Role of protein-S-glutathionylation in endothelial dysfunction and atherosclerosis

项目摘要 血管氧化应激与几乎所有主要危险因素的发病密切相关 心血管疾病(CVD)是全球主要的死亡原因。经典抗氧化剂(β-胡萝卜素、维生素A 和E)旨在清除短暂的自由基，对心血管疾病患者的益处有限。发展 对抗血管氧化应激的不同治疗方法似乎至关重要。蛋白质S- 谷胱甘肽基化(Pr-SSG)是氧化剂诱导的可逆翻译后修饰的普遍形式， 近年来，作为一种重要的氧化还原调节机制出现在心血管疾病中。我们发现，在内皮细胞(ECs)中 从2型糖尿病患者中分离出的Pr-SSG水平显著升高。这一点很重要 在高脂血症小鼠模型中进一步证实了这一发现，该模型显示Pr-SSG伴随显著增加 在动脉粥样硬化病变中，随着Glrx-1的下调，一种特定的谷胱甘肽脱氢酶， 尤其是在欧洲经济共同体。转基因Glrx-1在ApoE-/-小鼠中的过表达对饮食诱导的保护作用 主动脉内皮细胞的高通透性和减轻动脉粥样硬化(AS)的发展。这些令人兴奋的东西 初步结果导致了这一赠款提案的中心假设，即EC Glrx-1在 代谢应激诱导的内皮细胞功能障碍和AS。特定目标1将在体内测试EC特异性的假说 Glrx-1的上调将通过改善代谢引起的EC功能障碍来减缓AS的进展 具有ApoE-/-背景的EC特异性Glrx-1转基因小鼠的异常。我们的氧化还原蛋白质组和 代谢应激内皮细胞的生化研究确认rac1是Glrx-1的特异性靶标，并证明 Glrx-1对固醇调节元件结合蛋白(SREBPs)蛋白水解性激活的抑制作用 脂类平衡和EC功能障碍的中心角色。因此，《特定目标2》将对这部小说进行体外测试。 假设Glrx-1通过对rac1/SREBP信号的氧化还原调节来改善EC功能，采用 包括来自Glrx-1 TG和KO小鼠的原代内皮细胞的综合方法，药理学和遗传学 Means(编码rac1野生型和抗氧化还原突变体的siRNA和腺病毒)，以及氧化还原生化 技术(生物素开关试验和Pr-SSG免疫检测)。具体目标3将在体内检验这一假说 补充Glrx-1基因和/或重组蛋白的血管内皮细胞可逆转血管 使用内皮靶向脂质体给药系统时的功能障碍和延迟。我们相信， 拟议的研究将提供有关Glrx-1和rac1的谷胱甘肽基化如何参与 EC功能障碍和代谢紊乱的复杂性，并将寻求将Glrx-1确立为预期的 糖尿病环境下血管损伤的治疗剂。

Project Abstract Vascular oxidative stress is strongly implicated in the pathogenesis of virtually all primary risk factors for cardiovascular diseases (CVD), a leading cause of death globally. Classic antioxidants (β-carotene, Vitamin A and E) aimed at scavenging the short-lived free radicals have limited benefits in patients with CVD. Development of different therapeutic approaches combating vascular oxidative stress appears critical. Protein S- glutathionylation (Pr-SSG), the prevalent form of oxidant-induced reversible post-translational modification, recently emerged as an important redox regulatory mechanism in CVD. We found that in endothelial cells (ECs) isolated from patients with type 2 diabetes mellitus, the level of Pr-SSG was significantly elevated. This important finding is further confirmed in a hyperlipidemic mouse model showing markedly increased Pr-SSG concomitant with a down-regulation of Glrx-1, a specific de-glutathionylation enzyme, in aortic atherosclerotic lesions, particularly in ECs. Transgenic overexpression of Glrx-1 in ApoE-/- mouse strain protects against diet-induced aortic endothelial hyper-permeability and attenuates atherosclerosis (AS) development. These exciting preliminary results lead to a central hypothesis of this grant proposal that EC Glrx-1 has a protective role in metabolic stress-induced EC dysfunction and AS. Specific Aim#1 will test in vivo the hypothesis that EC-specific up-regulation of Glrx-1 will attenuate AS progression by improving EC dysfunction caused by metabolic abnormalities using EC specific Glrx-1 transgenic mice with ApoE-/- background. Our redox proteomic and biochemical studies in metabolically stressed ECs identify Rac1 as a specific target of Glrx-1 and demonstrate inhibitory effect of Glrx-1 on proteolytic activation of sterol regulatory element binding proteins (SREBPs), the central players in lipid homeostasis and EC dysfunction. Therefore, specific Aim#2 will test in vitro the novel hypothesis that Glrx-1 improves EC function through redox regulation of Rac1/SREBP signaling, employing comprehensive approaches including primary ECs from Glrx-1 TG and KO mice, pharmacological and genetic means (siRNA and adenovirus encoding Rac1 wild type and redox-resistant mutants), and redox biochemical techniques (Biotin-switch assay and immunodetection of Pr-SSG). Specific Aim#3 will test in vivo the hypothesis that supplementation of vascular ECs with Glrx-1 gene and/or recombinant protein can reverse vascular dysfunction and retard AS employing endothelium-targeted liposomal drug delivery system. We believe that the proposed studies will provide novel information about how Glrx-1 and glutathionylation of Rac1 are involved in EC dysfunction and AS complicated in metabolic disorders, and will seek to establish Glrx-1 as a prospective therapeutic agent for vascular injury in the setting of diabetes.