Abstract Body: Atherosclerosis is characterized by the accumulation of lipid-laden macrophages and cholesterol-filled plaques in the arteries. High-density lipoprotein (HDL) prevents atherosclerosis due to its role in facilitating the excretion of peripheral cholesterol via reverse cholesterol transport (RCT). However, the failure of HDL-cholesterol (HDL-C)-raising therapies to show clinical benefits suggests that HDL function may be a better indicator of cardiovascular risk. HDL can be rendered dysfunctional by reactive aldehydes that result from oxidative stress, causing HDL to lose its antiatherogenic properties. We have demonstrated that HDL modified by reactive aldehydes loses its cardioprotective functions in macrophages along with impaired ability to facilitate key steps in RCT in cultured cells. Based on these data, we aim to determine the in vivo physiological relevance of modified HDL and hypothesize that modified HDL is unable to participate in efficient RCT and, instead, supports progression of atherosclerosis. To test this hypothesis, we chose to use apoA-I knock-out (KO) mice as a model that will allow us to measure the impact of exogenously-injected modified forms of HDL on macrophage-to-feces RCT and atherosclerotic plaque progression due to their low circulating levels of HDL-C. First, we determined that the half-life of HDL in apoA-I KO mice was around 6 hours, as compared to 9 hours in wild-type (WT) mice. To assess RCT ability, plasma and fecal lipid radioactivity from WT and apoA-I KO mice were measured after intraperitoneal (ip) injection of exogenous HDL and [³H]cholesterol-loaded J774 macrophages. We showed that apoA-I KO mice had 63% and 39% decreases in plasma and fecal radioactivity, respectively, as compared to WT mice and that injection of HDL into apoA-I KO mice increased the movement of radiolabeled cholesterol into the plasma and feces. To assess atherosclerosis, apoA-I KO mice were injected ip with AAV-CRISPR-Ldlr and fed a Western diet for 8 or 12 weeks. We show that, compared to mice without AAV-CRISPR-Ldlr that had no plaque, apoA-I KO mice that received AAV-CRISPR-Ldlr had an average aortic sinus lesion area of 0.18 mm² and 5% arch lesion area of total arch after only 8 weeks of Western diet feeding. We now have the tools necessary to move forward with testing the impact reactive aldehyde modification of HDL have on RCT and atherosclerotic plaque progression.