Project summary My research is focused on the interactions between mineralocorticoid receptor (MR) signaling in perivascular adipose tissue (PVAT), dysregulation of immunity and vascular dysfunction in the setting of high-fat high- fructose diet (Western Diet [WD]). WD leads to insulin resistance. Importantly, insulin resistance leads to aggressive cardiovascular disease in females. I hypothesize that in females, WD-induced insulin resistance results in loss of T regulatory (Treg) function leading to macrophage MR activation and subsequent M1 polarization in PVAT. Further, I hypothesize that these events increase vascular stiffness via activation of Tissue transglutaminase-2 (TG2), in part, through oxidative stress and decreased bioavailable nitric oxide (NO). To test my hypothesis, specific Aim 1 is to determine the effect of MR activation in PVAT macrophages on the pathogenesis of vascular stiffness as it relates to impaired Treg function and M1 macrophage polarization. Myeloid-specific MR knockout (MyMRKO) male and female mice will be treated with a WD or aldosterone and we will measure aortic stiffness. In PVAT, we will measure macrophage polarization and inflammation. We will measure cellular stiffness in primary endothelial cells (EC) and vascular smooth muscle cells (VSMC) cultured in PVAT-conditioned media from each cohort. In Specific Aim 2, we will determine the role of Tregs on PVAT macrophage polarization and aortic stiffness. In this set of experiments, we will use adoptive Treg transfer from male and female C57Bl/6J mice fed a normal diet to the mice treated with WD or slow pressor doses of aldosterone. We will measure macrophage polarization, inflammation and aortic stiffness, along with EC/VSMC stiffness measurement in PVAT-conditioned media. I have assembled a team of mentors and collaborators that will guide me throughout this project and promote my transition as an independent researcher. My career development plan has an initial training period (years 1-3) in which I will complete the proposed work, and a transitional period (years 4-5) during which I will focus on an R01 proposal. My mentoring team has the necessary infrastructure, expertise and resources and my institution will provide the necessary support to enable me successfully achieve my goals. My intermediate goal is to successfully complete the work I propose. My ultimate goal is to establish a distinct line of work addressing the mechanisms by which MR activation and dysregulated immunity lead to vascular disease.