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）：低氧肺血管收缩（HPV）有助于优化肺部气体交换，但它会导致低氧肺病的肺动脉高压。已经出现了两个相反的模型来解释HPV中O2感应的潜在机制。1提出缺氧减少活性氧（ROS）的产生，将细胞质转移到更还原的状态。另一种假说认为，缺氧刺激活性氧，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了阻碍。在Aim 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）。我们将针对线粒体基质或细胞质中抗氧化酶的过度表达来确定哪些室室参与氧化还原信号传导。目的2将通过使用短发夹干扰RNA （shRNA）抑制ETC关键亚基的表达来确定哪些ETC复合物有助于ROS的产生。我们预测，当生成活性氧所需的亚基被抑制时，氧化信号将被减弱。目的3将测试线粒体ROS生成与肺动脉（PA）肌细胞（胞质Ca2+增加）和肺动脉内皮细胞（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（通过靶向过表达抗氧化酶）或阻止它们的产生（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，这些研究将测试在PA肌细胞和内皮细胞中是否存在一种共同的O2感应机制，以触发它们在HPV中的不同反应。