Role of Cytokine-Induced Inflammation in Endothelial Dysfunction in Diabetes

描述（由申请人提供）：本提案的总体目标是验证中心假设：2型糖尿病小鼠的冠状动脉功能障碍是由树突状细胞（DC）的激活和随后的干扰素γ (IFN)的表达产生的。有三个具体的目标，每个目标有两个假设。第一个目标是确定树突状细胞（DC）激活在2型糖尿病冠状动脉内皮功能障碍中的作用。我们提出：1)2型糖尿病小鼠（db/db）与CD11c-DTR小鼠（DC“条件零”小鼠）的杂交将保留冠状动脉内皮功能。在该模型中，白喉毒素处理后DC被耗尽，并将与同窝对照（低Db/ Db或野生型[WT]，但DC有条件为零）或未接受白喉毒素处理的小鼠（因此包含DC的常住种群）进行比较；2)炎症细胞因子（干扰素γ IFN和TNF）水平在白喉毒素治疗后的db/db × CD11c-DTR中会降低，与瘦窝的db/db或WT × CD11c-DTR相当；3)在dc （db/db x CD11c-DTR）缺失的糖尿病小鼠中，T细胞和巨噬细胞的激活与db/db小鼠相比降低，与瘦鼠（db/db或WT x CD11c-DTR）相当。第二个目的是确定2型糖尿病患者的dc激活是否会进展为T细胞和巨噬细胞的激活，从而导致冠状动脉中炎症细胞因子的释放。我们提出，在T细胞基因缺失的糖尿病模型中（db/db x CD4+ -/-；这是一个C57BL/6背景的小鼠模型，Cd4tm1Mak靶向突变显著阻断CD4+ T细胞发育），内皮依赖性血管扩张反应更大，氧化应激标志物（超氧化物产生，蛋白质硝化）减少，与2型糖尿病db/db小鼠相比。db/db × CD4+ -/-的血管舒张反应将与同窝、瘦对照（db/db × CD4+ -/-）小鼠相当。我们还提出，在白喉毒素诱导巨噬细胞缺失（db/db x CD11b-DTR）的糖尿病模型中，这是一个在CD11b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（超氧化物产生，蛋白质硝化）减少。db/db × CD11b-DTR小鼠的反应与同窝、瘦对照（db/db × CD11b-DTR）小鼠的反应相当。我们将使用方法方法的组合，主要包括体外显微镜，荧光和电子顺磁共振（EPR）分析超氧化物（O2）的产生，实时pcr用于RNA和DNA分析，以及Western Blotting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型糖尿病及相关疾病/并发症的血管损伤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，这些炎症会导致2型糖尿病的血管功能障碍。这些研究将有助于开发治疗策略，提供的结果磨练我们对DC激活如何影响冠状动脉损伤的理解，并改善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。

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.