PROJECT SUMMARY Obesity is a major risk factor for cardiovascular diseases (CVD) such as atherosclerosis, hypertension, type 2 diabetes and stroke. Although it is well documented that EC dysfunction is present in most forms of CVD, the mechanisms by which obesity shapes the development of EC dysfunction remains poorly understood. Further complicating the understanding of EC dysfunction is the heterogeneity of ECs throughout the vasculature. Recent studies have demonstrated a large degree of divergence between EC subtypes in the macrocirculation (aorta) that can be further altered with obesogenic diets over a short period of time. Our lab and others have shown that the microvasculature is particularly susceptible to obesity induced dysfunction and is a key predictor of end-organ damage. In the microvasculature of obese mice, we have shown a striking increase in NADPH oxidase 1 (NOX1) and histone deacetylase 9 (HDAC9), two molecules that have been shown to be important contributors to endothelial dysfunction and phenotypic changes, respectively. As of the writing of this proposal, no studies have been conducted to elucidate the heterogeneity of EC in the microcirculation, not to mention the effect of obesity on these populations or whether or not NOX1 and HDAC9 are preferentially expressed in certain populations over others. To perform these experiments in a time and cost effective manner, we have developed an AAV approach to deliver AgRP, an orexigenic peptide, specifically to the brain to induce hyperphagia and obesity. This novel approach in conjunction with endothelial specific lineage tracing mice and endothelial specific HDAC9 knockout mice will facilitate testing of our central hypothesis that obesity alters EC heterogeneity in the microcirculation to favor inflammatory EC phenotypes expressing NOX1. Aim 1 will define, through an obese endothelial specific lineage tracing model, the transcriptomic and phenotypic changes underlying loss of endothelial function in the microcirculation of obese mice. Using single cell RNA sequencing of fate mapped ECs from lean and obese mice, we will elucidate the subtypes of ECs expressing NOX1 and interrogate co-expression patterns. Aim 2 will test the hypothesis that HDAC9 has a causal role in increased expression of NOX1 and microvascular endothelial dysfunction and that weight loss reverses the expression of HDAC9 and NOX1 and restores endothelial function. We will assess in vitro and in vivo, whether HDAC9 drives the expression of NOX1 and whether its deletion ameliorates endothelial dysfunction in obesity. We will also investigate the effects of weight loss on microvascular endothelial function using a novel floxed AgRP AAV that enables cessation of AgRP expression with the goal of promoting weight loss. We anticipate the findings of this proposal will reveal new mechanisms contributing to endothelial dysfunction in obesity and will provide insight into potential therapeutic targets to improve cardiovas...