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1 Rayne Institute University College London, London, United Kingdom; Institute of Child Health, London, United Kingdom
* To whom correspondence should be addressed. E-mail: b.wojciak-stothard{at}ich.ucl.ac.uk.
Hypoxia/reoxygenation-induced changes in endothelial permeability are accompanied by remodelling of the endothelial actin cytoskeleton and adherens junctions but the mechanisms involved are uncertain. We therefore measured the activities of the Rho GTPases Rac1, RhoA and Cdc42 during hypoxia/reoxygenation and correlated them with the changes in endothelial permeability, remodelling of the actin cytoskeleton and adherens junctions and the production of reactive oxygen species (ROS). Dominant negative forms of Rho GTPases were introduced into the cells by adenoviral gene transfer and transfection and the inhibitors of NADPH oxidase, PI3 kinase and Rho kinase were used to characterize the signalling pathways involved. In some experiments constitutively activated forms of RhoA and Rac1 were also used. We show for the first time that hypoxia/reoxygenation-induced changes in endothelial permeability result from coordinated actions of the Rho GTPases Rac1 and RhoA. Rac1 and RhoA rapidly respond to changes in oxygen tension and their activity depends on NADPH oxidase- and PI3 kinase-dependent production of ROS. Rac1 acts upstream of RhoA and its transient inhibition by acute hypoxia leads to the activation of RhoA followed by stress fibre formation, dispersion of adherens junctions and increased endothelial permeability. Reoxygenation strongly activates Rac1 and restores cortical localization of F-actin and VEcadherin. This effect is a result of Rac1-mediated inhibition of RhoA and can be prevented by activators of RhoA, L63RhoA and lysophosphatidic acid (LPA). Cdc42 activation follows the RhoA pattern of activation but has no effect on actin remodelling, junctional integrity or endothelial permeability. Our results show that Rho GTPases act as mediators coupling cellular redox state to endothelial function.
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