Metabolic regulation of Ca2+ entry and endothelial-mesenchymal transition in pulmonary arterial hypertension

肺动脉高压（PAH）是一种致死性疾病，其特征是异常的 肺远端血管中微血管内皮细胞（MVEC）的增殖。有 目前尚无针对PAH潜在内皮功能障碍的治疗方法。线粒体功能障碍， 细胞内钙（[Ca 2 +]i）增加和内皮-间质转化（EndMT）是重要的 在PAH患者分离的MVEC中观察到的致病性异常， 这些细胞异常是未知的。在从经历Sugen/缺氧（SuHx）的大鼠分离的MVEC中， PAH的实验形式（SuHx-MVECs），我们以前的工作和目前的初步数据表明a） 线粒体功能障碍重现了在人PAH EC中观察到的那些，B）增加线粒体反应性 氧物种（mtROS）和β-羟基丁酸（BOHB），c）增加瞬时受体的激活 潜在的香草酸-4（TRPV 4）通道和增加的细胞内钙（[Ca 2 +]i），d）EndMT和e）增加 增殖此外，我们还观察到代谢的特定变化（随着呼吸的增加， 以及脂肪酸氧化的增加），这表明SuHx中线粒体功能障碍的代谢基础- MVEC。我们的初步数据表明，脂肪酸增加的两种代谢副产物的具体作用 氧化- BOHB和mtROS -分别敏化和激活TRPV 4。因此，我们假设， 在PAH中，MVEC线粒体代谢从糖酵解到脂肪酸氧化的转变促进mtROS， BOHB的产生，导致TRPV 4活化，并因此导致EndMT的Ca 2+依赖性活化， 增殖使用分离自大鼠、小鼠和人的MVEC（有和无PAH）以及体内 利用各种新型转基因大鼠和小鼠的实验，我们提出了三个独立的目标， 1）增加脂肪酸氧化如何促进TRPV 4活化， MVEC; 2）TRPV 4激活是否是诱导EndMT所必需和充分的;以及3） TRPV 4损失或BOHB补充对体内EndMT发育的影响。为了实现这些目标，我们计划 利用多种方法，从分离自WT的MVEC的荧光活细胞成像， 转基因小鼠和大鼠，PAH患者（和对照）人MVEC的体外研究，以及体内研究 谱系追踪研究，以检查PAH啮齿动物模型中EndMT的发育。实现这些目标 将为脂肪酸代谢，Ca 2+稳态和EndMT之间的相互作用提供新的见解， 正常MVEC和代谢功能障碍的作用，[Ca 2 +]I和EndMT增加PAH。

PROJECT SUMMARY Pulmonary arterial hypertension (PAH) is a lethal disease characterized by abnormal proliferation of microvascular endothelial cells (MVECs) in the distal blood vessels of the lung. There are currently no therapies that target the underlying endothelial dysfunction in PAH. Mitochondrial dysfunction, increased intracellular calcium ([Ca2+]i) and endothelial-mesenchymal transition (EndMT) are important pathogenic abnormalities observed in MVECs isolated from patients with PAH, but the mechanisms that link these cellular abnormalities is unknown. In MVECs isolated from rats undergoing Sugen/Hypoxia (SuHx), an experimental form of PAH (SuHx-MVECs), our prior work and current preliminary data suggest a) mitochondrial dysfunction recapitulating those seen in human PAH ECs, b) increased mitochondrial reactive oxygen species (mtROS) and β-hydroxybutyrate (BOHB), c) increased activation of the transient receptor potential vanilloid-4 (TRPV4) channel and increased intracellular calcium ([Ca2+]i), d) EndMT and e) increased proliferation. Further, we also observe specific shifts in metabolism (increased use of anaerobic respiration as well as an increase in fatty acid oxidation), suggesting a metabolic basis for mitochondrial dysfunction in SuHx- MVECs. Our preliminary data now suggest specific roles for two metabolic byproducts of increased fatty acid oxidation – BOHB and mtROS - in sensitizing and activating TRPV4, respectively. Thus, we hypothesize that, in PAH, a shift in MVEC mitochondrial metabolism from glycolysis to fatty acid oxidation promotes mtROS and BOHB generation, leading to TRPV4 activation and consequently, Ca2+-dependent activation of EndMT and proliferation. Using MVECs isolated from rats, mice and humans with and without PAH, as well as in vivo experiments utilizing various novel transgenic rats and mice, we propose the three independent aims centered around the following questions: 1) How does increased fatty acid oxidation promote TRPV4 activation in MVECs; 2) Is TRPV4 activation necessary and sufficient to induce EndMT; and 3) What is the impact of TRPV4 loss or BOHB supplementation on EndMT development in vivo. To accomplish these aims, we plan on utilizing a variety of methods ranging from fluorescent live-cell imaging of MVECs isolated from WT and transgenic mice and rats, in vitro studies in human MVECs from PAH patients (and controls), and in vivo lineage-tracing studies to examine EndMT development in rodent models of PAH. Completion of these aims will provide novel insight into the interplay between fatty acid metabolism, Ca2+ homeostasis and EndMT in normal MVECs and role of the metabolic dysfunction and increased [Ca2+]I and EndMT in PAH.