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）是一种影响儿童和成人的危及生命的肺循环疾病。越来越多的证据表明，导致血管疾病（包括多环芳烃）发病的一个关键因素是活性氧的增加，如超氧化物（O2-），超过抗氧化能力。血管壁上一种至关重要的抗氧化剂是细胞外超氧化物歧化酶（EC-SOD），它是抵抗细胞外O2-的唯一酶。正常情况下，EC-SOD是血管中表达量最高的SOD异构体，在肺或血管损伤动物模型和几种人类疾病中，EC-SOD表达降低，其中一项研究显示8例IPAH患者支气管中EC-SOD蛋白表达降低。通过转基因小鼠或通过腺病毒基因传递增强EC- SOD活性可减轻肺血管重构和肺动脉高压。尚未研究EC-SOD在IPAH中的调控作用。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，特别是鸟苷核苷酸附近的胞嘧啶（CpG岛）。新数据发现EC-SOD启动子内的CpG岛可以甲基化，并表明EC-SOD启动子的超甲基化抑制人类癌细胞中EC-SOD的转录，有助于增强肿瘤细胞的增殖。基于这些数据，我们假设EC-SOD启动子的DNA甲基化介导了这一保护性基因的沉默，并参与了特发性肺动脉高压的发病机制。Aim 1将利用器官移植时获取的IPAH患者、其他原因导致PAH的疾病特异性对照者、非PAH患者的肺和肺动脉组织及血清，确定IPAH患者EC-SOD基因、蛋白及酶活性是否降低，并检测EC-SOD表达对肺动脉平滑肌细胞增殖的影响。随后，Specific Aim 2将使用亚硫酸盐基因组测序来检测IPAH患者肺动脉组织和肺动脉平滑肌细胞中的EC-SOD启动子是否超甲基化，以及甲基化逆转是否能恢复EC-SOD的表达和正常的细胞增殖。本研究中使用的组织和细胞样本将通过肺动脉高压突破倡议研究人员的努力获得和处理。我们的研究结果将为随后提交全面的RO1申请提供强有力的初步数据，以调查EC-SOD在IPAH中的调节作用。长期目标是建立血管壁中这种关键抗氧化酶调控的重要新途径，确定细胞外超氧化物在促进肺血管重塑的增殖、炎症和纤维化中的作用，并为改善这种影响儿童和成人的致命疾病的治疗提供新的治疗工具的基础。

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)