Cardiovascular disease (CVD) is a public health challenge with a major economic and quality-of-life impact on its victims and their families. Impressive inroads have been made in the management of some CVD lipid risk factors; the statin class of hypolipidemic drugs reduces the number of CVD events by reducing plasma levels of low density lipoprotein-cholesterol. Reduction in CVD via the modification of other lipid-risk factors has been a challenge, especially raising the plasma concentrations of high density lipoproteins-cholesterol (HDL-C), in a cardioprotective way; attempts to reduce CVD via raising HDL-C have not been uniformly successful. Many scientists now appreciate that the mechanistic connections between HDL and atheroprotection are more complex than once thought and that a greater focus on HDL function, rather that HDL-C concentrations alone, is needed. HDL plays a role in reverse cholesterol transport (RCT), the transport of cholesterol in peripheral tissues, including macrophages in the subendothelial space of the arterial wall, to the liver for disposal. The major RCT steps are macrophage cholesterol efflux, HDL-C esterification in plasma, and hepatic disposal, which directs some cholesterol to excretion thereby reducing the total body cholesterol burden. Recent evidence is that not all HDL are the same, i.e., HDL are speciated according to composition, structure, and likely function. Several studies have shown cholesterol efflux to apo B-depleted plasma is lower in CVD patients, independent of HDL-C concentrations. This difference must be due to differences in the ?quality? of HDL in patients vs. those without CVD. Some HDL species may be better cholesterol acceptors; apo AI may play a direct role in efflux. Moreover, mimetics based on an optimized apo AI structure may be a promising therapeutic strategy. The aims of this application focus on three aspects of HDL and apo AI structure and/or function? speciated biogenesis of HDL with intact signal peptides (SP); the mechanism of efflux from macrophages giving nascent HDL and microsolubilization as a surrogate for nascent HDL formation, and optimization of apo AI structure for RCT. This background and rationale gave rise to three aims: Aim 1: To test the hypothesis that the biogenesis of HDL containing apos with intact signal peptides (SPs) is unique and speciated, and forms unique HDL species with distinct compositions and functions. Aim 2: To test a novel, hypothetical ?pancake? model of phospholipid microsolubilization by apo AI, which yields rHDL, the in vitro analog of nascent HDL produced by hepatocytes and M? cholesterol efflux. Aim 3: To test the hypothesis that conserved amino acid residues in apo AI are essential to its structure and function. Completion of these aims will reveal new molecular targets for cardioprotective HDL therapies. These aims will be addressed by multidisciplinary in vitro and in vivo approaches that includes chemical kinetics, cell biology, lipid and protein analyses, AAV-mediated gene delivery in vivo, high performance thin-layer chromatography, immune-blotting and precipitation methods, chemical cross-linking, MALDI MS, site-directed mutagenesis, circular dichroism, and kinetic turbidimetry.