DNA methylation of extracellular superoxide dismutase in pulmonary hypertension

摘要特发性肺动脉高压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，导致经济衰退的一个关键因素 血管疾病的发病机制，包括PAH，是活性氧物种的增加，如 超氧化物(O2-)，超过抗氧化能力。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)，唯一的酶防御细胞外O2-。EC-SOD 是正常情况下血管系统中表达最高的超氧化物歧化酶亚型，而EC-超氧化物歧化酶 在肺或血管损伤的动物模型和几种人类疾病中表达减少，包括 一项研究显示，8例IPAH患者的支气管EC-SOD蛋白减少。增强EC- 转基因小鼠体内或通过腺病毒基因传递的超氧化物歧化酶活性减轻肺血管重构 和肺动脉高压。EC-SOD在IPAH中的调节作用尚未见报道。现在是时候了 认识到基因表达可以通过包括胞嘧啶甲基化在内的表观遗传修饰来调节 在启动子区域内，特别是与鸟苷核苷酸(CpG岛)相邻的胞嘧啶。新数据 确定EC-SOD启动子中可以甲基化的CpG岛，并表明高甲基化 EC-SOD启动子的缺失抑制了人类癌细胞中EC-SOD的转录，促进了肿瘤的生长 细胞增殖。基于这些数据，我们假设EC-SOD启动子的DNA甲基化 介导这一保护基因的沉默，并参与特发性肺疾病的发病机制 动脉高血压。目标1将利用在以下时间获得的肺和肺动脉组织和血清 IPAH患者的器官移植，其他原因引起的PAH的疾病特异性对照，或患者 在没有PAH的情况下确定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。特定的 然后，AIM 2将使用亚硫酸氢盐基因组测序来测试EC-SOD启动子在肺动脉中是否 来自患有IPAH的人的组织和肺动脉平滑肌细胞高甲基化，如果逆转 甲基化可以恢复EC-SOD的表达和正常的细胞增殖。在这个实验中使用的组织和细胞样本 这项研究将通过肺部调查人员的努力获得和处理 高血压突破性倡议。我们的发现将作为后续研究的强有力的初步数据 提交一份全面的RO1申请，调查Ipah对EC-SOD的调节。《长河》 学期的目标是建立新的途径，在调节这种关键的抗氧化酶在 血管壁，确定细胞外超氧化物在促进增殖、炎症和纤维化中的作用 肺血管重塑，并为新的治疗手段改善治疗提供了理论基础 影响儿童和成人的致命疾病。

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.