RhoA inactivation enhances endothelial barrier function

The modulation of endothelial barrier function is thought to be a function of contractile tension mediated by the cell cytoskeleton, which consists of actomyosin stress fibers (SF) linked to focal adhesions (FA). We tested th is hypothesis by dissociating SF/FA with Clostridium botulinum exoenzyme C3 transferase (C3), an inhibitor of the small GTP-binding protein RhoA. Bovi ne pulmonary artery endothelial cell (EC) monolayers given C3, C3 + thrombi n, thrombin, or no treatment were examined using a size-selective permeabil ity assay and quantitative digital imaging measurements of SF/FA. C3 treatm ent disassembled SF/FA, stimulated diffuse myosin II immunostaining, and re duced the phosphotyrosine (PY) content of paxillin and 130- to 140-kDa prot eins that included p125(FAK). CS-treated monolayers displayed a 60-85% decl ine in F-actin content and a 170-300% increase in EC surface area with enha nced endothelial barrier function. This activity correlated with reorganiza tion of F-actin and PY protein(s) to beta-catenin-containing cell-cell junc tions. Because C3 prevented the the thrombin-induced formation of myosin ri bbons, SF/FA, and the increased PY content of proteins, these characteristi cs were Rho dependent. Our data show that C3 inhibition of Rho proteins lea ds to cAMP-like characteristics of reduced SF/FA and enhanced endothelial b arrier function.