Regulation of extracellular superoxide dismutase in human pulmonary arterial hype

描述（由申请人提供）：特发性肺动脉高压（IPAH）是一种危及生命的肺循环疾病，影响儿童和成人。越来越多的证据表明，导致血管疾病（包括PAH）发病的一个关键因素是活性氧（如超氧化物（O2-））的增加，超过了抗氧化能力。血管壁中一种至关重要的抗氧化剂是细胞外超氧化物歧化酶（EC-SOD），它是对抗细胞外O2-的唯一酶防御。EC-SOD是在正常条件下血管系统中表达最高的SOD同种型，并且在肺或血管损伤的动物模型和几种人类疾病中EC-SOD表达降低，包括一项显示8名IPAH患者支气管中EC-SOD蛋白降低的研究。在转基因小鼠中或通过腺病毒基因递送增强EC-SOD活性可减轻肺血管重构和肺动脉高压。EC-SOD在IPAH中的调节尚未被研究。现在认识到，基因表达可以通过表观遗传修饰来调节，包括启动子区域内的胞嘧啶甲基化，特别是与鸟苷核苷酸相邻的胞嘧啶（CpG岛）。新的数据确定了EC-SOD启动子内可以甲基化的CpG岛，并表明EC-SOD启动子的超甲基化抑制了人类癌细胞中的EC-SOD转录，有助于增强肿瘤细胞增殖。基于这些数据，我们假设EC-SOD启动子的DNA甲基化介导了该保护基因的沉默，并有助于特发性肺动脉高压的发病机制。目的1将利用IPAH患者、其他原因所致PAH的疾病特异性对照或非PAH患者器官移植时获得的肺和肺动脉组织和血清，确定IPAH中EC-SOD基因和蛋白以及酶活性是否降低，并检测EC-SOD表达对肺动脉平滑肌细胞增殖的影响。然后，特异性目标2将使用亚硫酸氢盐基因组测序来测试来自患有IPAH的人的肺动脉组织和肺动脉平滑肌细胞中的EC-SOD启动子是否过度甲基化，以及甲基化的逆转是否恢复EC-SOD表达和正常细胞增殖。本研究中使用的组织和细胞样本将通过肺动脉高压突破计划研究者的努力获得和处理。我们的研究结果将作为强有力的初步数据，为随后提交的全面RO 1申请调查EC-SOD在IPAH中的调节。长期目标是建立在血管壁中调节这种关键抗氧化酶的新途径，确定细胞外超氧化物在增殖，炎症和纤维化中的作用，从而促进肺血管重塑，并为新型治疗工具提供理论基础，以改善这种影响儿童和成人的致命疾病的治疗。 公共卫生相关性：这项研究计划将探讨DNA甲基化是否调节特发性或家族性肺动脉高压患者肺动脉中一种至关重要的抗氧化酶细胞外超氧化物歧化酶的转录，DNA甲基化越来越被认为是调节基因表达的关键表观遗传机制。这项工作将提供一个坚实的基础，以了解机制，负责损失这种重要的抗氧化剂在人类肺动脉高压，并提供了一个合理的新的治疗策略，以改善治疗这种致命的疾病影响儿童和成人。(End摘要）

DESCRIPTION (provided by applicant): Idiopathic pulmonary arterial hypertension (IPAH) is a life-threatening disease of the pulmonary circulation affecting children and adults. Accumulating evidence indicates that one key factor that contributes to the pathogenesis of vascular diseases, including PAH, is an increase in reactive oxygen species, such as superoxide (O2-), that exceed antioxidant capabilities. One critically important antioxidant in the vessel wall is extracellular superoxide dismutase (EC-SOD), the sole enzymatic defense against extracellular O2-. EC-SOD is the most highly expressed SOD isoform in the vasculature under normal conditions, and EC-SOD expression is decreased in animal models of lung or vascular injury and several human diseases, including one study showing a decrease in EC-SOD protein in the bronchus of 8 patients with IPAH. Augmenting EC- SOD activity in transgenic mice or through adenoviral gene delivery attenuates pulmonary vascular remodeling and pulmonary hypertension. The regulation of EC-SOD in IPAH has not been investigated. It is now recognized that gene expression can be regulated by epigenetic modifications including cytosine methylation within the promoter region, specifically cytosines adjacent to guanosine nucleotides (CpG islands). New data identify CpG islands within the EC-SOD promoter that can be methylated, and indicate that hypermethylation of the EC-SOD promoter inhibits EC-SOD transcription in human cancer cells, contributing to enhanced tumor cell proliferation. Based on these data, we hypothesize that DNA methylation of the EC-SOD promoter mediates silencing of this protective gene and contributes to the pathogenesis of idiopathic pulmonary arterial hypertension. Aim 1 will utilize lung and pulmonary artery tissue and serum procured at the time of organ transplantation in patients with IPAH, disease-specific controls with PAH due to other causes, or patients without PAH to determine whether EC-SOD gene and protein as well as enzyme activity are decreased in IPAH and test the impact of EC-SOD expression on pulmonary artery smooth muscle cell proliferation. Specific Aim 2 will then use bisulfite genomic sequencing to test whether the EC-SOD promoter in pulmonary artery tissue and pulmonary artery smooth muscle cells from humans with IPAH is hypermethylated, and if reversal of methylation restores EC-SOD expression and normal cell proliferation. The tissue and cell samples used in this study will have been procured and processed through the efforts of the investigators of the Pulmonary Hypertension Breakthrough Initiative. Our findings will serve as strong preliminary data for the subsequent submission of a comprehensive RO1 application investigating the regulation of EC-SOD in IPAH. The long term goals are to establish new pathways important in the regulation of this pivotal antioxidant enzyme in the vessel wall, identify the role of extracellular superoxide in proliferation, inflammation and fibrosis contributing to pulmonary vascular remodeling, and provide a rationale for novel therapeutic tools to improve treatment of this lethal disease affecting children and adults. PUBLIC HEALTH RELEVANCE: This research proposal examines whether DNA methylation, increasingly recognized as a key epigenetic mechanism to regulate gene expression, regulates the transcription of a critically important antioxidant enzyme, extracellular superoxide dismutase, in the pulmonary artery of patients with idiopathic or familial pulmonary artery hypertension. This work will provide a strong foundation to understand the mechanisms responsible for loss of this essential antioxidant in human pulmonary arterial hypertension and provide a rationale for novel therapeutic strategies to improve treatment of this lethal disease affecting children and adults. (End of Abstract)