Mechanisms of PRMT regulation of pulmonary endothelial cell function

 描述（由申请方提供）：本提案的总体目标是确定蛋白质精氨酸甲基转移酶（PRMT）（负责蛋白质精氨酸甲基化和甲基精氨酸合成的酶）在慢性缺氧诱导的肺血管疾病中观察到的肺内皮功能障碍发病机制中的作用。尽管血清中不对称二甲基精氨酸（ADMA）（一种内源性一氧化氮合酶抑制剂）水平升高已被确定为心血管疾病（包括与慢性缺氧诱导的肺动脉高压（PH）相关的心血管疾病）的独立危险因素，我们实验室的证据表明，在疾病中观察到的血浆ADMA增加可能是PRMT活性变化的生物标志物，归因于ADMA直接表现为PRMT活性的改变。在这方面，我们的初步数据支持我们的总体假设，即除了ADMA竞争性抑制eNOS活性外，通过PRMT介导的直接蛋白质甲基化事件调节eNOS-NO的产生。具体而言，我们证明，eNOS是PRMT的底物，eNOS蛋白在缺氧反应中的甲基化增加可能在内皮功能障碍中发挥关键作用，如通过降低NO产生来测量。本研究的目的是：1）明确PRMT在缺氧诱导的肺内皮功能失调中的作用; 2）确定PRMT在缺氧诱导的肺内皮功能失调中调节eNOS活性的机制; 3）确定PRMT活性失调在缺氧发病机制中的作用-诱导PH。该建议的目的将通过使用细胞，分子，生物物理，和生理学方法来表征PRMT在内皮功能中的作用以及使用体外和体内模型通过缺氧对其进行调节。这些研究的结果将提供PRMT调节内皮功能以及调节PH易感性和进展的基本机制模型。

 DESCRIPTION (provided by applicant): The overall objective of this proposal is to identify the role of protein arginine methyltransferases (PRMT), the enzymes responsible for protein-arginine methylation and methylarginine synthesis, in the pathogenesis of chronic hypoxia induced pulmonary endothelial dysfunction as seen in pulmonary vascular disease. Although increased serum levels of asymmetric dimethyl arginine (ADMA), an endogenous inhibitor of nitric oxide synthase, have been identified as an independent risk factor for cardiovascular diseases including those associated with chronic hypoxia-induced pulmonary hypertension (PH), evidence from our laboratory indicates that increased plasma ADMA observed in disease may be a biomarker for changes in PRMT activity and that many of the endothelial effects attributed to ADMA are directly manifested through altered activity of PRMT. In this regard, our preliminary data support our overall hypothesis that in addition to competitive inhibition of eNOS activity by ADMA, modulation of eNOS-NO production occurs through direct protein-methylation events mediated by PRMT. Specifically, we demonstrate that eNOS is a substrate of PRMT and that increased methylation of eNOS protein in response to hypoxia may play a critical role in endothelial dysfunction as measured by decreased NO production. The goals of the current proposal are to: 1) Define the role played by the PRMT in hypoxia induced pulmonary endothelial dysregulation; 2) Identify the mechanism(s) by which the PRMT regulates eNOS activity in hypoxia induced pulmonary endothelial dysfunction; 3) Determine the role of dysregulated PRMT activity in the pathogenesis of hypoxia-induced PH. The aims in this proposal will be accomplished through the use of a combination of cellular, molecular, biophysical, and physiological approaches to characterize the role of the PRMT in endothelial function and its regulation by hypoxia using both in vitro and in vivo models. Results from these studies will provide fundamental mechanistic models through which PRMT modulates endothelial function as well as modulates susceptibility to, and progression of, PH.