Rac and Rho play opposing roles in the regulation of hypoxia/reoxygenation-induced permeability changes in pulmonary artery endothelial cells

• Published in: American journal of physiology. Lung cellular and molecular physiology 2005-04, Vol.288 (4), p.L749-L760
• Main Authors: Wojciak-Stothard, Beata, Tsang, Lillian Yen Fen, Haworth, Sheila G
• Format: Article
• Language: English
• Subjects: Actins - metabolism; Adenoviridae - genetics; Adherens Junctions - drug effects; Animals; Antigens, CD; Cadherins - metabolism; cdc42 GTP-Binding Protein - genetics; cdc42 GTP-Binding Protein - metabolism; Cell Membrane Permeability - drug effects; Cytoskeleton - metabolism; Endothelial Cells - cytology; Endothelial Cells - drug effects; Endothelial Cells - metabolism; Genes, Dominant; Hypoxia; Lysophospholipids - pharmacology; NADPH Oxidases - metabolism; Oxygen - metabolism; Phosphatidylinositol 3-Kinases - metabolism; Pulmonary Artery - cytology; Pulmonary Artery - drug effects; Pulmonary Artery - metabolism; rac1 GTP-Binding Protein - genetics; rac1 GTP-Binding Protein - metabolism; Reactive Oxygen Species - metabolism; rhoA GTP-Binding Protein - genetics; rhoA GTP-Binding Protein - metabolism; Swine; Tight Junctions - drug effects
• ISSN: 1040-0605; 1522-1504
• DOI: 10.1152/ajplung.00361.2004

British Heart Foundation Laboratories, Department of Medicine, University College London; and Institute of Child Health, London, United Kingdom Submitted 24 September 2004 ; accepted in final form 2 December 2004 Hypoxia/reoxygenation-induced changes in endothelial permeability are accompanied by endothelial actin cytoskeletal and adherens junction remodeling, 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 changes in endothelial permeability, remodeling of the actin cytoskeleton and adherens junctions, and production of ROS. Dominant negative forms of Rho GTPases were introduced into cells by adenoviral gene transfer and transfection, and inhibitors of NADPH oxidase, PI3 kinase, and Rho kinase were used to characterize the signaling 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 activation of RhoA followed by stress fiber formation, dispersion of adherens junctions, and increased endothelial permeability. Reoxygenation strongly activates Rac1 and restores cortical localization of F-actin and VE-cadherin. This effect is a result of Rac1-mediated inhibition of RhoA and can be prevented by activators of RhoA, L63RhoA, and lysophosphatidic acid. Cdc42 activation follows the RhoA pattern of activation but has no effect on actin remodeling, junctional integrity, or endothelial permeability. Our results show that Rho GTPases act as mediators coupling cellular redox state to endothelial function. reactive oxygen species; adherens junctions; actin cytoskeleton Address for reprint requests and other correspondence: B. Wojciak-Stothard, BHF Laboratories, Department of Medicine, UCL, 5 University St., WC1 E6JJ London, UK (E-mail: B.Wojciak-Stothard{at}ucl.ac.uk )