P258	Rho GTPases Rac1and RhoA act as regulators of endothelial phenotype and function in hypoxia-induced pulmonary hypertension of the newborn.
1B.Wojciak-Stothard, 1L.Yen Fen Tsang, 2S.Hall, 2S.Haworth
1University College London Department of Medicine, London, GB; 2Institute of Child Health, London, GB.

Chronic hypoxia is a common cause of pulmonary hypertension in the newborn (PPHN), a condition associated with endothelial dysfunction and abnormal pulmonary vascular remodelling. The GTPase RhoA has been implicated in the pathogenesis of PPHN but its contribution to endothelial remodelling and function is not known. We studied pulmonary artery endothelial cells (PAECs) taken from piglets with chronic hypoxia-induced PPHN and from healthy animals and analysed the roles of Rho GTPases in the regulation of endothelial phenotype and function in basal normoxic conditions, acute hypoxia and reoxygenation. The activities of RhoA, Rac1 and Cdc42 were correlated with changes in the endothelial cytoskeleton, adherens junctions, permeability, reactive oxygen species (ROS) production, VEGF levels and the activity of transcription factors HIF-1α and NFkB. Adenoviral gene transfer was used to express dominant negative GTPases, kinase dead p21-activated kinase (PAK-1) and V12Rac1 in cells. We show that PAECs from hypertensive animals show a sustained reduction in Rac1 activity and an increase in RhoA activity which correlate with an increase in stress fibre formation, increased permeability and a decrease in VEGF and ROS production. Cells from pulmonary hypertensive animals also show high activities of HIF1α and NFkB likely to result from changes in the activities of Rho GTPases. Activation of Rac1 and its effector PAK-1 as well as inhibition of RhoA restore normal phenotype and permeability in cells from pulmonary hypertensive animals.

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