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

基本信息

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
项目类别：
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

项目摘要

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)，全球主要死亡原因经典抗氧化剂（β-胡萝卜素、维生素 A）。 E) 旨在清除短命自由基的方法对 CVD 患者的益处有限。对抗血管氧化应激的不同治疗方法似乎至关重要。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，我们在内皮细胞（EC）中发现了一种重要的氧化还原调节机制。从 2 型糖尿病患者中分离出来的 Pr-SSG 水平显着升高，这一点很重要。这一发现在高脂血症小鼠模型中得到进一步证实，该模型显示 Pr-SSG 伴随显着增加在主动脉粥样硬化病变中，Glrx-1（一种特定的去谷胱甘肽酶）下调，特别是在 EC 中，ApoE-/- 小鼠品系中 Glrx-1 的转基因过度表达可以防止饮食诱导的影响。主动脉内皮细胞渗透性过高并减弱动脉粥样硬化 (AS) 的发展。初步结果得出了该拨款提案的一个中心假设，即 EC Glrx-1 在以下方面具有保护作用：代谢应激诱导的 EC 功能障碍和 AS 具体目标#1 将在体内测试 EC 特异性的假设。 Glrx-1 的上调将通过改善代谢引起的 EC 功能障碍来减轻 AS 进展使用具有 ApoE-/- 背景的 EC 特异性 Glrx-1 转基因小鼠的异常。代谢应激 EC 中的生化研究将 Rac1 确定为 Glrx-1 的特定靶标，并证明 Glrx-1 对甾醇调节元件结合蛋白 (SREBP) 的蛋白水解激活的抑制作用，因此，特定的 Aim#2 将在体外测试该新型药物。假设 Glrx-1 通过 Rac1/SREBP 信号传导的氧化还原调节改善 EC 功能，采用综合方法，包括来自 Glrx-1 TG 和 KO 小鼠的原代 EC、药理学和遗传学手段（编码 Rac1 野生型和氧化还原抗性突变体的 siRNA 和腺病毒）和氧化还原生化技术（生物素开关测定和 Pr-SSG 免疫检测）将在体内测试该假设。补充血管内皮细胞Glrx-1基因和/或重组蛋白可以逆转血管内皮细胞我们认为，采用内皮靶向脂质体药物递送系统可以改善功能障碍并延缓 AS。拟议的研究将提供有关 Glrx-1 和 Rac1 谷胱甘肽化如何参与的新信息 EC功能障碍和AS在代谢紊乱中复杂化，并将寻求将Glrx-1建立为前瞻性药物糖尿病血管损伤的治疗剂。