Abstract 466: The p21-activated kinase-1 (PAK1) Promotes Proliferation of Pulmonary Artery Smooth Muscle Cells involving PAI-1 and HIF-1alpha: Role of Calcium and Reactive Oxygen Species
Pulmonary hypertension (PH) is frequently aggravated by enhanced proliferation of pulmonary artery smooth muscle cells (PASMC) and a prothrombotic state due to increased levels of thrombin and plasminogen activator inhibitor-1 (PAI-1). Although mainly known as an inhibitor of fibrinolysis, PAI-1 plays also a role in proliferation and its expression can be activated by thrombin. Thus, we aimed to investigate the molecular link by which thrombin connects proliferation of PASMC via PAI-1. We showed that stimulation of PASMC with thrombin activated the GTP-binding protein Rac1 and its downstream kinase p21-activated kinase-1 (PAK1) suggesting that PAK1 may link thrombin to PAI-1 expression and proliferation of PASMC. The thrombin-dependent activation of Rac1 and PAK1 was inhibited by the calcium chelator BAPTA-AM and antioxidants. BAPTA-AM and the dominant-negative mutants RacT17N and PAK-R295 prevented thrombin-stimulated generation of reactive oxygen species (ROS). Thrombin also enhanced PAI-1 promoter activity, mRNA and protein expression, whereas RacT17N, PAK-R295, BAPTA-AM and antioxidants prevented upregulation of PAI-1 by thrombin. Furthermore, thrombin induced expression and activity of the transcription factor HIF-1alpha involving Rac1, PAK1, calcium and ROS whereas targetting of HIF-1alpha by siRNA prevented thrombin-stimulated PAI-1 expression. Finally, thrombin increased proliferation of PASMC which was prevented by an inhibitory antibody against PAI-1 as well as by PAK-R295 and HIF-1alpha siRNA. Consistently, the active mutant PAKT423E increased HIF-1alpha and PAI-1 levels as well as proliferation. Together, these data show that thrombin stimulates proliferation of PASMC via PAI-1 involving calcium- and ROS-dependent activation of Rac1, PAK1 and HIF-1alpha. This novel pathway may play an important role in linking a prothrombotic state to advanced pulmonary vascular remodeling in PH and may provide a molecular explanation for the beneficial effects of anticoagulation in this disease.