Antibodies to VE-cadherin inhibit VE-cadherin clustering after Ca⁺⁺ switch.

(A) Endothelial cell monolayers were exposed to 5 mM EGTA for 30 minutes. EGTA was then removed and Ca⁺⁺ restored adding fresh culture medium either in the presence or in absence of mAbs. One hour later, cells were fixed and processed for immunofluorescence microscopy. The presence of VE-cadherin at junctions was detected by adding a polyclonal antibody VECR1-5. EGTA induced disappearance of VE-cadherin from cell-to-cell contacts. Addition of Ca⁺⁺restored junctional localizaton of VE-cadherin in cells cultured in absence of mAbs (not shown) or in the presence of Hec 1.2 or TEA 1.31. VE-cadherin remained in large part outside the intercellular junctions when cells were cultured in the presence of BV6, BV9, and Cad 5 (bar = 20 μm). (B) Endothelial cells grown on Transwell filters were incubated with EGTA for 30 minutes. To allow junction reorganization, Ca⁺⁺ was then restored in the presence or absence of mAbs (see above). FITC-dextran was added 1 hour after Ca⁺⁺ restoration, and permeability was measured 30 minutes later. Data are expressed as percentage increase in permeability in comparison to cells maintained in the absence of mAbs and are means ± SEM of 4 replicates from a typical experiment out of 3 performed.