O205	Nitric oxide limits activation of Rac by thrombin: role in the redox-sensitive regulation of angiogenesis by HIF-1 and PAI-1.
1R.S.Belaiba, 1T.Djordjevic, 2T.Kietzmann, 1J.Hess, 1A.Görlach
1Experimentelle Kinderkardiologie, Deutsches Herzzentrum München an der TU München, München, DE; 2Fachbereich Chemie, Technische Universität Kaiserslautern, Kaiserslautern, DE.

Atherosclerosis is associated with a prothrombotic state, endothelial dysfunction with enhanced levels of reactive oxygen species (ROS) and reduced nitric oxide (NO) bioavailability as well as with increased levels of plasminogen activator inhibitor–1, PAI-1, a major inhibitor of fibrinolysis. Recently, enhanced formation of new vessels (angiogenesis) has been associated with the progression of atherothrombosis. However, the signaling pathways linking angiogenesis, coagulation and thrombosis in the setting of atherosclerosis and atherothrombosis are not well understood. Thrombin has been shown to modulate ROS and NO levels in endothelial cells, and the hypoxia-inducible transcription factor HIF-1 has been shown to promote angiogenesis under hypoxic conditions. We therefore investigated whether the GTPase Rac, known to activate NADPHoxidases and NO are involved in the regulation of HIF-1, PAI-1 and angiogenesis by thrombin in microvascular endothelial cells. Stimulation with thrombin enhanced ROS and NO production, increased Rac activity and protein expression as well as expression of endothelial NO synthase (eNOS). Transfection of the active RacG12V mutant elevated ROS production, whereas the dominant-negative RacT17N had opposite effects. In contrast, inhibition of NO production by N-nitro-L-arginine (L-NNA) enhanced ROS production and Rac activity whereas antioxidants had opposite effects. Thrombin increased HIF-1alpha expression and activity and subsequently PAI-1 promoter activity and expression in a HIF-dependent manner. These responses were enhanced by RacG12V and L-NNA, but diminished by RacT17N or antioxidants. Finally, thrombin and RacG12V stimulated in vitro angiogenesis. Antioxidants, RacT17N and an inhibitory antibody against PAI-1 diminished the angiogenic response whereas L-NNA further promoted angiogenesis. These data show that Rac increases PAI-1 expression and HIF-1 activity by a redox-sensitive pathway in thrombin-stimulated endothelial cells leading to enhanced angiogenesis and that NO prevents these responses. Thus, activated Rac may play an important role in linking oxidative stress with a procoagulant state and angiogenesis in atherosclerosis. Concomitant inhibition of Rac by NO may be a critical step in limiting disease progression and preventing a putative vicious circle.

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