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Summary

Endothelial permeability induced by thrombin and histamine is accompanied by actin stress fibre assembly and intercellular gap formation. Here, we investigate the roles of the Ρ family GTPases Rho1, Rac1 and Cdc42 in regulating endothelial barrier function, and correlate this with their effects on F-actin organization and intercellular junctions. RhoA, Rac1 and Cdc42 proteins were expressed efficiently in human umbilical vein endothelial cells by adenovirus-mediated gene transfer. We show that inhibition of Ρ prevents both thrombin- and histamine-induced increases in endothelial permeability and decreases in transendothelial resistance. Dominant-negative RhoA and a Ρ kinase inhibitor, Y-27632, not only inhibit stress fibre assembly and contractility but also prevent thrombin- and histamine-induced disassembly of adherens and tight junctions in endothelial cells, providing an explanation for their effects on permeability. In contrast, dominant-negative Rac1 induces permeability in unstimulated cells and enhances thrombin-induced permeability, yet inhibits stress fibre assembly, indicating that increased stress fibre formation is not essential for endothelial permeability. Dominant-negative Cdc42 reduces thrombin-induced stress fibre formation and contractility but does not affect endothelial cell permeability or responses to histamine. These results demonstrate that Ρ and Rac act in different ways to alter endothelial barrier function, whereas Cdc42 does not affect barrier function.