Nitric Oxide in Pulmonary Hypertension

摘要 这是我们R37优点(2009-2019年)的竞争性更新，旨在定义肺的机制 动脉高压(PAH)。我们的工作带来了新的知识、共享的资源和方法，包括关键的 人肺血管内皮细胞(PAEC)的获取和培养研究进展我们发现了硝酸盐的损失 氧化物(NO)的产生及其机制：(I)内皮型一氧化氮合酶(ENOS)的磷酸化失活； 和(Ii)eNOS底物精氨酸(Arg)利用率降低与线粒体精氨酸酶2增加有关 (ARG2)。我们发现，避免高原缺氧性肺动脉高压的高原原住民 (HAPH)，具有增加Arg、NO和降低ARG2的适应。同时，我们发现PAH PAEC 线粒体呼吸作用比对照细胞少，并转变为有氧糖酵解。活体、肺和心脏 18F-脱氧葡萄糖正电子发射断层扫描测定PAH患者的葡萄糖摄取 高于对照组。在初步数据中，摄取FDG最高的患者Arg水平最低 和最严重的疾病，表明Arg代谢的命运影响PAH不仅仅是血管扩张剂的丧失 不是的。我们发现线粒体精氨酸代谢与生物能量学有关，包括对燃料的依赖 三羧酸循环(TCA)和线粒体呼吸作用。在小鼠研究中，Arg2基因敲除(Arg2KO)具有 与野生型(WT)相比，线粒体呼吸能力更大，心脏葡萄糖摄取更少，并且缺乏 低氧可引起WT大鼠肺内压力和促红细胞生成素(EPO)的升高。因此，我们假设 线粒体Arg通过ARG2的代谢与TCA循环和生物能量学的异常有关， 促进PAEC病理性增殖、发展为PAH和右室功能不全。目标 1确定疾病代谢机制和途径，参与病理性内皮功能和 PAH病理生理学。为了找到与PAH相关的途径，我们执行差异表达(RNA，蛋白质， 代谢产物)以及多环芳烃和控制多环芳烃的整合网络分析，然后测量细胞生物能量学和 阻断富含多环芳烃的通路后的功能(Aim1A)。PAH患者，分为低心或高心 利用代谢和转录分析将FDG摄取组与对照组进行比较，以揭示 然后纵向跟踪，以确定死亡、移植和/或恶化的RV收缩 高摄取组(Aim1B)的压力(RVSP)较大。目标2确定线粒体Arg是否减少 新陈代谢对低氧相关的PH具有保护作用。高海拔阿姆哈拉人的代谢谱，他们有 高NO、低精氨酸酶和抵抗HAPH与低海拔对应的患者和PAH患者相比， 确定与数量性状有关的保护途径，如EPO和RVSP(目标2A)。在机械学中 研究，我们确定与WT相比，Arg2KO是否有生物能量变化，以及Arg2KO是否受到保护 低氧引起的促红细胞生成素和肺压力升高(目标2B)。这些研究将提供新的 了解多环芳烃的代谢，并提供新的潜在治疗靶点。

ABSTRACT This is a competitive renewal of our R37 MERIT (2009-2019), which aims to define mechanisms of Pulmonary Arterial Hypertension (PAH). Our work led to new knowledge, shared resources and methods, including a critical advance in harvest and culture of human pulmonary artery endothelial cells (PAEC). We identified loss of nitric oxide (NO) production, and mechanisms: (i) phosphorylation inactivation of endothelial NO synthase (eNOS), and (ii) decreased eNOS substrate arginine (arg) availability related to increased mitochondrial arginase 2 (ARG2). We discovered that high-altitude natives, who avoid high-altitude hypoxic pulmonary hypertension (HAPH), have adaptations that increase arg, NO, and decrease ARG2. In parallel, we found that PAH PAEC have less mitochondrial respiration than control cells, and a shift to aerobic glycolysis. In vivo, lung and cardiac glucose uptake in PAH patients measured by 18F-fluorodeoxyglucose-positron emission tomography (FDG-PET) was higher than controls. In preliminary data, patients with the highest FDG uptake have the lowest arg levels and most severe disease, suggesting that arg metabolic fate impacts PAH beyond just the loss of vasodilator NO. We show that mitochondrial arg metabolism is interconnected to bioenergetics, including fuel dependency of tricarboxylic acid cycle (TCA) and mitochondrial respiration. In murine studies, Arg2 knockout (Arg2KO) have greater capacity for mitochondrial respiration and less cardiac glucose uptake than wildtype (WT), and lack the hypoxia-induced increases of pulmonary pressures and erythropoietin (Epo) found in WT. Thus, we hypothesize that mitochondrial arg metabolism via ARG2 is interconnected to abnormalities of TCA cycle and bioenergetics, and promotes pathologic proliferation of PAEC, development of PAH and right ventricular (RV) dysfunction. Aim 1 identifies disease metabolic mechanisms and pathways that participate in pathologic endothelial functions and PAH pathophysiology. To find pathways associated with PAH, we perform differential expression (RNA, protein, metabolites) and integrative network analyses of PAH and control PAEC, then measure cell bioenergetics and functions after blocking pathways enriched in PAH (Aim1A). PAH patients, dichotomized into low or high cardiac FDG uptake groups, are compared with controls using metabolomic and transcriptomic analyses to uncover pathways in vivo, then followed longitudinally to determine if death, transplant, and/or worsening RV systolic pressure (RVSP) is greater in the high uptake group (Aim1B). Aim 2 determines if decreasing mitochondrial arg metabolism is protective against hypoxia-associated PH. Metabolomic profiles of high-altitude Amhara, who have high NO, low arginase, and resist HAPH, are compared to their low-altitude counterparts and PAH patients, to identify protective pathways in relation to quantitative traits, such as Epo and RVSP (Aim 2A). In mechanistic studies, we determine if Arg2KO have bioenergetic changes as compared to WT, and if Arg2KO are protected from hypoxia-induced elevations in Epo and pulmonary pressures (Aim 2B). These studies will provide new metabolic understanding of PAH, and offer new potential therapeutic targets.