Project Summary: Despite antiretroviral therapy (ART), people living with HIV (PLWH) have a two-fold increased risk of cardiovascular disease (CVD) compared with the general population. While persistent systemic inflammation and immune activation appear to predict this risk, it has been challenging to identify the pathophysiologic pathways that can be targeted to reduce inflammation and cardiovascular risk in treated HIV infection. Extracellular vesicles (EVs) play a significant role in regulating pathophysiologic pathways like inflammation and angiogenesis, and they are implicated in key aspects of heart disease. To date, limited data exist on the molecular mechanisms underlying the effect of EVs on atherosclerosis progression, leaving open the question of how EVs might impact the resolution of inflammation to influence CVD risk in HIV. This proposal is innovative in that it will employ high-throughput testing platforms for characterizing EVs and their cargo on longitudinal samples from well-characterized cohorts of persons with HIV and CVD and will utilize a novel mouse model of HIV atherosclerosis and cutting-edge in vitro models to identify and validate novel inflammatory mediators carried by EVs and provide potential novel targets for intervention. In Aim 1, we will test the hypothesis that 1) Immunologically active EVs derived from HIV+ adults on ART will carry pro- inflammatory mediators that will be associated with carotid artery intima-media thickness progression; and 2) EVs from HIV+ adults with higher CVD risk will carry a unique miRNA signature and will be associated with a specific inflammatory pathway (e.g. NLRP3 inflammasome signaling/caspase-1 activation). In Aim 2, we will develop a novel accelerated atherosclerosis mouse model associated with HIV infection to test the hypothesis that 1) Systemic administration of EVs derived from HIV-1 Tg26/ApoE−/− mice will more strongly induce production of proinflammatory mediators when infused into recipient mice and will transfer molecules that alter exosomal miRNA expression in recipient cells; 2) DNA-containing EVs from HIV-1 Tg26/ApoE−/−mice will induce epigenetic changes in recipient cells; and 3) the administration of EVs derived from VX-765 or MCC950-treated atherosclerotic mice will produce circulating EVs that lack the ability to affect the exosomal miRNA expression in recipient cells. In Aim 3, we will test the hypothesis that 1) EVs from those with high CVD risk will directly cause cardiomyocyte and endothelial cell dysfunction and promote increased cell death compared to controls; 2) Caspase inhibitors (caspase-1/inflammasome inhibition) will mitigate apoptosis in cardiomyocytes and endothelial cells mediated by EVs derived from those with high CVD risk; and 3) EV signaling will be dependent on EV RNA content for delivery of a pro-inflammatory signal. We also will focus on the development of genome-wide CRISPR screens to identify the causal variants, genes, and molecular mechanis...