PROJECT SUMMARY Obesity causes microvascular dysfunction. The mechanisms that underpin the initiation and progression of microvascular dysfunction are not known. This fundamental gap is a problem because microvascular dysfunction precedes, and likely contributes to, the development of insulin resistance, hypertension, and type-2 diabetes. The perivascular adipose tissue (PVAT) that surrounds vessels plays a physiological role in regulating vascular function. Preliminary data from the applicant’s lab show that proinflammatory macrophages within the PVAT mediate microvascular dysfunction in obesity, and that macrophages release redox species that impinge on microvascular function by modulating crosstalk between vessel nitric oxide and the gaseous signaling molecule hydrogen sulfide. The objective of the current proposal is to define the role of PVAT macrophages in the initiation and progression of microvascular dysfunction during obesity. The objective will be accomplished by testing the central hypothesis that proinflammatory macrophages drive microvascular disease during obesity by infiltrating the PVAT and releasing reactive nitrogen species, which limits nitric oxide bioavailability and increase contractility by depleting vessel hydrogen sulfide. Three specific aims are proposed: aim 1 will define the contribution of PVAT inflammation to the initiation and progression of microvascular disease; aim 2 identify the paracrine signal that mediates communication between PVAT- macrophages and the microvasculature, and aim 3 will define mechanisms by which PVAT-macrophages affect microvascular signal transduction. A diet-induced mouse model of obesity will be used in combination with macrophage depletion and tissue-specific knockouts. A comprehensive in vivo and in vitro analysis of the interaction between PVAT, macrophages, and microvasculature will be performed in both male and female animals by integrating data from intravital microscopy, pressure myography, confocal imaging of live mesenteric arteriolar segments, and metabolic analyses. Completion of these aims will establish a new paradigm in which recruitment of proinflammatory macrophages to the PVAT is a major catalyzing event in the development and progression of microvascular dysfunction.