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

描述(由申请人提供)：NIH指导临床科学家研究职业发展奖提案描述了一个为期5年的学术心血管医学职业发展培训计划。首席研究员(PI)完成了博士后基础科学研究奖学金(2005-2008)，并于2011年7月加入布里格姆妇女医院/哈佛医学院(BWH/HMS)心血管医学系。PI继续进行密集的研究计划(2010-)，旨在促进作为心血管疾病独立科学家的职业生涯。拟议的研究计划为PI提供了细胞生物学、液质联用(LC-MS)和体内心肺血流动力学评估方面的严格经验。约瑟夫·洛斯卡尔佐博士和简·利奥波德博士将在此期间指导国际和平组织的科学发展。洛斯卡尔佐博士在氧化还原生物学和肺动脉高压(PAH)领域是国际公认的领导者，他在成功指导年轻的内科科学家方面拥有丰富的经验。利奥波德博士是医学副教授(HMS)，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。这项建议侧重于氧化还原生物化学，以调查ALDO在PAH肺血管功能障碍发展中的作用；因此，这项建议的共同指导是唯一适合于PI的。洛斯卡佐/利奥波德心血管研究实验室(BWH/HMS)是该项目的所在地，拥有确保PI成功实现学术医学独立的良好环境和必要资源。 PAH是一种致命性疾病，其特征是肺血管内皮细胞活性氧物种(ROS)生成增加，生物可利用的一氧化氮(NO)水平降低，肺内皮细胞依赖性血管扩张受损。氧化还原功能必需蛋白半胱氨酸硫醇参与NO氧化还原状态的ROS介导的修饰。信令可能会影响 不利的血管紧张度。在人肺动脉内皮细胞(HPAECs)中，内皮素-B(ETB)受体Cys405在功能上与ETB激活内皮型一氧化氮合酶产生NO有关。在这里，我们提供了新的证据来证明在体内PAH中存在高ALDO，它增加了HPAECs中的ROS水平，从而诱导ETB二硫键/磺酸的形成，并降低了ETB依赖的NO。综合。因此，这一建议的中心假设是：在PAH中，由高ALDO诱导的ROS形成对Cys405的氧化修饰起到了分子“开关”的作用，破坏了ETB依赖的NO。产生并损害肺血管扩张。本研究的具体目的是：(1)体外研究羟醛诱导的ETB半胱氨酸硫醇氧化修饰(S)的功能作用；(2a)确定高醛脱氧作用在体内PAH肺血管反应性受损中的作用；(2b)观察Aldo拮抗剂对PAH的治疗作用。我们将使用两种不同的大鼠体内多环芳烃模型和LC-MS来鉴定ETB半胱氨酰硫醇氧化产物(S)。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.