PROJECT SUMMARY The objectives of this NRSA application are to 1) develop research skills that will allow the candidate to become a successful physician-scientist and 2) investigate the mechanisms by which hypoxia-inducible factor 2 (HIF-2) causes pulmonary arterial hypertension (PAH). PAH is a highly morbid disease despite recent advancements in therapy. Clinically, increased pulmonary artery pressure, caused in part by hyperproliferative lung microvascular endothelial cells (MVEC), leads to right ventricular failure and death. Rigorous preclinical studies have uncovered a critical role of HIF-2 in PAH development, but the specific mechanisms are unknown. In tumor cells, HIF-2 causes mitochondrial dysfunction and augmented cell growth. In pulmonary MVECs, our lab showed that mitochondrial dysfunction causes activation of the Ca2+ channel transient receptor potential vanilloid type 4 (TRPV4), increased intracellular Ca2+ concentration ([Ca2+]i) and MVEC hyperproliferation. The causes of mitochondrial dysfunction in PAH MVEC – and whether HIF-2 plays any role in the observed abnormalities – are not known, and will be the focus of this application. The long-term goal of this proposal is to identify pathways of HIF-2-induced MVEC dysfunction that can be targeted for the treatment of PAH. Thus, the purpose of this project is to examine the mechanisms by which HIF-2 causes MVEC dysfunction. The candidate’s preliminary studies have shown that non-specific HIF stabilization in pulmonary MVECs causes mitochondrial fragmentation. In Specific Aim 1, the candidate will assess the effects of HIF-2 over-expression and deletion on several measures of mitochondrial dysfunction, including fragmentation and ROS production. In Specific Aim 2, using live Ca2+ imaging, immunoblots, RT-PCR and biotinylation assays, the candidate will assess the role of HIF-2 in modulating MVEC [Ca2+]i. In Aim 3, the candidate will test whether HIF-2 regulates MVEC hyperproliferation and, if so, whether this occurs via mitochondrial dysfunction or changes in [Ca2+]i. The goal of these experiments is to determine whether HIF-2 causes mitochondrial dysfunction, increased [Ca2+]i and hyperproliferation in PAH MVECs; completion of this project will provide a training platform to gain skills that will prove invaluable in the candidate’s transition to independence at the end of fellowship.