DNA methylation of extracellular superoxide dismutase in pulmonary hypertension

肺动脉高压细胞外超氧化物歧化酶 DNA 甲基化

• 批准号: 8335465
• 负责人: Eva S. Nozik
• 金额: $ 7.65万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-23 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8335465
• 关键词: Address; Adult; Affect; Animal Model; Antioxidants; Attenuated; Azacitidine; Biology; Blood Vessels; Bronchi; Cell Proliferation; Cells; Child; CpG Islands; Cytosine; DNA Methylation; Data; Development; Diagnosis; Disease; Disease Progression; Enzymes; Epigenetic Process; Event; Fibrosis; Foundations; Freezing; Gene Delivery; Gene Expression; Gene Proteins; Genes; Genetic Transcription; Genomics; Goals; Guanosine; Human; Human Development; Hypermethylation; Hypertension; Inflammation; Injury; Lead; Life; Lung; Lung Transplantation; Lung diseases; Measures; Mediating; Messenger RNA; Methylation; Modification; Molecular; National Heart, Lung, and Blood Institute; Normal Cell; Nucleotides; Organ Transplantation; Pathogenesis; Pathway interactions; Patients; Process; Promoter Regions; Protein Isoforms; Proteins; Pulmonary Circulation; Pulmonary Hypertension; Pulmonary artery structure; Reactive Oxygen Species; Regulation; Research; Research Personnel; Research Proposals; Role; Sampling; Serum; Smooth Muscle Myocytes; Superoxide Dismutase; Superoxides; Testing; Therapeutic; Time; Tissue Banking; Tissue Banks; Tissues; Transgenic Mice; Vascular Diseases; Vascular remodeling; Work; attenuation; base; bisulfite; cancer cell; demethylation; disorder control; enzyme activity; extracellular; human disease; improved; inhibitor/antagonist; neoplastic cell; novel; novel therapeutics; promoter; protein expression; pulmonary arterial hypertension; tool

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.

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，促成这一现象的一个关键因素是 包括 PAH 在内的血管疾病的发病机制是活性氧的增加，例如 超氧化物（O2-），超出抗氧化能力。血管壁中一种极其重要的抗氧化剂是 细胞外超氧化物歧化酶 (EC-SOD)，对抗细胞外 O2- 的唯一酶防御。超氧化物歧化酶 正常情况下，EC-SOD 是脉管系统中表达最高的 SOD 亚型 在肺或血管损伤以及多种人类疾病（包括 一项研究显示 8 名 IPAH 患者的支气管中 EC-SOD 蛋白减少。增强EC- 转基因小鼠中的 SOD 活性或通过腺病毒基因传递减弱肺血管重塑 和肺动脉高压。 EC-SOD 在 IPAH 中的调节尚未被研究。现在是 认识到基因表达可以通过表观遗传修饰（包括胞嘧啶甲基化）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 识别 EC-SOD 启动子内可甲基化的 CpG 岛，并表明超甲基化 EC-SOD 启动子抑制人类癌细胞中的 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 介导该保护性基因的沉默并有助于特发性肺病的发病机制 动脉高血压。目标 1 将利用当时获得的肺和肺动脉组织和血清 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 不含PAH以确定EC-SOD基因和蛋白质以及酶活性是否降低 IPAH并检测EC-SOD表达对肺动脉平滑肌细胞增殖的影响。具体的 目标2随后将使用亚硫酸氢盐基因组测序来测试EC-SOD启动子是否存在于肺动脉中 患有 IPAH 的人的组织和肺动脉平滑肌细胞是高甲基化的，如果逆转 甲基化恢复 EC-SOD 表达和正常细胞增殖。本实验中使用的组织和细胞样本 该研究将通过肺科研究人员的努力获得和处理 高血压突破计划。我们的研究结果将为后续研究提供有力的初步数据 提交一份全面的 RO1 申请，调查 IPAH 中 EC-SOD 的监管。长的 长期目标是建立在调节这种关键抗氧化酶方面重要的新途径 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，并为改善肺血管重塑治疗的新型治疗工具提供理论依据 影响儿童和成人的致命疾病。