Lipid Droplets of Human Carotid Atherosclerotic Plaques: Cholesteryl Arachidonate Core and Cytoskeletal Fibrotic Cover

Lipid droplets (LDs) within atherosclerotic plaques are central to cardiovascular disease, yet their molecular composition remains largely unknown. More than 20 years experiences of developing LD purification method allowed us recently to successfully purify LDs from atherosclerotic plaques of patients undergoing carotid artery surgery, including three types, soft (lipid), mixed, and hard (calcified). In addition, the subpopulations of large LDs and small LDs were purified and studied, respectively. We report the first comprehensive proteo-lipidomic analysis of these LDs. Unexpectedly, about 19.3% of the plaque LD proteins belonged to cytoskeleton, while the content of lipid metabolism enzymes on plaque LDs was low. Our results show that the compositions of lipids and proteins were similar between two population LDs. Compared to soft (lipid) plaque LDs, LDs from mixed and hard (calcified) plaques had fewer metabolic enzymes and more cytoskeletal proteins. Lipidomics of the LD core uncovered an unexpected enrichment of polyunsaturated fatty acid cholesteryl esters (PUFA-CEs), which constituted 88.3% of all cholesterol esters. Notably, esters of the pro-inflammatory precursor arachidonic acid (AA) were among the most abundant species (16.5%). In addition, plaque LD were uniquely enriched in phospholipids including phosphatidic acid (PA), phosphatidylethanolamine plasmagens (PE-P), sphingomyelin (SM), and phosphatidylserine (PS). Our findings redefine the plaque LD as a dual-function “bad” organelle: a metabolically inert container for cholesterol ester, coated with cytoskeletons, and a massive reservoir for inflammatory precursors.