P332	Rac and PAK promote plasminogen activator inhibitor-1 expression and proliferation of pulmonary artery smooth muscle cells involving hypoxia-inducible factor-1.
1T.Djordjevic, 1R.S.Belaiba, 1I.Diebold, 2T.Kietzmann, 1J.Hess, 1A.Görlach
1Experimentelle Kinderkardiologie, Deutsches Herzzentrum München an der Technischen Universität München, München, DE; 2Fachbereich Chemie, Technische Universität Kaiserslautern, Kaiserslautern, DE.

A prothrombotic state due to enhanced levels of thrombin and plasminogen activator inhibitor-1 (PAI-1), an inhibitor of fibrinolysis, is frequently found in pulmonary hypertension and has been associated with remodeling processes in the vascular wall. However, the molecular mechanisms possibly connecting these factors with proliferation of pulmonary artery smooth muscle cells (PASMC), the main cell type involved in pulmonary vascular remodeling, are not resolved. Reactive oxygen species (ROS) have been implicated to play an important role in vascular signaling processes. We therefore investigated whether the GTPase Rac which contributes to vascular signaling and ROS production may link thrombin to PAI-1 expression and proliferation of PASMC. Thrombin rapidly activated Rac and ROS production and stimulated Rac protein expression as well as PAI-1 promoter activity, mRNA and protein expression. These responses were prevented by pre-treatment with the calcium chelator BAPTA-AM and antioxidants. Expression of constitutively active RacG12V or dominant-negative RacT17N increased or decreased, respectively, thrombin-stimulated ROS production as well as thrombin-induced PAI-1 promoter activity, mRNA and protein expression. Thrombin also stimulated the phosphorylation of p-21-activated kinase (PAK) in a Rac-dependent manner. Expression of an active PAK mutant (PAK-T423) or a dominant-negative mutant of PAK (PAK-R295) increased or decreased, respectively, PAI-1 promoter activity. Furthermore, thrombin increased expression and activity of the hypoxia-inducible transcription factor HIF-1alpha, and this response was inhibited by antioxidants, BAPTA-AM, RacT17N and PAK-R295. Depletion of HIF-1alpha by siRNA prevented upregulation of PAI-1 by thrombin. Finally, thrombin stimulated proliferation of PASMC whereas antioxidants, BAPTA-AM, RacT17N, PAK-R295, HIF-1alpha siRNA and an inhibitory antibody against PAI-1 inhibited proliferation of PASMC. These data show that thrombin sitmulates PASMC proliferation via calcium-dependent activation of Rac and PAK, subsequent ROS formation and induction of PAI-1 via activation of HIF-1. This mechanism may play an important role in promoting vascular remodeling processes in pulmonary hypertension.

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