O2 Sensing in Hypoxic Pulmonary Vasoconstriction

描述（由申请人提供）：低氧性肺血管收缩（HPV）有助于优化肺气体交换，但它有助于低氧性肺疾病的肺动脉高压。已经出现了两种相反的模型来解释HPV中O2传感的潜在机制。1提出缺氧减少了活性氧（ROS）的产生，使细胞溶质转变为更还原的状态。另一种认为缺氧刺激ROS，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，这一争论的解决一直受到阻碍。在目的1中，我们将使用新的氧化还原依赖性荧光共振能量转移（HSP-FRET）和RoGFP 1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的抗氧化酶的过表达，以确定哪些隔间参与氧化还原信号。目的2将确定哪些ETC复合物有助于ROS的产生，通过使用短发夹干扰RNA（shRNA）抑制关键ETC亚基的表达。我们预测，当ROS生成所需的亚基被抑制时，氧化剂信号将被减弱。目的3将检测肺动脉（PA）心肌细胞（胞浆Ca 2+增加）和肺动脉内皮细胞（低氧诱导因子-1的活化增加和内皮素-1的表达增加）中线粒体ROS产生与对低氧的功能反应之间的关系。我们预测，抑制ROS信号从线粒体到胞质溶胶的传播（通过抗氧化酶的靶向过表达）或防止其产生（通过关键ETC亚基的shRNA抑制）将消除两种细胞类型中对缺氧的功能反应。总的来说，这些研究将测试是否有一个共同的O2传感机制在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。

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.