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

蛋白质-S-谷胱甘肽化在内皮功能障碍和动脉粥样硬化中的作用

• 批准号: 9544365
• 负责人: JINGYAN HAN
• 金额: $ 41.15万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9544365
• 关键词: Actins; Adenovirus Vector; Adenoviruses; Antioxidants; Apolipoprotein E; Applications Grants; Area; Arterial Fatty Streak; Atherosclerosis; Attenuated; Beta Carotene; Binding Proteins; Biochemical; Biological Assay; Biological Response Modifier Therapy; Biotin; Blood Vessels; Cardiovascular Diseases; Cardiovascular system; Carotid Arteries; Cause of Death; Cell physiology; Cysteine; Cytoskeleton; Data; Development; Diabetes Mellitus; Diet; Down-Regulation; Drug Delivery Systems; Encapsulated; Endothelial Cells; Endothelium; Enzymes; Excision; Free Radicals; Functional disorder; Gene Proteins; Genes; Genetic; Goals; Grx1 protein; Homeostasis; Human; Hyperlipidemia; Impairment; Inflammation; Injury; Knockout Mice; Lead; Link; Lipids; Liposomes; Mediating; Metabolic; Metabolic Diseases; Metabolic stress; Modeling; Modification; Morbidity - disease rate; Mouse Strains; Mus; Natural regeneration; Non-Insulin-Dependent Diabetes Mellitus; Oxidants; Oxidation-Reduction; Oxidative Stress; Pathogenesis; Patients; Permeability; Pharmacology; Phenotype; Post-Translational Protein Processing; Predisposition; Process; Production; Protein S; Proteins; Proteomics; Reactive Oxygen Species; Recombinant Proteins; Regulation; Regulatory Element; Research Personnel; Resistance; Risk Factors; Role; Signal Pathway; Signal Transduction; Signaling Protein; Small Interfering RNA; Sterols; Supplementation; Techniques; Testing; Therapeutic; Therapeutic Agents; Therapeutic Effect; Transgenic Mice; Transgenic Organisms; Up-Regulation; Vascular Diseases; Vascular Endothelial Cell; Vitamin A; Vitamin E; antibody conjugate; cardiovascular risk factor; combat; diabetic patient; endothelial dysfunction; improved; in vitro testing; in vivo; mortality; mouse model; mutant; novel; novel therapeutics; overexpression; prospective; protective effect; protein function; rho GTP-Binding Proteins; targeted delivery; targeted treatment; virtual

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.

项目摘要 血管氧化应激与几乎所有主要危险因素的发病机制密切相关， 心血管疾病（CVD）是全球死亡的主要原因。经典抗氧化剂（β-胡萝卜素、维生素A 和E）旨在清除短寿命自由基的药物对CVD患者的益处有限。发展 不同的治疗方法对抗血管氧化应激似乎是至关重要的。蛋白S- 谷胱甘肽化（Pr-SSG），氧化剂诱导的可逆翻译后修饰的普遍形式， 最近出现作为一个重要的氧化还原调节机制，在CVD。我们发现，在内皮细胞（EC） 从2型糖尿病患者中分离，Pr-SSG水平显著升高。这一重要 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 在主动脉粥样硬化病变中， 尤其是在EC中。转基因过表达Glucose-1在ApoE-/-小鼠品系中保护免受饮食诱导的 主动脉内皮通透性过高，并减弱动脉粥样硬化（AS）的发展。这些令人兴奋 初步结果导致了这项拨款提案的中心假设，即EC Glycoprotein-1在 代谢应激诱导的EC功能障碍和AS。特异性目标#1将在体内测试EC特异性 Glucose-1的上调将通过改善代谢性血管紧张素转换酶引起的EC功能障碍来减轻AS的进展。 使用具有ApoE-/-背景的EC特异性Glycoprotein-1转基因小鼠进行检测。我们的氧化还原蛋白质组学和 在代谢应激的EC中的生物化学研究将Rac 1鉴定为Glycoprotein-1的特异性靶点，并证明 Glycoprotein-1对固醇调节元件结合蛋白（SREBP）的蛋白水解活化的抑制作用， 脂质稳态和EC功能障碍的中心参与者。因此，特定的目标#2将在体外测试新化合物。 假设Glycoprotein-1通过Rac 1/SREBP信号的氧化还原调节改善EC功能， 包括来自Glycoprotein-1 TG和KO小鼠的原代EC、药理学和遗传学 方法（编码Rac 1野生型和氧化还原抗性突变体的siRNA和腺病毒），和氧化还原生物化学 技术（生物素开关测定和Pr-SSG的免疫检测）。具体目标#3将在体内检验假设 用Glycoprotein-1基因和/或重组蛋白补充血管内皮细胞可以逆转血管内皮细胞增殖， 通过内皮靶向脂质体给药系统，我们认为 拟议的研究将提供关于Glucose-1和Rac 1的谷胱甘肽化如何参与 EC功能障碍和AS并发代谢紊乱，并将寻求建立Glycoprotein-1作为一种有前景的 用于糖尿病背景下血管损伤的治疗剂。