DNA methylation of extracellular superoxide dismutase in pulmonary hypertension

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，一个关键因素，有助于 包括PAH在内的血管疾病的发病机制是活性氧的增加，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 胞外超氧化物歧化酶（EC-SOD）是对抗胞外O2-的唯一酶防御。EC-sod 在正常情况下，EC-SOD是血管中表达最高的SOD同工型， 在肺或血管损伤的动物模型和几种人类疾病中， 一项研究显示8例IPAH患者支气管中EC-SOD蛋白降低。增强EC- SOD活性在转基因小鼠或腺病毒介导的肺血管重建中的作用 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相邻的胞嘧啶（CpG岛）。新数据 鉴定了EC-SOD启动子内可以甲基化的CpG岛，并表明超甲基化 EC-SOD启动子抑制EC-SOD在人癌细胞中的转录，有助于增强肿瘤细胞的增殖。 细胞增殖基于这些数据，我们推测EC-SOD启动子的DNA甲基化可能与EC-SOD基因启动子的甲基化有关。 介导这种保护性基因的沉默，并有助于特发性肺动脉高压的发病机制。 动脉高血压目标1将利用在 IPAH患者、因其他原因导致PAH的疾病特异性对照或患者的器官移植 以确定EC-SOD基因和蛋白以及酶活性是否在 检测EC-SOD表达对肺动脉平滑肌细胞增殖的影响。具体 然后目标2将使用亚硫酸氢盐基因组测序来测试肺动脉中EC-SOD启动子是否存在 来自患有IPAH的人的组织和肺动脉平滑肌细胞是高甲基化的，并且如果逆转 甲基化恢复EC-SOD表达和正常细胞增殖。用于此的组织和细胞样本 研究将通过肺研究者的努力进行采购和处理。 高血压突破倡议。我们的研究结果将作为后续研究的有力的初步数据。 提交一份全面的RO 1申请，调查IPAH中EC-SOD的调节。长 长期的目标是建立新的途径，在调节这种关键的抗氧化酶， 血管壁，确定细胞外超氧化物在增殖、炎症和纤维化中的作用，从而导致 肺血管重塑，并提供了新的治疗工具，以改善这种治疗的基本原理 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.