Although high plasma concentrations of LDL-C (“bad cholesterol”) are associated with atherosclerotic cardiovascular disease (ACVD), statins reduce plasma LDL-C and with it ACVD. In contrast, high density lipoprotein-cholesterol (HDL-C; “good cholesterol”) varies inversely with ACVD. However attempts to reduce ACVD via increased plasma HDL-C levels have failed. New evidence suggests that HDL quality is more important than quantity and that its ability to remove free cholesterol (FC) from macrophages (MΦ), an important cell type in ACVD, is its most important atheroprotective quality. This process, MΦ-FC efflux, initiates the FC transfer to the intestine for disposal—an atheroprotective process. Paradoxically, patients with very high plasma HDL-C levels are at high ACVD risk; the underlying mechanism is unknown, and currently there are no interventions that reverse high HDL-C levels in a cardioprotective way. We hypothesize that the underlying cause of ACVD in patients with very high plasma HDL-C levels is too much HDL that contains high amounts of FC, which transfers freely among cells and lipoproteins. This state makes FC highly bioavailable so that rather than removing FC from the arterial wall, FC-rich HDL transfers FC to arterial-wall MΦ—an atherogenic process. Using a mouse model of ACVD with underlying high HDL-C levels (SR-B1-/- mouse) we plan to identify HDL-FC bioavailability as a driver of ACVD and show that treatment with an HDL-lowering bacterial protein (serum opacity factor), delivered with an adeno-associated virus prevents/reverses ACVD. Within this ACVD-HDL axis, we propose the following specific aims: Aim 1—To compare the plasma clearance kinetics of wild-type and SR-B1-/- HDL-[3H]FC and cholesteryl ester (CE) in wild-type and SR-B1-/- mice, simultaneously identifying the tissue sites of [3H]FC and [3H]CE accretion, and the effects of AAVSOF vs. AAVGFP on these kinetics and tissue distributions. Aim 2a—To test the hypothesis that FC flux between HDL and J774 MΦ switches from efflux to influx with increasing HDL-FC bioavailability, which is a function of HDL particle concentration and HDL-FC content (mol% FC). Aim 2b—Concurrently with Aim 1, to test the hypothesis that HMGCoA reductase and ACAT activities decrease and increase, respectively, MΦ-FC content as effected by increasing HDL-FC bioavailability. Aim 2c— To test the hypothesis that increased HDL-FC bioavailability induces foam cell formation in J774 MΦ. Aim 3—To test the hypothesis that reduction of HDL-FC by AAVSOF vs. AAVGFP delivery prevents and/or reverses atherosclerosis in SR-B1-/- mice. Completion of these aims will provide a compelling rationale ●for studies to determine whether high plasma HDL- FC is associated with ACVD in patients with high HDL-C and ●for the development of drugs that lower HDL-FC.