O2 Sensing in Hypoxic Pulmonary Vasoconstriction

描述(由申请人提供)：缺氧性肺血管收缩(HPV)有助于优化肺气体交换，但在缺氧性肺部疾病中它会导致肺动脉高压。已经出现了相反的模型来解释HPV中O2感应的潜在机制。1提出，低氧减少了活性氧(ROS)的产生，使细胞质处于更低的状态。另一种观点认为，低氧刺激ROS，在胞浆中产生氧化信号。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。在目标1中，我们将使用新的氧化还原依赖的荧光共振能量转移(HSP-FRET)和RoGFP1探针来评估常氧和低氧肺血管细胞的氧化还原。我们假设ROS的增加来自线粒体电子传输链(ETC)。我们将针对抗氧化酶在线粒体基质或胞浆中的过度表达来确定哪些隔室参与氧化还原信号。目的2通过使用短发夹状干扰RNA(ShRNA)抑制关键的ETC亚基的表达，确定哪些ETC复合体有助于ROS的产生。我们预测，当ROS产生所需的亚基被抑制时，氧化信号将被减弱。目的研究肺动脉(PA)心肌细胞线粒体ROS生成与功能反应(细胞内钙离子浓度升高)和肺动脉内皮细胞(缺氧诱导因子-1活化和内皮素-1表达增加)之间的关系。我们预测，抑制ROS信号从线粒体到胞浆的传播(通过靶向过表达抗氧化酶)或阻止它们的产生(通过shRNA抑制关键的ETC亚基)将在两种类型的细胞中消除对缺氧的功能反应。总的来说，这些研究将测试在PA肌细胞和内皮细胞中是否有共同的氧气感知机制发挥作用，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.