrombosis.Platelets in the lumen frequently formed groups or loose platelet aggregates. Individual platelets and platelet aggregates, both loose and dense, were often in contact with the inner vascular lining in focal areas. The underlying intimal tissue, including the endothelial cells, showed varying degrees of injury. Occasionally, however, platelet material was in contact even with structurally normal endothelial cells. The platelet- endothelial cell contact appeared to be an expression of adherence.Small mural thrombi were less frequently observed. They comprised platelets and fibrin and were mostly attached to subendothelial structures. Underneath the mural thrombi, the internal elastic membrane was often ruptured.The observed phenomena fit best with the theory that the primary event in mural thrombosis in aorta of mice is platelet aggregation under the influence of local flow irregularities. The next steps could be adherence of platelets to the vessel wall and injury of the intimai tissue through the release of vasodestructive agents from the platelets. Through this injury a focus of mural thrombosis could be established.