Pulmonary Arterial Hypertension (PAH) is a fatal disease characterized by impaired regulation of pulmonary hemodynamics and vascular growth. The excessive growth and dysfunction of endothelial cells that line the blood vessels in PAH is the continuing focus of our research. In the current award period, we established primary cell culture methods from PAH lungs; mechanistically linked low levels of nitric oxide (NO) to PAH in humans; identified pathologic expression of HIF-1? and its role in the metabolic shift to aerobic glycolysis in PAH; and defined a novel myelopulmonary disease paradigm in PAH pathogenesis. In this extension, the hypotheses and aims remain well within the scope of the award. In Aim 1, we define mechanisms of low NO production by PAH pulmonary artery endothelial cells (PAEC). We hypothesize that low NO production is due to reduced activity of the endothelial NO synthase (eNOS) and plan to uncover mechanisms. We will measure intracellular NO production in PAEC in real time and define functional endophenotypes. Because eNOS activity depends on tetrahydrobiopterin (H4B), we will quantify H4B in PAEC and serum of patients in comparison to controls. Likewise, we investigate intracellular mechanisms that regulate eNOS phosphorylation states, i.e. kinase and phosphatase pathways. In Aim 2, we identify HIF-1? expression in PAH and the mechanisms accounting for expression. We hypothesize that HIF-1? expression is fundamental in the pathologic angiogenesis and preferential energy generation by glycolysis in PAH. We determine mitochondrial and glycolytic bioenergetics in PAH PAEC and pulmonary artery smooth muscle cells. We extend mechanistic studies to iron metabolism, which is important to understanding HIF-1? and bioenergetics. We measure hepcidin in PAH patients in comparison to healthy people to test the hypothesis that higher hepcidin accounts for lower iron in PAH. In Aim 3, we determine consequences and relevance of HIF-expression and myeloproliferative processes to pathophysiology of PAH. We hypothesize that HIF- expression is mechanistically important to PAH pathogenesis by establishing a pathologic myeloproliferative process that consequently promotes and sustains the proliferative panvasculopathy of PAH. We test .the effects of transplantation of hematopoietic and mesenchymal stem cells isolated from PAH'bone marrow into NOD-SCID mice. Similarly, we plan mechanistic transplantation experiments using marrows from Caveolin-1 deficient mice, which develop pulmonary hypertension when exposed to hypoxia. We will quantitate lung vasculature, activation and injury of vascular endothelium, and cardiac function in the Wild type mice that receive Caveolin-1 deficient stem cells. Overall, in this extension of the MERIT, our long-term goal remains the same: to define the pathophysiology of abnormal pulmonary vascular growth and endothelial dysfunction, and in so doing, apply the knowledge to improve the care of patients with PAH. RELEVANCE (See instructions): Overall our goals are to define the pathophysiology of the abnormal vascular growth in Pulmonary Arterial Hypertension (PAH), and in so doing, apply the knowledge to improve the care of patients.