Role of RhoA and Rho kinase in lysophosphatidic acid-induced endothelial barrier dysfunction

In the present study, the roles of the small GTPase RhoA and its target Rho kinase in endothelial permeability were investigated in vitro. We have sho wn previously that, in addition to a rise in the intracellular Ca2+ concent ration ([Ca2+](i)), RhoA is involved in the prolonged thrombin-induced barr ier dysfunction. To study the role of RhoA and Rho kinase more specifically , endothelial cells were stimulated with lysophosphatidic acid (LPA), a com monly used RhoA activator. LPA induced a 2- to 3-fold increase in the passa ge of horseradish peroxidase (HRP) across endothelial monolayers that laste d for several hours, whereas thrombin induced a 5- to 10-fold increase. Com parable to: the thrombin-induced barrier dysfunction, the LPA-induced barri er dysfunction was accompanied by a reorganization of the F-actin cytoskele ton and the formation of focal attachment sites. LPA induced only a transie nt increase in myosin light-chain (MLC) phosphorylation, which returned to basal level within 10 minutes. In endothelial cells, [Ca2+](i) was; not ele vated by LPA. Chelation of Ca-i(2+) ions by 1,2-bis(2-aminophenoxy)ethane-N ,N,N',N'-tetraacetic acid did not prevent the LPA-induced passage of HRP. A pparently, a low degree of MLC kinase activation occurred, because the MLC kinase inhibitor KT5926 reduced the levels of both basal and LPA-stimulated HRP passage. Inhibition of RhoA by the C3 transferase from Clostridium bot ulinum inhibited the LPA-induced cytoskeletal changes and prevented the LPA -induced HRP passage. Inhibition of Rho kinase by Y-27632 completely preven ted the LPA-induced increase in HRP passage without affecting basal permeab ility. These data indicate that LPA-induced endothelial hyperpermeability o ccurs without a change in [Ca2+](i) and requires activation of RhoA and Rho kinase.