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.
Our proposed studies will investigate the causes of vascular inflammation initiating the spiral of endothelial dysfunction and culminating in diabetic vascular and microvascular disease by deciphering mechanisms of coronary microvascular dysfunction in type 2 diabetes. Specifically, our goal is to explore the central hypothesis that the activationof dendritic cells (DCs) with subsequent expression of interferon gamma (IFN) drives the events (T cell activation, macrophage activation, vascular inflammation) that result in endothelial dysfunction in type 2 diabetes. The results of this study are expected to contribute to the development of novel adjunctive therapies by identifying mechanisms and key targetable elements within them that activate vascular dendritic cells to produce endothelial dysfunction, which if untreated culminates in diabetic vascular and microvascular disease. New therapies that target vascular inflammation will aid in maintaining proper endothelial function and prevent initiation of the dysfunctional pathological spiral that inexorably evolves towards the pathologica manifestations of diabetes in the macro- and microcirculation.
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