Protein kinase C‐mediated endothelial barrier regulation is caveolin 1‐dependent

Abstract

Protein kinase C (PKC) is activated in response to various inflammatory mediators and contributes significantly to the endothelial barrier breakdown. However, the mechanisms underlying PKC-mediated permeability regulation are not well understood. We prepared microvascular myocardial endothelial cells (MyEnd) cells from both wild-type (WT) and caveolin-1-deficient (Cav-1 −/−) mice. Activation of PKC by phorbol myristate acetate (PMA, 100 nM) for 30 min induced intercellular gap formation and fragmentation of VE-cadherin immunoreactivity in WT but not in Cav-1(−/−) cells. To test the effect of PKC activation on VE-cadherin-mediated adhesion, we allowed VE-cadherin-coated microbeads to bind to the endothelial cell surface and probed their adhesion by laser tweezers. PMA (100 nM, 30 min) significantly reduced bead binding to 78 ± 6 % of controls in WT endothelial cells without any effect in Cav-1(−/−) cells. The latter also exhibited higher baseline values of VE-cadherin-mediated bead adhesion. In WT cells, PMA caused an 86 ± 18 % increase in FITC-dextran permeability whereas no increase in permeability was observed in Cav-1(−/−) monolayers. Inhibition of PKC by staurosporine (50 nM, 30 min) did not affect barrier functions in both WT and Cav-1(−/−) MyEnd cells. Theses data indicate that PKC activation reduces endothelial barrier functions at least in part by reduction of VE-cadherin-mediated adhesion and demonstrate that PKC-mediated permeability regulation depends on caveolin-1. DFG SFB688