巯基一氧化氮调控小G蛋白RhoA活性在肺动脉高压中的作用及机制研究

Pulmonary hypertension is characterized by increased vascular resistance with vascular structural remodeling. At the cellular level, activation of small GTPase RhoA and its downstream signaling pathways have been shown to play an important role in each of these events. S-nitrosothiols (SNO) derived from endothelial nitric oxide synthase are known to lead to vasodilation as well as inhibition of smooth muscle cell growth. There are two cysteine residues in the RhoA GTP binding domain, nitrosylation of these critical thiols may alter the ability of RhoA for GTP binding, thus regulate RhoA function. We have made several novel observations which strongly suggest S-nitrosothiols exert some of their vascular activities via RhoA modulation, implying that these endogenous compounds oppose mechanisms dependent on RhoA activation and might prevent progression of pulmonary hypertension. The objective of this proposal is to investigate mechanisms involved in S-nitrosothiols-mediated inhibition of RhoA particularly as they relate to regulation of vascular smooth muscle relaxation and vascular remodeling. Data from these studies may establish an important but unexplored mechanism for fine tuning vascular tone by endogenous NO derivatives. Importantly these studies may also provide new therapeutic options in which S-nitrosothiols or agents that increase levels of these mediators are used to treat pulmonary hypertension.

肺动脉高压主要的病理生理改变是肺血管阻力增加和血管重构。小G蛋白RhoA对这两个环节均有不良影响，其活性在肺动脉高压中可见增高，但机制不清楚。序列分析显示RhoA在其GTP结合域有两个半胱氨酸残基，我们推测该部位巯基亚硝基化可能对RhoA与GTP结合产生影响，进而对其功能产生作用，并提出肺动脉高压时巯基一氧化氮（SNO）不足导致RhoA脱抑制这一新发病机制。本项目将在蛋白、细胞以及动物水平上，利用基因突变、基因敲除等方法，探讨SNO对RhoA蛋白的修饰作用与功能调节作用，阐明肺动脉高压时RhoA巯基亚硝基化水平的改变、巯基亚硝基化与RhoA活性之间的关系，以及调节内源性SNO水平对RhoA活性、肺动脉压力、肺血管增生重构和右心功能的影响；并在此基础上，探索外源性补充SNO抑制RhoA后对实验动物肺动脉高压的治疗作用。研究结果将为肺动脉高压发病机制提供新学说，为防治策略提供新的思路及方法。

肺动脉高压是由多种病因引起的，以肺血管内皮细胞功能失常、肺小动脉收缩、平滑肌增生重构从而导致肺血管阻力持续增高为主要特征的慢性致死性疾病。本研究探讨了SNO对RhoA蛋白的修饰作用与功能调节作用，平滑肌细胞中RhoA巯基亚硝基化水平的改变、巯基亚硝基化与RhoA活性之间的关系、对细胞张力平衡的影响，以及外源性补充SNO抑制RhoA对实验动物肺动脉高压的治疗作用。在蛋白质水平，我们证实了SNO可以通过巯基亚硝基化修饰RhoA而抑制其活性，并成功鉴定了其分子靶标。在平滑肌细胞中，我们发现SNO可以通过巯基亚硝基化修饰RhoA降低其活性，并且影响下游信号通路中MYPT1和MLC磷酸化表达，这提示CSNO可以抑制平滑肌细胞中RhoA活性，具有扩张血管的功能。我们进一步构建了大鼠肺动脉高压模型，验证外源性补充SNO对肺动脉高压血流动力学及病理生理改变的影响。我们发现外源性补充SNO可以有效降低肺动脉压力、肺动脉血管阻力，缓解右心室肥厚。除此之外，SNO可以降低肺组织纤维化，肺血管周纤维化水平以及右心室纤维化。进一步的研究发现，SNO降低肺动脉压力，延缓肺动脉高压进展的一系列改变可能与抑制JAK2/STAT3和MEK/ERK信号通路有关。这一系列的研究结果将为肺动脉高压发病机制提供新方向，为防治策略提供新的思路及方法。