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）有助于优化肺气交换，但有助于低氧肺部疾病中的肺动脉高压。 2个相反的模型已出现，以解释HPV中O2传感的潜在机制。 1提出，缺氧会降低活性氧（ROS）的产生，将细胞质转移到更降低的状态。另一个提出缺氧会刺激ROS，从而在细胞质中产生氧化剂信号。缺乏评估细胞内氧化还原的工具，阻碍了这一辩论的解决。在AIM 1中，我们将使用新型的氧化还原依赖性荧光共振能量转移（HSP-FRET）和ROGFP1探针来评估常氧和低氧肺血管细胞中的氧化还原。我们假设ROS增加来自线粒体电子传输链（ETC）。我们将靶向抗氧化酶的过表达对线粒体基质或细胞质，以确定哪些隔室参与氧化还原信号传导。 AIM 2将通过使用短发夹干扰RNA（shRNA）来抑制关键ECT亚基的表达来确定哪些ETC络合物有助于ROS产生。我们预测，当抑制ROS生成所需的亚基时，氧化信号将被减弱。 AIM 3将测试线粒体ROS的产生与对肺动脉（PA）肌细胞缺氧的功能反应之间的关系（胞质CA2+的增加）和PA内皮细胞（增加了缺氧诱导因子-1的激活增加，以及内皮素-1的表达增加）。我们预测，抑制线粒体向细胞质的ROS信号传播（通过靶向抗氧化剂酶的过表达）或防止其产生（通过shRNA抑制临界等亚基）将消除两种细胞类型中缺氧的功能性反应。总的来说，这些研究将测试PA肌细胞和内皮细胞中常见的O2感应机制是否在HPV中触发其多种反应。