Role of Cytokine-Induced Inflammation in Endothelial Dysfunction in Diabetes

细胞因子诱导的炎症在糖尿病内皮功能障碍中的作用

• 批准号: 8451911
• 负责人: Michael A HILL
• 金额: $ 35.57万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-02-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8451911
• 关键词: Ablation; Affect; Aftercare; Antigens; Blood Vessels; CD4 Positive T Lymphocytes; Cells; Coronary; DNA; Dendritic Cells; Dendritic cell activation; Development; Diabetes Mellitus; Diabetic Angiopathies; Diabetic mouse; Diphtheria Toxin; Disease; Electron Spin Resonance Spectroscopy; Elements; Endothelium; Event; Fluorescence Microscopy; Functional disorder; Funding; Genetic; Goals; Heart Diseases; Human; ITGAM gene; ITGAX gene; Immune; Impairment; In Vitro; Inflammation; Inflammatory; Injury; Interferon Type II; Knockout Mice; Knowledge; Lead; Link; Macrophage Activation; Mediating; Microcirculation; Modeling; Morbidity - disease rate; Mus; Mutation; Myocardial Ischemia; Natural Immunity; Nitric Oxide; Non-Insulin-Dependent Diabetes Mellitus; Outcome; Oxidative Stress; Play; Population; Production; Proteins; RNA; Receptor Activation; Research Personnel; Risk Factors; Role; Smooth Muscle Myocytes; Societies; Stroke; Superoxides; T-Cell Activation; T-Cell Development; T-Lymphocyte; Testing; Therapeutic; Toxin; Tumor Necrosis Factor-alpha; Tunica Adventitia; Vascular Diseases; Vasodilator Agents; Western Blotting; Work; arteriole; cell type; combat; cytokine; db/db mouse; design; diabetes risk; diabetic; diabetic cardiomyopathy; diphtheria toxin receptor; experience; improved; insight; macrophage; monocyte; mortality; mouse model; neutralizing antibody; nitration; novel; novel strategies; prevent; promoter; research study; response; therapeutic development; therapy design; vascular inflammation

DESCRIPTION (provided by applicant): The overarching goal of this proposal is to test the central hypothesis that: coronary dysfunction in type 2 diabetic mice is produced by the activation of dendritic cells (DC) with subsequent expression of interferon gamma (IFN). There are 3 specific aims and each aim has 2 hypotheses. The first aim will causally determine the role of dendritic cell (DC) activation in endothelial dysfunction in coronary arterioles in type 2 diabetes. We propose that 1) Endothelial function will be preserved in coronary arterioles in a cross between type 2 diabetic (db/db) and CD11c-DTR mice, a mouse "conditionally null" for DC. In this model, DCs are depleted after treatment with Diptheria toxin and comparisons will be made to both littermate controls (lean Db/db or wild type [WT], but conditionally null for DC) or mice not treated with Diptheria toxin (and thus containing the resident population of DCs); and 2) Levels of inflammatory cytokines (interferon gamma IFN and TNF) will be reduced in db/db x CD11c-DTR following treatment with Diptheria toxin and comparable to lean littermates, Db/db or WT x CD11c-DTR; and 3) In diabetic mice depleted of DCs (db/db x CD11c-DTR), activation of T cells and macrophages are reduced compared to db/db mice and comparable to lean littermates, Db/db or WT x CD11c-DTR. The second aim will determine if activation of DCs progresses to activation of T cells and macrophages leading to release of the inflammatory cytokines in coronary arterioles in type 2 diabetes. We propose that in a diabetic model with genetic deletion of T cells (db/db x CD4+ -/-; this is a mouse model on a C57BL/6 background with a targeted mutation in Cd4tm1Mak that significantly blocks in CD4+ T-cell development) endothelium-dependent vasodilatory responses are greater, and markers of oxidative stress (superoxide production, protein nitration) are reduced, compared to that in type 2 diabetic db/db mice. Vasodilatory responses of db/db x CD4+ -/- will be comparable to that of littermate, lean control (Db/db x CD4+ -/-) mice. We also propose that in a diabetic model with Diptheria toxin induced deletion of macrophages (db/db x CD11b-DTR; this is a mouse model which expresses the human diphtheria toxin receptor [DTR] under the control of the CD11b promoter. Treatment with diphtheria toxin results in the complete ablation of circulating monocytes), endothelium-dependent vasodilatory responses are greater, and markers of oxidative stress (superoxide production, protein nitration) are reduced, compared to that in type 2 diabetic db/db mice. Responses of db/db x CD11b-DTR will be comparable to that in the littermate, lean control (Db/db x CD11b-DTR) mice. We will use a combination of methodological approaches principally consisting of in vitro microscopy, fluorescence and electron paramagnetic resonance (EPR) analysis of superoxide (O2¿¿) production, real timePCRfor RNA and DNA analyse s, and Western Blotting to evaluate expression of key proteins in all strains and crosses. We believe that this study will provide a new understanding of, and new approaches for, the treatment of vascular injury in type 2 diabetes and related disorders/complications. This work will increase our knowledge of the role of dendritic cells, T cells and macrophage-induced vascular inflammation, which causes vascular dysfunction in type 2 diabetes. These studies will aid development of therapeutic strategies by providing results that hone our understanding of how DC activation affects coronary impairment and improve how we develop optimal therapeutic strategies for harnessing the adaptive impact of latent, innate immunity to combat inflammation.

