Glycation inactivation of human CD59 and diabetic complications

人 CD59 的糖基化失活和糖尿病并发症

• 批准号: 8234691
• 负责人: JOSE A HALPERIN
• 金额: $ 28.84万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-12-15 至 2012-07-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8234691
• 关键词: Accounting; Active Sites; Acute; Adult; Animal Model; Animals; Antibodies; Aorta; Apolipoprotein E; Atherosclerosis; Attenuated; Backcrossings; Biochemical Pathway; Biological Response Modifiers; Blood Glucose; Blood Vessels; Budgets; Cardiovascular Diseases; Cardiovascular system; Cells; Chronic; Clinical; Clinical Research; Complement; Complications of Diabetes Mellitus; Deposition; Development; Diabetes Mellitus; Diabetic Angiopathies; Diabetic mouse; Disease; Endothelial Cells; Endothelium; Engineering; Enzyme-Linked Immunosorbent Assay; Epidemiologic Studies; Erythrocytes; Ethics; Evans blue stain; Exhibits; Fatty acid glycerol esters; Fructosamine; Future; Generations; Genes; Genetic; Glucose; Glycosylated Hemoglobin; Goals; Group Identifications; Health; Hemolytic Anemia; Histologic; Human; Hyperglycemia; Immune system; Individual; Inflammation; Injection of therapeutic agent; Insulin; Insulin-Dependent Diabetes Mellitus; Kidney Diseases; Knock-out; Laboratories; Life; Link; Measurement; Mediating; Metabolic Pathway; Modeling; Molecular; Morbidity - disease rate; Mus; Myocardial Ischemia; Neuropathy; Organ; Pathogenesis; Pathology; Peripheral Vascular Diseases; Plant Roots; Plasma; Play; Population; Prevention; Prevention therapy; Protein Isoforms; Proteins; Publications; Publishing; Relative (related person); Reporting; Research; Resistance; Retinal Diseases; Risk; Role; Severities; Staining method; Stains; Streptozocin; Stroke; Testing; Tissues; Transgenic Mice; United States National Institutes of Health; VWF gene; Vascular Diseases; Work; atherogenesis; complement system; diabetic; diabetic cardiomyopathy; follow-up; genetic regulatory protein; glycation; mortality; mouse model; mutant; novel; preclinical study; trait

DESCRIPTION (provided by applicant): Chronic high blood sugar levels (hyperglycemia) are responsible for diabetic complications including nephropathy, retinopathy, neuropathy and accelerated atherosclerosis, all major causes of morbidity and mortality in the adult population. However, how hyperglycemia causes diabetic complications is still poorly understood. Extensive clinical studies by us and others indicate that the complement system, a major effector of the immune system and mediator of inflammation, may play a significant role in the pathogenesis of these complications. In particular, we have established that human CD59, a key complement regulatory protein that protects cells from complement-mediated damage, is inhibited by glycation, the non-enzymatic attachment of glucose to proteins. This is because human CD59 contains a glycation motif, formed by its H44 residue at H 5A0 from its K41 residue in the active site of the protein. Interestingly, this glycation motif is not present in CD59 from other species. Our hypothesis is that glycation-inactivation of hCD59 due to its unique H44 residue could represent the elusive link between hyperglycemia and the vascular proliferative complications of human diabetes. However, practical and ethical considerations preclude conducting clinical studies to test this hypothesis comprehensively in humans. Therefore, our strategy to determine the cellular and molecular mechanism by which complement damages the target organs of diabetic complications has focused on developing molecular engineered mice that are now available in our laboratory. We will make these mice diabetic by STZ injection (type 1 diabetes model), and investigate the role of glycation-inactivation of hCD59 in diabetic cardiovascular disease taking advantage of our previous generation of mCD59KO mice backcrossed into the Apoe-/- background. Specifically, we will compare diabetic hCD59 wild type (glycation-inactivation sensitive) transgenic mice with diabetic hCD59Gln44 mutant (glycation- inactivation resistant) transgenic mice, both in a mCd59KO and Apoe-/-background. We will also use an available anti-mC5 antibody to block the terminal complement cascade and provide direct mechanistic evidence for the contribution of complement to diabetic vascular diseases. Successful accomplishment of this work will provide: 1) clear evidence for the role of complement and glycation-inactivation of hCD59 in the pathogenesis of the cardiovascular complications of diabetes; 2) needed animal models to study mechanism, therapy and prevention of diabetic complications; and 3) strong evidence to support future studies on complement and diabetes in humans. PUBLIC HEALTH RELEVANCE: The major goal of this proposal is to test our hypothesis that glycation-inactivation of the complement regulatory protein CD59 plays a critical role in the pathogenesis of diabetic complications with particular focus on the cardiovascular disease commonly seen in diabetic individuals. To this end, we will use STZ-induced diabetic mice that lack mCD59 (mCD59KO), are backcrossed into an Apoe deficient background, and transgenetically express human CD59 in either the glycation-senstive wild type form or as a glycation-insensitive mutant, all generated and available in our laboratory. The proposed studies are highly translational; their relevance is highlighted by the following facts: 1) Diabetes and its complications are a leading cause of morbidity and mortality in the adult population, 2) vascular pathology causing ischemic heart disease, stroke, and peripheral vascular disease account for more than 50% of the mortality rate in the diabetic population, 3) treatment of diabetic cardiovascular disease accounts for close to 65% of the more than 100 billion dollars annually spent in the US to treat chronic diabetic complications (H 1 out 10 dollars of the overall US health budget), and 4) successful accomplishment of our aims will provide a much needed animal model for pre-clinical studies on the pathogenesis, treatment and prevention of diabetic complications.

