Mechanisms of HIF-Induced Microvascular Endothelial Dysfunction in Pulmonary Arterial Hypertension

项目摘要 这个NRSA应用程序的目标是1）发展研究技能，使候选人成为 一个成功的医生科学家和2）研究缺氧诱导因子2（HIF-2）的机制， 导致肺动脉高压（PAH）。PAH是一种高度病态的疾病，尽管最近在 疗法临床上，肺动脉压升高，部分由肺微血管过度增生引起， 内皮细胞（MVEC）的死亡导致右心室衰竭和死亡。严格的临床前研究已经发现 HIF-2在PAH的发展中起着关键作用，但具体机制尚不清楚。在肿瘤细胞中，HIF-2 导致线粒体功能障碍和细胞生长增强。在肺MVEC中，我们的实验室表明， 线粒体功能障碍导致钙通道瞬时受体电位香草酸4型激活 （TRPV 4），增加细胞内Ca 2+浓度（[Ca 2 +]i）和MVEC过度增殖。的原因 PAH MVEC中的线粒体功能障碍-以及HIF-2是否在观察到的异常中起任何作用-是 这是未知的，并将是本申请的重点。该提案的长期目标是确定 HIF-2诱导的MVEC功能障碍可作为PAH治疗的靶点因此，本项目的目的 目的是研究HIF-2引起MVEC功能障碍的机制。候选人的初步研究 已经表明肺MVEC中非特异性HIF稳定化导致线粒体断裂。在 具体目标1，候选人将评估HIF-2过表达和缺失对几项指标的影响 线粒体功能障碍，包括断裂和ROS的产生。在特定目标2中，使用活性Ca 2 + 成像，免疫印迹，RT-PCR和生物素化测定，候选人将评估HIF-2在 调节MVEC [Ca 2 +]i.在目标3中，候选人将测试HIF-2是否调节MVEC过度增殖， 如果是，这是否通过线粒体功能障碍或[Ca 2 +]i的变化发生。这些实验的目的是 以确定HIF-2是否导致PAH中线粒体功能障碍、[Ca 2 +]i增加和过度增殖 MVEC;该项目的完成将提供一个培训平台，以获得将在未来几年中被证明是非常宝贵的技能。 候选人在研究金结束时向独立过渡。

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.