O2 Sensing in Hypoxic Pulmonary Vasoconstriction

缺氧肺血管收缩中的 O2 传感

• 批准号: 7074609
• 负责人: PAUL T SCHUMACKER
• 金额: $ 32.16万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7074609
• 关键词: RNA interference; cellular respiration; electron transport; endothelin; fluorescence resonance energy transfer; free radical oxygen; green fluorescent proteins; hypoxia inducible factor 1; laboratory rat; lung ischemia /hypoxia; mitochondria; oxidation reduction reaction; oxygen tension; pulmonary artery; respiratory circulation; tissue /cell culture; vascular endothelium; vascular smooth muscle; vasoconstriction

DESCRIPTION (provided by applicant): Hypoxic pulmonary vasoconstriction (HPV) helps to optimize lung gas exchange, but it contributes to pulmonary hypertension in hypoxic lung disease. 2 opposing models have emerged to explain the underlying mechanism of O2 sensing in HPV. 1 proposes that hypoxia decreases reactive oxygen species (ROS) generation, shifting the cytosol to a more reduced state. The other proposes that hypoxia stimulates ROS, generating an oxidant signal in the cytosol. Resolution of this debate has been hindered by a lack of tools to assess intracellular redox. In Aim 1 we will use novel redox-dependant Fluorescence Resonance Energy Transfer (HSP-FRET) and RoGFP1 probes to assess redox in normoxic and hypoxic pulmonary vascular cells. We hypothesize that increased ROS come from the mitochondrial electron transport chain (ETC). We will target overexpression of antioxidant enzymes to mitochondrial matrix or the cytosol to determine which compartments participate in redox signaling. Aim 2 will determine which ETC complexes contribute to ROS generation by using short hairpin interfering RNA (shRNA) to suppress expression of critical ETC subunits. We predict that oxidant signals will be attenuated when the subunits required for ROS generation are suppressed. Aim 3 will test the relationship between mitochondrial ROS generation and functional responses to hypoxia in pulmonary artery (PA) myocytes (increase in cytosolic Ca2+) and in PA endothelial cells (increased activation of Hypoxia Inducible Factor-1 and increased expression of endothelin-1). We predict that inhibiting the propagation of ROS signals from mitochondria to cytosol (by targeted overexpression of antioxidant enzymes) or preventing their generation (by shRNA suppression of critical ETC subunits) will abrogate the functional responses to hypoxia in both cell types. Collectively, these studies will test whether a common O2 sensing mechanism functions in PA myocytes and endothelial cells to trigger their diverse responses in HPV.

描述（由申请人提供）：缺氧性肺血管收缩（HPV）有助于优化肺部气体交换，但在缺氧性肺病中会导致肺动脉高压。已经出现了两种相反的模型来解释 HPV 中 O2 感应的基本机制。图 1 表明缺氧会减少活性氧 (ROS) 的产生，从而将细胞质转变为更还原的状态。另一种观点认为，缺氧会刺激活性氧，在细胞质中产生氧化信号。由于缺乏评估细胞内氧化还原的工具，这一争论的解决受到阻碍。在目标 1 中，我们将使用新型氧化还原依赖性荧光共振能量转移 (HSP-FRET) 和 RoGFP1 探针来评估常氧和缺氧肺血管细胞中的氧化还原。我们假设增加的 ROS 来自线粒体电子传递链 (ETC)。我们将抗氧化酶的过度表达靶向线粒体基质或细胞质，以确定哪些区室参与氧化还原信号传导。目标 2 将通过使用短发夹干扰 RNA (shRNA) 抑制关键 ETC 亚基的表达来确定哪些 ETC 复合物有助于 ROS 生成。我们预测，当 ROS 生成所需的亚基受到抑制时，氧化剂信号将会减弱。目标 3 将测试线粒体 ROS 生成与肺动脉 (PA) 肌细胞（胞浆 Ca2+ 增加）和 PA 内皮细胞（缺氧诱导因子 1 激活增加和内皮素 1 表达增加）对缺氧的功能反应之间的关系。我们预测，抑制 ROS 信号从线粒体到细胞质的传播（通过抗氧化酶的靶向过度表达）或阻止其产生（通过 shRNA 抑制关键 ETC 亚基）将消除两种细胞类型对缺氧的功能反应。总的来说，这些研究将测试一种常见的 O2 传感机制是否在 PA 肌细胞和内皮细胞中发挥作用，以触发它们在 HPV 中的不同反应。