ROS-induced endothelial dysfunction in pulmonary arterial hypertension

项目概要K 08提案的目标有两个方面：1）促进基本 科学技能，这将使候选人成为一个独立的医生，科学家专注于作用 ROS和细胞内钙在异常内皮功能和2）研究机制 肺动脉高压（PAH）中的异常内皮迁移/增殖。通过实验室 经验，课程和同行评审，苏雷什博士和他的导师在约翰霍普金斯大学，拉里萨博士 下田，设计了一个具体的培训计划，将提供苏雷什博士所需的研究技能， 对肺疾病（包括PAH）中的内皮功能进行独立研究。PAH是一种致命的 以远端血液中内皮细胞（EC）异常迁移和增殖为特征的疾病 肺的血管。目前尚无针对PAH潜在内皮功能障碍的治疗方法。 活性氧（ROS）和细胞内钙（Ca 2+）是迁移和增殖的重要介质。 内皮细胞的增殖和这两者都是已知的PAH升高，但连接ROS和Ca 2+的机制， 正常内皮细胞转化为PAH中所见的异常表型的流入尚不清楚。我们事先 已发表的工作和从PAH患者和接受PAH治疗的大鼠中分离的EC（hPAH-EC）的初步数据 Sugen/Hypoxia（SuHx）是PAH（rPAH-ECs）的一种实验形式，表明在ECs中：1）ROS升高 通过激活钙通道TRPV 4增加[Ca 2 +]i; 2）通过Src调节TRPV 4磷酸化 激酶Fyn通过其锚CD 36栓系到细胞膜，对于TRPV 4的活化至关重要; 3）基线 rPAH-和hPAH-EC中ROS水平、胞浆Ca 2+、迁移和增殖升高， 通过猝灭ROS或抑制TRPV 4; 4）rPAH-EC表现出线粒体功能障碍的证据， 可能是PAH中ROS升高的来源; 5）CD 36或Fyn的缺失减弱了PAH的发展， 小鼠SuHx模型中的PAH。因此，我们假设TRPV 4通过CD 36-tethered Fyn的磷酸化， 由于线粒体功能障碍引起的细胞溶质ROS升高激活该通道所需的， 促进EC迁移和增殖。使用大鼠和人类PAH-EC（和常氧对照），我们提出 以下目的：1）确定CD 36和Fyn是否是增加基础Ca 2+水平、迁移 2）评估线粒体活性氧猝灭是否能恢复正常EC 3）鉴定ROS诱导的Ca 2+的CD 36/Fyn/TRPV 4途径的贡献 体内PAH发生/进展的流入。研究这些目标的方法包括荧光 细胞内Ca 2+和ROS的活细胞成像、遗传敲低技术、体外迁移和 增殖测定，以及PAH的体内模型与生理学和组织学测量。完成 这些目标将为Suresh博士提供严格的培训计划，并揭示内皮细胞的机制。 PAH的功能障碍，可以转化为这种和其他血管疾病的未来疗法。

PROJECT SUMMARY The objectives of this K08 proposal are two-fold: 1) foster the development of essential scientific skills that will allow the candidate to become an independent physician-scientist focused on the role of ROS and intracellular calcium in abnormal endothelial function and 2) investigate mechanisms underlying abnormal endothelial migration/proliferation in pulmonary arterial hypertension (PAH). Through laboratory experience, coursework and peer review, Dr. Suresh and his mentor at Johns Hopkins University, Dr. Larissa Shimoda, have designed a specific training plan that will provide Dr. Suresh with the research skills needed to pursue independent investigation of endothelial function in lung diseases including PAH. PAH is a lethal disease characterized by abnormal migration and proliferation of endothelial cells (ECs) in the distal blood vessels of the lung. There are currently no therapies that target the underlying endothelial dysfunction in PAH. Reactive oxygen species (ROS) and intracellular calcium (Ca2+) are important mediators of migration and proliferation in ECs and both are known to be elevated in PAH, but the mechanisms that link ROS and Ca2+ influx to the transformation of normal ECs to the abnormal phenotype seen in PAH is unknown. Our prior published work and preliminary data in ECs isolated from humans with PAH (hPAH-ECs) and rats undergoing Sugen/Hypoxia (SuHx), an experimental form of PAH (rPAH-ECs), suggest that in ECs: 1) elevations in ROS increase [Ca2+]i by activating the calcium channel TRPV4; 2) regulation of TRPV4 phosphorylation by the Src kinase Fyn tethered to the cell membrane by its anchor, CD36, is critical for activation of TRPV4; 3) baseline ROS levels, cytosolic Ca2+, migration and proliferation are elevated in rPAH- and hPAH-ECs, and attenuated by quenching of ROS or inhibition of TRPV4; 4) rPAH-ECs exhibit evidence of mitochondrial dysfunction that may represent the source of ROS elevations in PAH; and 5) loss of CD36 or Fyn attenuates development of PAH in a murine SuHx model. Thus, we hypothesize that phosphorylation of TRPV4 by CD36-tethered Fyn is required for activation of this channel by elevated cytosolic ROS that occur due to mitochondrial dysfunction, promoting EC migration and proliferation. Using rat and human PAH-ECs (and normoxic controls) we propose the following aims: 1) Determine whether CD36 and Fyn are required for increased basal Ca2+ levels, migration and proliferation in PAH-EC in vitro; 2) Evaluate whether quenching mitochondrial ROS restores normal EC function in PAH-ECs; and 3) identify the contribution of the CD36/Fyn/TRPV4 pathway of ROS-induced Ca2+ influx towards development/progression of PAH in vivo. Methods for studying these aims include fluorescent live cell imaging of intracellular Ca2+ and ROS, genetic knockdown techniques, in vitro migration and proliferation assays, and an in vivo model of PAH with physiologic and histologic measurements. Completing these aims will provide a rigorous training program for Dr. Suresh and uncover mechanisms of endothelial dysfunction in PAH that could be translated into future therapies for this and other vascular diseases.