Patients with diabetes suffer from increased incidence and severity of atherosclerotic cardiovascular diseases. To study the mechanisms through which diabetes accelerates vascular disease, we have identified mouse models of insulin resistance, i.e. C57BL/6 apoE-/- mice fed a fat-rich western diet and insu|in receptor and insulin receptor substrate-1 double heterozygous (IR+/-IRS1+/-) mice, which we have successfully crossed into the apolipoprotein E-deficient (apoE-/-) mouse on the C57BL/6 background. ApoE-/- mice, like humans at elevated risk for atherosclerotic cardiovascular disease, show monocyte-derived macrophages and foam cells and T-helper 1 cells (Th1) in lesions. The role B cells and other lymphocytes in diabetesaccelerated atherosclerosis is unknown. We have developed a flow cytometry-based method to analyze immune cell content of the mouse aortic wall. We propose to determine why and how atherosclerosis results in chronic inflammation of the vascular wall that does not resolve, and how insulin resistance and type 2 diabetes modify this process.
Specific aim 1 is to determine the aortic wall content of B, T, and antigen presenting cells in atherosclerotic, insulin resistant and type 2 diabetic mice and pigs. We propose to compare the composition of the aortic walls in apoE-/- mice on chow diet, apoE-/- mice on western diet (insulin resistant), IR+/- IRS1+/- mice (insulin resistant or diabetic, depending on age), and IR+/- IRS1+/- apoE-/- mice (insulin resistant and atherosclerosis-prone) with and without western diet. All these mice are on the C57BL/6 background.
Specific aim 2 is to identify the molecular mechanisms by which lymphocytes enter the artery wall in these mouse models of atherosclerosis, insulin resistance, T2D or both. We will use short-term homing assays using fluorescently labeled splenocytes from various knockout mice and long-term homing assays using green fluorescent protein (GFP)-expressing splenocytes.
Specific aim 3 is to directly investigate the mechanisms by which lymphocytes interact with the endothelium in carotid and femoral arteries of apoE-/- mice with and without insulin resistance/T2D in vivo. Molecular insights from these intravital microscopy studies and the homing studies described in aim 2 will guide the generation pf double knockout mice and the design of antibody blocking experiments. Disease outcome will be assessed by mapping lesion sizes en face and in cross sections by histology and immunostaining. Together with the other projects described in this PPG renewal application, we expect that our cumulative research will impact the understanding and treatment of diabetic cardiovascular disease.
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