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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:e127.)
© 2000 American Heart Association, Inc.

Vascular Biology

Role of RhoA and Rho Kinase in Lysophosphatidic Acid–Induced Endothelial Barrier Dysfunction

Geerten P. van Nieuw Amerongen; Mario A. Vermeer; Victor W. M. van Hinsbergh

From the Gaubius Laboratory TNO-PG (G.P.v.N.A., M.A.V., V.M.M.v.H.), Leiden, and the Department of Physiology (G.P.v.N.A., V.M.M.v.H.), Institute for Cardiovascular Research, Vrije Universiteit, Amsterdam, The Netherlands.

Correspondence to Prof Dr V.W.M. van Hinsbergh, Gaubius Laboratory TNO-PG, PO Box 2215, 2301 CE Leiden, the Netherlands. E-mail vwm.vanhinsbergh{at}pg.tno.nl

Abstract—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 shown previously that, in addition to a rise in the intracellular Ca²⁺ concentration ([Ca²⁺]_i), RhoA is involved in the prolonged thrombin-induced barrier dysfunction. To study the role of RhoA and Rho kinase more specifically, endothelial cells were stimulated with lysophosphatidic acid (LPA), a commonly used RhoA activator. LPA induced a 2- to 3-fold increase in the passage of horseradish peroxidase (HRP) across endothelial monolayers that lasted for several hours, whereas thrombin induced a 5- to 10-fold increase. Comparable to the thrombin-induced barrier dysfunction, the LPA-induced barrier dysfunction was accompanied by a reorganization of the F-actin cytoskeleton and the formation of focal attachment sites. LPA induced only a transient increase in myosin light-chain (MLC) phosphorylation, which returned to basal level within 10 minutes. In endothelial cells, [Ca²⁺]_i was not elevated by LPA. Chelation of Ca²⁺_i ions by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid did not prevent the LPA-induced passage of HRP. Apparently, 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 botulinum inhibited the LPA-induced cytoskeletal changes and prevented the LPA-induced HRP passage. Inhibition of Rho kinase by Y-27632 completely prevented the LPA-induced increase in HRP passage without affecting basal permeability. These data indicate that LPA-induced endothelial hyperpermeability occurs without a change in [Ca²⁺]_i and requires activation of RhoA and Rho kinase.

Key Words: human endothelial cells • RhoA • calcium • myosin light-chain phosphorylation • myosin light-chain kinase

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