Role of Cytokine-Induced Inflammation in Endothelial Dysfunction in Diabetes

描述(申请人提供)：这项建议的主要目标是测试中心假设：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(干扰素)造成的。有3个具体目标，每个目标有2个假设。第一个目的将因果地确定树突状细胞(DC)激活在2型糖尿病冠状动脉小动脉内皮功能障碍中的作用。我们认为1)在2型糖尿病(db/db)和CD11c-DTR小鼠之间的杂交中，冠状动脉内皮细胞的功能将得到保护，CD11c-DTR小鼠是DC的“条件零”小鼠。在这个模型中，DC在白喉毒素处理后被耗尽，并将与两种仔鼠对照(瘦肉型DB/db或野生型[WT]，但DC条件为零)或未经白喉毒素处理的小鼠(因此包含DC的常驻种群)进行比较；2)在db/db x CD11c-DTR中，炎性细胞因子(干扰素-γ干扰素和肿瘤坏死因子)的水平将在白喉毒素处理后降低，与瘦肉型仔鼠、DB/db或WT x CD11c-DTR相似；3)在DC耗竭的糖尿病小鼠(db/db x CD11c-DTR)中，T细胞和巨噬细胞的激活比db/db小鼠减少，与瘦子、DB/db或WT x CD11c-DTR相似。第二个目的将确定在2型糖尿病中，DC的激活是否进展到T细胞和巨噬细胞的激活，从而导致冠状动脉小动脉中炎性细胞因子的释放。我们认为，与2型糖尿病db/db小鼠相比，在T细胞基因缺失(db/db x CD4+/-；这是一个以C57BL/6为背景的小鼠模型，CD4tm1Mak具有显著阻止CD4+T细胞发育的靶向突变)中，内皮依赖的血管扩张反应更强，氧化应激标志(超氧化物产生，蛋白质硝化)减少。Db/db x CD4+-/-的血管扩张反应将与产仔瘦身对照(db/db x CD4+/-)小鼠的反应相当。我们还提出，在白喉毒素诱导的巨噬细胞(db/db x CD11b-DTR)缺失的糖尿病模型中，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，内皮依赖性血管扩张反应更强，氧化应激标志(超氧化物产生，蛋白质硝化)减少。Db/db x CD11b-DTR的反应将与产仔瘦身对照(DB/db x CD11b-DTR)小鼠的反应相当。我们将结合使用多种方法，主要包括体外显微镜、超氧化物(O2)产生的荧光和电子顺磁共振(EPR)分析、实时PCR核糖核酸和脱氧核糖核酸分析、S和Western blotting，以评估关键蛋白在所有菌株和杂交中的表达。我们相信，这项研究将为2型糖尿病血管损伤和相关疾病/并发症的治疗提供新的理解和新的方法。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了解，这些炎症导致2型糖尿病患者的血管功能障碍。这些研究将通过提供结果来帮助开发治疗策略，从而磨练我们对DC激活如何影响冠状动脉损害的理解，并改进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抗击炎症。

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.