This project is designed to investigate the mechanisms by which cholesterol accumulates in atherosclerotic vessels. Earlier studies of this problem in our laboratory led to the discovery of two types of previously unrecognized cholesterol-containing lipid particles that accumulate in the extracellular spaces of atherosclerotic lesions. One particle is a liposome enriched in unesterified cholesterol and the other particle is a droplet enriched in cholesteryl ester. They are both much larger than HDL and LDL, the major carriers of cholesterol in plasma. Findings of the past year suggest a new hypothesis about the origins of lipid accumulation in atherosclerosis. Certain proteases can induce fusion of LDL to produce larger lipid droplets similar to the cholesteryl ester lipid particles that accumulate in atherosclerotic lesions. Experiments indicate that the protein moiety of LDL is often lost from its cholesteryl ester core in lesions but infrequently lost in normal regions of aorta. Proteases can also destabilize the small nascent HDL particles produced by macrophages as they process lesion cholesterol. The destabilized nascent HDL form larger unesterified cholesterol-rich liposomes similar to those in lesions. Unlike the small nascent HDL, the liposomes are too large to transport cholesterol out of the vessel wall. Thus, protease activity generated within atherosclerotic lesions may transform LDL and HDL into larger lipid particles that become trapped. These cholesterol-rich particles may not only be constituents of a frustrated reverse cholesterol transport process, but also may be a link to the subsequent calcification that occurs in lesions. Calcification promotes lesion rupture resulting in thrombosis and myocardial infarction. We have obtained experimental evidence that these particles nucleate the deposition of hydroxyapatite, the specific form of calcium crystal which accumulates in lesions. Continued studies of the mechanisms that cause LDL to deposit its cholesteryl ester core and impair its resolubilization will suggest new ways to treat atherosclerotic vascular disease.