ecSOD derived peroxide in pulmonary adaptation to hypoxia

描述（由申请人提供）：本项目的中心假设是细胞外超氧化物歧化酶（ecSOD）活性通过过氧化氢的产生及其对cGMP蛋白激酶（PKG）的刺激，在控制慢性缺氧引起的肺动脉高压表达方面具有潜在的有益作用。也有假设认为，ecSOD的表达增加有助于血红素氧合酶-1 （HO-1）升高对肺动脉高压的保护作用。虽然在一些动物模型中有大量证据表明HO-1和ecSOD有利于减轻肺动脉高压的发展，但以前的研究似乎没有考虑到在这一应用中所研究的机制。我们最近的研究表明，过氧化氢可以通过硫醇氧化介导的PKG亚基二聚化机制促进PKG的活化，该机制参与了肺动脉对细胞外过氧化氢的松弛和对急性缺氧的反应。初步数据表明，体内慢性缺氧可促进PKG二聚化激活，并通过过氧化氢酶可逆的过程抑制离体小鼠肺动脉的力产生。这些研究的重点是利用缺乏ecSOD或HO-1 （PKG敲入小鼠被修饰以防止硫醇氧化激活PKG）的小鼠，以及有益的肺动脉高压治疗（增加ecSOD或HO-1表达并促进PKG二聚体）来研究它们在控制小鼠暴露于慢性缺氧引起的肺血管功能适应中的作用。Aim 1中的研究通过使用ecSOD过表达或缺乏的小鼠，确定了ecSOD表达变化对参与缺氧诱导的肺动脉高压发生和逆转的血管功能和重塑控制过程的影响。目的2侧重于定义ecSOD表达如何通过增加PKG的过氧化物调节和/或通过增加超氧化物清除来增加一氧化氮（NO），利用PKG敲入小鼠（具有阻止PKG二聚化的修饰）和内皮型一氧化氮合酶（eNOS）缺乏的小鼠。Aim 3的研究重点是利用缺乏HO-1或ecSOD的小鼠以及诱导HO-1和ecSOD的治疗方法，确定ecSOD表达变化对HO-1保护作用的影响。研究将包括心肺功能的echo -多普勒血流分析，孤立肺动脉的机制功能研究，PKG相关的信号研究以及影响氧化还原调控的过程src激酶介导的STAT-3磷酸化促进miR-204的表达，miR-204是控制肺动脉高压平滑肌重塑发展和逆转的主控基因调控系统。这些研究应该为慢性缺氧如何促进肺动脉高压的发展提供明确的新机制信息，这可能对治疗COPD和睡眠呼吸暂停等疾病的进展有益。

DESCRIPTION (provided by applicant): The central hypothesis of this project is that extracellular SOD (ecSOD) activity has potentially beneficial important roles in controlling the expression of pulmonary hypertension resulting from exposure to chronic hypoxia through the generation of hydrogen peroxide and its stimulation of cGMP protein kinase (PKG). It is also hypothesized that increased expression of ecSOD is a process that contributes to the protective effects of heme oxygenase-1 (HO-1) elevation in pulmonary hypertension. While there is substantial evidence for HO-1 and ecSOD being beneficial in attenuating the development of pulmonary hypertension in several animal models, previous studies do not appear to have considered the mechanisms investigated in this application. Our recent studies document that peroxide can promote activation of PKG by a thiol oxidation-mediated PKG subunit dimerization mechanism which participates in relaxation of pulmonary arteries to extracellular hydrogen peroxide and responses to acute hypoxia. Preliminary data provide evidence that chronic hypoxia in vivo promotes both a dimerization activation of PKG and a suppression of force generation in isolated mouse pulmonary arteries by a process that is reversible by catalase. The studies proposed focus on using mice deficient in ecSOD or HO-1, a PKG knockin mouse that is modified to prevent thiol oxidation-activation of PKG by peroxide together with beneficial pulmonary hypertension therapies that increase ecSOD or HO-1 expression and promote PKG dimerization to investigate their roles in controlling the adaptation of pulmonary vascular function caused by exposure of mice to chronic hypoxia. Studies in Aim 1 define the influence of changes in ecSOD expression on processes contributing to the control of vascular function and remodeling involved in the development and reversal of hypoxia-induced pulmonary hypertension by utilizing mice either overexpressing or deficient in ecSOD. Aim 2 focuses on defining how ecSOD expression functions through either increased peroxide regulation of PKG and/or through increased superoxide scavenging to increase nitric oxide (NO) utilizing PKG knockin mice which have a modification preventing PKG dimerization and mice deficient in endothelial nitric oxide synthase (eNOS). The focus of studies in Aim 3 is defining the influence of changes in ecSOD expression on the protective effects of HO-1 utilizing mice deficient in HO-1 or ecSOD and therapies to induce HO-1 and ecSOD. Studies will include ECHO-Doppler flow analysis of cardiopulmonary function, mechanistic functional studies in isolated pulmonary arteries, signaling studies related to PKG and processes influencing the redox regulation src kinase-mediated STAT-3 phosphorylation promoting expression of miR-204, a master gene regulatory system controlling the development and reversal of smooth muscle remodeling in pulmonary hypertension. These studies should provide definitive new mechanistic information on how the effects of chronic hypoxia promote the development of pulmonary hypertension, which could be beneficial in treating the progression of diseases such as COPD and sleep apnea.