描述（由申请方提供）：本提案的总体目标是检验中心假设：2型糖尿病小鼠的冠状动脉功能障碍是由树突状细胞（DC）激活并随后表达干扰素γ（IFN）引起的。有3个具体目标，每个目标有2个假设。第一个目标是确定树突状细胞（DC）激活在2型糖尿病冠状小动脉内皮功能障碍中的作用。我们提出：1）在2型糖尿病（db/db）和CD 11 c-DTR小鼠（DC“条件性无效”小鼠）之间的交叉中，冠状动脉中的内皮功能将得到保留。在该模型中，DCs在用白喉毒素处理后耗尽，并将与两个同窝对照进行比较（瘦Db/db或野生型[WT]，但DC条件性无效）或未用白喉毒素处理的小鼠（从而包括区议会的居住人口）;和2）炎性细胞因子的水平（干扰素γ IFN和TNF）在用白喉毒素处理后的db/db x CD 11 c-DTR中将降低，并且与瘦的同窝仔相当，Db/db或WT x CD 11 c-DTR;和3）在DCs耗尽的糖尿病小鼠（db/db x CD 11 c-DTR）中，T细胞和巨噬细胞的活化与db/db小鼠相比降低，并且与瘦的同窝出生小鼠（Db/db或WT x CD 11 c-DTR）相当。第二个目标将确定DC的活化是否进展为T细胞和巨噬细胞的活化，从而导致2型糖尿病冠状动脉中炎性细胞因子的释放。我们提出，在T细胞基因缺失的糖尿病模型中，（db/db x CD4+ -/-;这是C57 BL/6背景下的小鼠模型，Cd 4 tm 1 Mak靶向突变显著阻断CD 4 + T细胞发育）内皮依赖性血管舒张反应更大，氧化应激标志物（超氧化物产生，蛋白质硝化）减少。db/db x CD 4 + -/-小鼠的血管舒张反应与同窝瘦对照（Db/db x CD 4 + -/-）小鼠的血管舒张反应相当。我们还提出，在白喉毒素诱导的巨噬细胞缺失的糖尿病模型中（db/db x CD 11b-DTR;这是一种在CD 11b启动子控制下表达人白喉毒素受体[DTR]的小鼠模型。与2型糖尿病db/db小鼠相比，白喉毒素治疗导致循环单核细胞完全消融，内皮依赖性血管舒张反应更大，氧化应激标志物（超氧化物生成，蛋白质硝化）减少。db/db x CD 11b-DTR的反应与同窝瘦对照（Db/db x CD 11b-DTR）小鼠的反应相当。我们将使用主要由体外显微镜、荧光和电子顺磁共振（EPR）分析超氧化物（O2）产生、用于RNA和DNA分析的真实的实时PCR以及蛋白质印迹法组成的方法学方法的组合来评估所有菌株和杂交中关键蛋白质的表达。我们相信，这项研究将为2型糖尿病血管损伤及相关疾病/并发症的治疗提供新的认识和新的方法。这项工作将增加我们对树突状细胞，T细胞和巨噬细胞诱导的血管炎症的作用的认识，这些炎症导致2型糖尿病的血管功能障碍。这些研究将通过提供结果来帮助开发治疗策略，这些结果磨练了我们对DC激活如何影响冠状动脉损伤的理解，并改善了我们如何开发最佳治疗策略，以利用潜在的先天免疫的适应性影响来对抗炎症。