ROS-induced endothelial dysfunction in pulmonary arterial hypertension

项目摘要本K08提案的目标有两个：1)促进Essential 科学技能将使候选人成为一名独立的内科医生-专注于这一角色的科学家 ROS和细胞内钙在内皮功能异常中的作用 肺动脉高压(PAH)时内皮细胞异常迁移/增殖。通过实验室 经验、课程和同行评议，苏雷什博士和他在约翰·霍普金斯大学的导师拉里萨博士 Shimoda已经设计了一个具体的培训计划，将为Suresh博士提供所需的研究技能 对包括肺高压在内的肺部疾病的内皮功能进行独立研究。多环芳烃是一种致命的 以远端血液中内皮细胞的异常迁移和增殖为特征的疾病 肺的血管。目前还没有针对PAH潜在的内皮功能障碍的治疗方法。 活性氧物种(ROS)和细胞内钙离子(Ca~(2+))是迁移和释放的重要介质 已知血管内皮细胞和两者的增殖在PAH中升高，但连接ROS和Ca2+的机制 正常内皮细胞向PAH异常表型转化的内流尚不清楚。我们的前辈 已发表的工作和初步数据从患有PAH的人(hPAH-ECs)和经历了 一种实验性的PAH(RPAH-ECs)SUGEN/HO2(SuHx)提示：在ECs中：1)ROS的抬高 通过激活钙通道TRPV4升高[Ca~(2+)]i；2)Src对TRPV4磷酸化的调节 通过其锚定CD36连接到细胞膜上的激酶Fyn对于激活TRPV4至关重要；3)基线 RPAH-和hPAH-ECs的ROS水平、细胞内钙离子、迁移和增殖水平升高，并减弱 通过抑制ROS或抑制TRPV4；4)RPAH-ECs表现出线粒体功能障碍的证据 可能代表PAH中ROS升高的来源；以及5)CD36或Fyn的丢失减弱了 小鼠SuHx模型中的PAH。因此，我们假设CD36拴系的Fyn对TRPV4的磷酸化是 线粒体功能障碍引起的胞浆ROS升高激活该通道所必需的， 促进EC的迁移和扩散。使用大鼠和人的PAH-ECs(和常氧对照)，我们建议 以下目标：1)确定CD36和Fyn是否是基础钙水平升高、迁移所必需的 2)评价线粒体ROS猝灭能否恢复正常的内皮细胞 在PAH-ECs中的作用；3)确定CD36/Fyn/TRPV4通路在ROS诱导的钙离子中的作用 向体内多环芳烃的发展/进展的内流。研究这些目的的方法包括荧光 细胞内钙离子和ROS的活细胞成像，基因敲除技术，体外迁移和 增殖分析，以及生理学和组织学测量的PAH体内模型。正在完成 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，可能被转化为未来对这种和其他血管疾病的治疗。

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.