描述(申请人提供)：慢性高血糖水平(高血糖)是糖尿病并发症的罪魁祸首，包括肾病、视网膜病变、神经病变和加速的动脉粥样硬化，所有这些都是成年人发病率和死亡率的主要原因。然而，高血糖是如何导致糖尿病并发症的，目前仍知之甚少。我们等人的广泛临床研究表明，补体系统作为免疫系统的主要效应者和炎症介质，可能在这些并发症的发病机制中发挥重要作用。特别是，我们已经确定，人类CD59是一种关键的补体调节蛋白，可以保护细胞免受补体介导的损伤，它受到糖基化的抑制，糖基化是葡萄糖与蛋白质的非酶结合。这是因为人CD59含有一个糖基化基序，由其在蛋白质活性部位的H5A0处的H44残基和K41残基形成。有趣的是，这个糖基化基序在其他物种的CD59中不存在。我们的假设是，hCD59由于其独特的H44残基而导致的糖基化失活可能代表了高血糖和人类糖尿病血管增生性并发症之间难以捉摸的联系。然而，出于实际和伦理方面的考虑，无法进行临床研究来在人体上全面检验这一假说。因此，我们的策略是确定补体损害糖尿病并发症靶器官的细胞和分子机制，重点是发展分子工程小鼠，现在我们的实验室可以使用这些小鼠。我们将通过注射STZ使这些小鼠糖尿病(1型糖尿病模型)，并利用我们的上一代mCD59KO小鼠回交到APOE-/-背景中的优势，研究hCD59糖基化失活在糖尿病心血管疾病中的作用。具体地说，我们将在mCD59KO和APOE-/-背景下比较糖尿病hCD59野生型(糖基化失活敏感)转基因小鼠和糖尿病hCD59Gln44突变(糖基化失活抵抗)转基因小鼠。我们还将使用可用的抗MC5抗体来阻断末端补体级联，并为补体在糖尿病血管疾病中的作用提供直接的机制证据。这项工作的成功完成将为：1)hCD59的补体和糖基化失活在糖尿病心血管并发症发病机制中的作用提供明确的证据；2)需要动物模型来研究糖尿病并发症的机制、治疗和预防；以及3)强有力的证据支持未来对补体和糖尿病的研究。 公共卫生相关性：这项提案的主要目标是检验我们的假设，即补体调节蛋白CD59的糖基化失活在糖尿病并发症的发病机制中发挥关键作用，特别是关注糖尿病患者常见的心血管疾病。为此，我们将使用缺乏mCD59的STZ诱导的糖尿病小鼠(MCD59KO)，回交到APOE缺乏的背景中，并以糖基化敏感的野生型或糖基化不敏感的突变体的形式转基因表达人CD59，所有这些都是在我们实验室产生和获得的。建议的研究具有很强的翻译性；以下事实突显了它们的相关性：1)糖尿病及其并发症是成人人口发病率和死亡率的主要原因，2)导致缺血性心脏病、中风和外周血管疾病的血管病变占糖尿病人口死亡率的50%以上，3)糖尿病心血管疾病的治疗占美国每年用于治疗糖尿病慢性并发症的1000亿美元的近65%(美国总体卫生预算的10美元中有10美元是H1)，以及4)我们目标的成功实现将为发病机制的临床前研究提供急需的动物模型。糖尿病并发症的治疗和预防。