Aldosterone impairs endothelin B-dependent synthesis of nitric oxide to promote p

描述（由申请人提供）：美国国立卫生研究院指导临床科学家研究职业发展奖的建议描述了一个为期5年的培训计划，在学术心血管医学的职业发展。主要研究者（PI）完成了博士后基础科学研究奖学金（2005-2008年），并于2011年7月加入布莱根妇女医院/哈佛医学院（BWH/HMS）心血管医学部。PI继续进行密集的研究计划（2010年-），旨在促进心血管疾病独立科学家的职业生涯。拟定的研究计划为PI提供了细胞生物学、液相色谱-质谱（LC-MS）和体内心肺血流动力学评估方面的严格经验。Joseph Loscalzo博士和Jane Leopold博士将在此期间指导PI的科学发展。Loscalzo博士是氧化还原生物学和肺动脉高压（PAH）领域的国际公认领导者，在成功指导年轻的医生科学家方面拥有丰富的经验。Leopold博士是医学（HMS）副教授，也是醛固酮（ALDO）介导的血管功能障碍领域的专家，在指导方面有着良好的记录。该提案侧重于氧化还原生物化学，以研究ALDO对PAH肺血管功能障碍发展的贡献;因此，该提案的共同指导仅适用于PI。Loscalzo/Leopold心血管研究实验室（BWH/HMS）是该项目的所在地，拥有必要的资源，确保PI成功实现学术医学的独立性。 PAH是一种致死性疾病，其特征为肺血管内皮活性氧（ROS）形成增加，生物可利用的一氧化氮（NO）水平降低，和肺内皮依赖性血管舒张受损。ROS介导的参与NO信号传导的功能必需蛋白半胱氨酰硫醇的氧化还原状态的修饰可能影响 对血管张力不利在人肺动脉内皮细胞（HPAECs）中，内皮素- B（ET B）受体Cys 405与ET B激活内皮一氧化氮合酶以产生NO功能相关。在这里，我们提供了新的证据来证明体内PAH中存在hyperALDO，其增加HPAEC中的ROS水平以诱导ETB二硫键/次磺酸形成并减少ETB依赖性NO合成。因此，该建议的中心假设是：在PAH中，通过高ALDO诱导的ROS形成对Cys 405的氧化修饰充当分子“开关”以破坏ETB依赖性NO生成并损害肺血管舒张。具体目标是：（1）研究ALDO诱导的ETB半胱氨酰巯基氧化修饰的体外功能作用;（2a）确定体内高ALDO对PAH中肺血管反应性受损的贡献;和（2b）研究体内ALDO拮抗PAH的治疗作用。我们将使用两种不同的大鼠体内PAH模型和LC-MS来鉴定ETB半胱氨酰硫醇氧化产物。这些研究旨在确定PAH和其他具有相似病理生物学的血管疾病的新治疗靶点。

DESCRIPTION (provided by applicant): The NIH Mentored Clinical Scientist Research Career Development Award proposal describes a 5-year training program for career development in academic cardiovascular medicine. The principal investigator (PI) completed a postdoctoral basic science research fellowship (2005-2008) and in July 2011 joined as Faculty in the Division of Cardiovascular Medicine at Brigham and Women's Hospital/Harvard Medical School (BWH/HMS). The PI continues an intensive research program (2010- ) designed to promote a career as an independent scientist in cardiovascular diseases. The proposed research plan affords the PI rigorous experience in cell biology, liquid chromatography-mass spectrometry (LC-MS), and cardiopulmonary hemodynamic assessment in vivo. Drs. Joseph Loscalzo and Jane Leopold will mentor the PI's scientific development during this period. Dr. Loscalzo is an internationally recognized leader in the fields of redox biology and pulmonary arterial hypertension (PAH), who has vast experience in mentoring successfully young physician-scientists. Dr. Leopold is an Associate Professor of Medicine (HMS) and an expert in the field of aldosterone (ALDO)-mediated vascular dysfunction, with a strong track record in mentorship. This proposal focuses on redox biochemistry to investigate the contribution of ALDO to the development of pulmonary vascular dysfunction in PAH; thus, co-mentorship for this proposal is uniquely suited for the PI. The Loscalzo/Leopold Cardiovascular Research Laboratory (BWH/HMS) is the site for this project and an excellent environment with the necessary resources to ensure the PI's success for achieving independence in academic medicine. PAH is a fatal disease characterized by increased pulmonary vascular endothelial reactive oxygen species (ROS) formation, decreased levels of bioavailable nitric oxide (NO.), and impaired pulmonary endothelium-dependent vasodilation. ROS-mediated modifications in the redox state of functionally essential protein cysteinyl thiols involved in NO. signaling may affect adversely vascular tone. In human pulmonary artery endothelial cells (HPAECs), endothelin- B (ETB) receptor Cys405 is functionally linked to activation of endothelial nitric oxide synthase by ETB to generate NO.. Here, we provide novel evidence to demonstrate that hyperALDO is present in PAH in vivo, which increases ROS levels in HPAECs to induce ETB disulfide bond/sulfenic acid formation and decrease ETB-dependent NO. synthesis. Thus, the central hypothesis of this proposal is that: In PAH, oxidative modification of Cys405 by hyperALDO- induced ROS formation acts as a molecular "switch" to disrupt ETB-dependent NO. generation and impair pulmonary vasodilation. The specific aims are: (1) investigate the functional effects of ALDO-induced ETB cysteinyl thiol oxidative modification(s) in vitro; (2a) determine the contribution of hyperALDO to impaired pulmonary vascular reactivity in PAH in vivo; and (2b) investigate the therapeutic effects of ALDO antagonism for PAH in vivo. We will use two different models of PAH in rats in vivo and LC-MS to identify ETB cysteinyl thiol oxidation product(s). These studies aim to identify novel treatment targets for PAH and other vascular diseases with similar pathobiology.