Project Summary Macrophages are ubiquitous innate immune cells that display tissue-specific function and phenotype. Microenvironmental factors derived from a tissue niche can regulate the development of tissue specific macrophages (TSM). The molecular basis of the specialization of TSM remains poorly understood. Perivascular macrophages (pvMAC) are TSMs that can regulate vascular permeability and immune responses to blood-borne pathogens. Whether and how factors unique to the vascular or perivascular microenvironment regulate pvMACs is currently unknown. This proposal will address these key knowledge gaps. Preliminary work in our laboratory showed that pvMACs in murine fibrosarcoma tumors promote angiogenesis and immunosuppression. We discovered that pvMACs have high intracellular iron and express the GPCR endothelin receptor-B (EdnrB). Macrophage-specific deletion of EdnrB led to reduced vascular density in tumors. I also found that heme induces EdnrB expression in macrophages by inhibiting the transcription factor Bach1. Outside of tumors, I have found that pvMACs express EdnrB in lung and adipose tissue. However, we do not know whether EdnrB expression is a common feature of all pvMACs and whether heme regulates EdnrB expression in these cells. The expression of EdnrB in pvMACs close to the source of the endothelin ligand (endothelial cells) and heme (circulating erythrocytes) leads us to hypothesize that increased heme availability in the perivascular niche drives EdnrB expression in pvMACs through degradation of Bach1 and EdnrB signaling in pvMACs regulates vascular function. In Aim 1, I will assess the iron content and transcriptional profile of pvMACs in adipose tissue through single cell RNA sequencing. In Aim 2, I will identify the function of EdnrB signaling in adipose tissue pvMACs using macrophage-specific EdnrB KO mouse and reductionist ex vivo approaches. In Aim 3, I will determine the role of Bach1 in pvMAC development and function using a Bach1 KO mouse and a model of intravascular hemolysis. Better understanding of the biology of pvMACs is essential given the central importance of the vascular system. This work has far-reaching implications for many pathological conditions, such as hemolysis and ischemia. Endothelin signaling can be targeted by existing small molecule inhibitors. Hence, this work also has the potential to be translated into appropriate clinical contexts.