Vascular–Endothelial Cadherin (CD144)– but Not PECAM–1 (CD31)–Based Cell–to–Cell Contacts Convey the Maintenance of a Quiescent Endothelial Monolayer

Background: In vivo, all blood vessels are lined by a single layer of flattened noncycling endothelial cells. We tested the hypothesis that the maintenance of such a quiescent endothelial monolayer depends on homotypic contacts between not yet defined growth–inhibitory molecules located at interendothelial junctions. Methods: ECV304 cells, which lack endogenous vascular endothelial cadherin (VE cadherin) or CD31 expression, were transfected with cDNA encoding for the respective proteins or with the empty vector. Results: In VE cadherin transfectants, β–catenin was targeted to junctional regions and the F–actin–based cytoskeleton formed parallel bundles reaching from one cell border to the other. In contrast, in CD31 transfectants and in empty vector cells, β–catenin was dispersed throughout the cytoplasm, and F–actin formed short, plump and criss–cross bundles. On a two–dimensional plastic matrix, both, VE cadherin and CD31 transfectants formed clusters of polygonal cells, whereas in three–dimensional gels, only VE cadherin cells were able to form tubes. Empty vector cells grew in a fibroblast–like pattern and neither formed clusters nor tubes. Most importantly, whereas CD31 and empty vector cells grew on top of each other, formed polylayers and maintained cycling even after reaching confluence, VE cadherin cells strictly maintained a single layer of flattened cells and the numbers of cycling cells dramatically dropped after reaching a continuous monolayer. Conclusion: The insertion of VE cadherin into ECV304 cells produces a cell type which mimics endothelial growth characteristics seen in vivo.