PROJECT SUMMARY. A great accomplishment of modern medicine is the increased lifespan of the population worldwide. Unfortunately, longevity also increased the healthcare burden due to comorbidities of aging such as atherosclerotic cardio- vascular disease (CVD). Overwhelming evidence indicates that hyperlipidemia is a main driver of CVD, which promotes an inflammatory response by vasculature-resident macrophages. Other inflammatory cells, chiefly among them T-cells, are also recruited to growing plaques and play a fundamental role in their remodeling and susceptibility to rupture. In women, it is well recognized that loss of ovarian hormones at menopause is an independent risk factor for cardiovascular events. Current therapies, such as statins, lower circulating LDL- cholesterol and have been clinically demonstrated to decrease mortality in the elderly. Yet, despite the use of statins for over five decades, atherosclerosis still remains the leading cause of death in men and women globally. These observations indicate that 1) there are additional, yet unidentified, lipid-independent mechanisms that contribute to atheroprogression and plaque rupture; and 2) additional therapies are needed to treat ASCVD. Unpublished preliminary data from our laboratories in mice show that aging in both sexes and loss of estrogen in females promotes T-cell mediated inflammation. We hypothesize that this age- and menopause-dependent inflammation exacerbates underlying CVD such that further lowering cholesterol does not provide added benefit. Here we will test the idea that promoting an anti-inflammatory, pro-resolving environment in the plaque is atheroprotective. To accomplish this, we will use treat mice with pulsed low-dose RANKL (pRL), which we show induces plaque resident regulatory CD8+FoxP3+CD25+CTLA4+ T-cells that secrete IL-10. If successful, pRL will be a novel therapeutic for treating ASCVD, that works by mechanism distinct from therapies currently in use. Additionally, the proposed studies will deliver two key innovations. First, we will compare atheromata in young (2-month-old) and old (18-month-old at beginning of intervention, corresponding to ~60-year-old human) male and female mice. Second, old females will be ovariectomized to mimic human menopause. Thus, in females we will segregate the effects of aging and menopause on plaque development. We will comprehensively map athero-prone proximal aorta transcriptomic profiles at a single-cell resolution, focusing on inflammatory cell clusters, cell-to-cell communication networks, T-cell-mediated dysfunction of smooth muscle cells and endothelial cells, markers linked to plaque stability/vulnerability, and transcripts encoding predicted secreted proteins (secretome). The integrated mechanistic data obtained with our unprecedented discovery approach will have a lasting impact in the field of vascular physiology and pathology because most published murine atherosclerosis studies have systematically ignored th...