Cholesterol Regulation of EGFR-dependent Vasoconstriction in Chronic Hypoxia-induced Pulmonary Hypertension

项目摘要 慢性低氧(CH)所致的肺动脉高压(PH)是发病率和 慢性阻塞性肺疾病患者的死亡率。人们普遍认为，血管收缩 是PH的关键调节因子，尽管所涉及的机制知之甚少。我们之前的研究 已经证明，脑出血后血管收缩敏感性的增强涉及到必要的减少 肺动脉平滑肌细胞(PASMC)膜胆固醇含量。我们还展示了 CH通过切换主要依赖钙的信号来增强血管收缩反应性 涉及表皮生长因子受体(EGFR)和 活性氧(ROS)。然而，CH降低膜胆固醇和 这如何揭开EGFR依赖的血管收缩的面纱还有待评估。 拟议的研究将调查中心假设，血管收缩刺激耦合到 脑出血后的EGFR信号通过ROS依赖的降低促进PASMC的超收缩 膜胆固醇。为了验证这一假设，方案将使用体内和体外方法，使用 从分子和单细胞成像研究到视频显微镜的各种实验准备 用CH诱导的大鼠肺动脉高压模型加压小肺动脉。 我们计划实现以下具体目标： 具体目标1：确定CH降低PASMC膜胆固醇的机制。 假设：在CH过程中ROS的产生增加可以减少膜胆固醇。 特定目标2：确定PASMC膜胆固醇降低的机制 血管收缩药敏感性。 假设：CHUNMASS对EGFR依赖的PASMC膜胆固醇降低的反应 通过调节NOX2和rac1的肺血管收缩。 申请者将沉浸在UNM血管生理学小组丰富的培训环境中 通过一个独特的、多赞助商的指导团队，将促进他在 确定ROS改变PASMC膜微环境影响细胞的新机制 在CH诱导的PH中的作用。拟议的培训计划将为申请者提供强化培训体验。 在各种新的实验方法中，他的口头和书面沟通技能的精炼，以及 职业发展培训，这将帮助他实现作为一名独立的学者的目标 肺部研究方面的内科医生兼科学家。

Project Summary Chronic Hypoxia (CH)-induced pulmonary hypertension (PH) is a significant source of morbidity and mortality in patients with chronic obstructive pulmonary diseases. It is widely recognized that vasoconstriction is a critical mediator of PH, although the mechanisms involved are poorly understood. Our previous studies have demonstrated that enhanced vasoconstrictor sensitivity following CH involves a requisite reduction in pulmonary arterial smooth muscle cell (PASMC) membrane cholesterol content. We have also demonstrated that CH augments vasoconstrictor reactivity by a switch in signaling from primarily calcium-dependent mechanisms to a Ca2+ sensitization pathway that involves the epidermal growth factor receptor (EGFR) and reactive oxygen species (ROS). However, the mechanisms by which CH decreases membrane cholesterol and how this unmasks EGFR-dependent vasoconstriction has yet to be assessed. The proposed studies will investigate the central hypothesis that coupling of vasoconstrictor stimuli to EGFR signaling following CH promotes PASMC hypercontractility through a ROS-dependent decrease in membrane cholesterol. To test this hypothesis, protocols will employ both in vivo and in vitro approaches using a variety of experimental preparations from molecular and single cell imaging studies to video-microscopy of pressurized small pulmonary arteries using a rat model of CH-induced PH. We plan to pursue the following specific aims: Specific Aim 1: Determine the mechanism by which CH decreases PASMC membrane cholesterol. Hypothesis: Elevated ROS production during CH diminishes membrane cholesterol. Specific Aim 2: Determine the mechanism by which decreased PASMC membrane cholesterol augments vasoconstrictor sensitivity following CH. Hypothesis: Decreased PASMC membrane cholesterol in response to CH unmasks EGFR-dependent pulmonary vasoconstriction through regulation of NOX2 and Rac1. The applicant will be immersed in a rich training environment in the Vascular Physiology Group at the UNM School of Medicine through a unique, multi-sponsor mentoring team that will facilitate his research training in defining novel mechanisms by which ROS alter the PASMC membrane microenvironment to affect cellular function in CH-induced PH. The proposed training plan will afford the applicant intensive training experiences in a variety of new experimental approaches, refinement of his oral and written communication skills, and professional development training that will aid him in achieving his goal as an independent, academic physician-scientist in pulmonary research.