Primary pulmonary hypertension (PPH) is a fatal disease of unknown etiology characterized by impaired regulation of both pulmonary hemodynamics and vascular growth. Our preliminary data show that primary pulmonary artery endothelial cells (PAEC) from PPH lung have enhanced proliferation, migration and abnormal tube formation in vitro. The signal transducer and activator of transcription (STAT) 3, recently identified as a critical regulator for angiogenesis, is persistently activated in PPH PAEC, but not in control cells. High-level expression of putative downstream target genes, arginase II and vascular endothelial growth factor (VEGF), are present only in PPH PAEC. Previously, we showed that diminished vasodilator nitric oxide (NO) is important in the pathophysiology of PPH, but NO synthases (NOS) expression are intact. Here, we propose a post-translational mechanism for low NO, i.e. arginase II, an enzyme that competes for the NOS substrate arginine, is increased. Thus, we hypothesize that the pathogenesis of PPH stems from abnormal endothelial cells, which have persistent STAT3 activation with consequent expression of VEGF and arginase II, that leads to increased proliferation, deregulated angiogenesis, and loss of NO. Initially, we will quantitate proliferation, migration and tube formation of PPH PAEC in comparison to healthy and disease controls. To identify mechanisms that account for the altered biology of PPH cells and low NO in PPH, we focus our investigations on arginase II using strategies of over-expression, RNA silencing or pharmacologic inhibition of arginase under conditions that assess enzyme substrate and product effects, and NO synthesis. To investigate mechanisms for high-level arginase expression in PPH, we plan to analyze STAT-mediated transcriptional activation of arginase II. Our studies also include evaluation for type II bone morphogenetic protein receptor (BMPR2) mutations and Human Herpesvirus 8 (HHV8) infection, both implicated in PPH pathogenesis, and use proteomic methods to discover novel differences between control and PPH. Our previous study showed an inverse correlation of NO to pulmonary artery pressure. Here, we propose a longitudinal study of PPH patients to test our hypothesis that determinants of NO synthesis, i.e. arginase and arginine, predict outcomes in PPH. Taken together, these studies will conclusively reveal fundamental and inherent alterations in PPH endothelial cells, identify the causal mechanisms, and lead to novel therapies for treatment of PPH.
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