Project Summary/Abstract Cardiovascular disease is a leading cause of morbidity and mortality in the United States. Atherosclerosis is an inflammatory disease of the large and mid-sized arteries that promotes the development of cardiovascular disease through plaque formation, restricted blood flow, and ultimately thrombotic events. Chronic exposure of the arterial endothelium to elevated (and potentially modified) serum cholesterol leads to the hallmarks of pathology; including altered blood flow patterns, endothelial activation, and recruitment of blood monocytes. Infiltrating monocytes differentiate into lipid-laden plaque macrophages and contribute to the necrotic core that is a hallmark of advanced lesions. Research has focused on the role of monocytes in the maintenance of atherosclerotic plaques however; relatively few resources have been dedicated to investigating the role of tissue-resident mononuclear phagocytes (MNPs), which reside in the aortic arch. Resident MNPs reside in the nascent aorta at birth in locations where plaques have propensity to develop, and the cells are even present there in animals not susceptible to disease. My preliminary studies definitively show these cells are macrophage lineage cells, not dendritic cells as previously reported and may facilitate the onset of early plaque by importing the first quantitative load of cholesterol into the intima. Thus, these cells may be pivotal in regulating atherosclerotic plaque burden. However, technical approaches to determine if intimal resident macrophages persist in plaques and carry out distinct roles have not emerged to address this possibility. I have established protocols to track and modulate the resident intimal macrophage population independent of infiltrating blood-derived precursors like monocytes. I find that resident macrophages are the first cells to take up lipid in the aortic wall and persist through plaque development. Resident macrophages reside inside regions known to develop into necrotic core, and also form a “boarder” around the plaque itself. Further, I have developed approaches to track proliferation of macrophages within the plaque, as well as use new technologies to isolate intimal macrophages without contamination from monocytes or adventitial macrophages. Primarily using a combination of fixed- and live-imaging approaches in murine atherosclerotic plaque and taking advantage of new mouse models, I will test the hypothesis that resident aortic intimal macrophages, independent of recruited monocyte-derived macrophages, play a unique and important role in atherosclerotic plaque development. To test my hypothesis, I will assess the role of proliferation and motility in the plaques, address the differential roles of resident versus recruited monocytes in disease progression, and further develop intravital approaches to understand how interactions between monocytes and intimal macrophages may contribute to disease severity. If true, the implications o...