ecSOD derived peroxide in pulmonary adaptation to hypoxia

描述(申请人提供)：该项目的中心假设是，细胞外超氧化物歧化酶(EcSOD)活性在控制慢性缺氧引起的肺动脉高压的表达中具有潜在的有益的重要作用，它通过产生过氧化氢并刺激cGMP蛋白激酶(PKG)来实现。推测ECSOD表达增加是血红素氧合酶-1(HO-1)升高在肺动脉高压中发挥保护作用的过程。虽然有大量证据表明HO-1和EcSOD在减轻几种动物模型中的肺动脉高压发展方面是有益的，但以前的研究似乎没有考虑到这一应用中所研究的机制。我们最近的研究证明，过氧化氢可以通过硫醇氧化介导的PKG亚单位二聚化机制促进PKG的激活，该机制参与了肺动脉对细胞外过氧化氢的松弛和对急性低氧的反应。初步数据提供证据表明，体内慢性低氧通过过氧化氢酶可逆的过程促进PKG的二聚化激活和抑制在分离的小鼠肺动脉中的力产生。这些研究建议以ECSOD或HO-1缺陷的小鼠为重点，研究它们在控制慢性低氧暴露小鼠肺血管功能适应中的作用。ECSOD或HO-1是一种PKG敲打小鼠，经过修饰以防止过氧化氢对PKG的硫醇氧化激活，结合有益的肺动脉高压治疗增加ECSOD或HO-1的表达并促进PKG二聚体的形成。目标1中的研究明确了EcSOD表达的变化对参与控制血管功能和重塑的过程的影响，这些过程涉及通过利用EcSOD过表达或缺乏的小鼠参与低氧诱导的肺动脉高压的发展和逆转。目的2利用PKG二聚化修饰小鼠和内皮型一氧化氮合酶(ENOS)缺陷小鼠，通过增加对PKG的过氧化调节和/或通过增加超氧化物歧化清除来增加一氧化氮(NO)，重点阐明EcSOD的表达如何发挥作用。目标3的研究重点是利用HO-1或EcSOD缺陷的小鼠，确定EcSOD表达的变化对HO-1保护作用的影响，以及诱导HO-1和EcSOD的治疗。研究将包括心肺功能的回声-多普勒血流分析，离体肺动脉的机械功能研究，与PKG相关的信号研究以及影响氧化还原调节的过程-src激酶介导的STAT-3磷酸化促进miR-204的表达，这是一个主要的基因调控系统，控制着肺动脉高压中平滑肌重塑的发展和逆转。这些研究应该提供明确的新的机制信息，说明慢性低氧如何促进肺动脉高压的发展，这可能有助于治疗COPD和睡眠呼吸暂停等疾病的进展。

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.