Objective-The purpose of this study was to define heterogeneity in the molecular profile of lipids, including sphingomyelin and sphingosine-1-phosphate, among physicochemically-defined HDL subpopulations and potential relevance to antiatherogenic biological activities of dense HDL3. Methods and Results-The molecular profile of lipids (cholesteryl esters, phospholipids, sphingomyelin, and sphingosine-1-phosphate) in physicochemically-defined normolipidemic HDL subpopulations was determined by high-performance liquid chromatography and gas chromatography. As HDL particle size and molecular weight decreased with increment in density, molar lipid content diminished concomitantly. On a % basis, sphingomyelin abundance diminished in parallel with progressive increase in HDL density from HDL2b (12.8%) to HDL3c (6.2%; PϽ0.001); in contrast, sphingosine-1-phosphate was preferentially enriched in small HDL3 (40 to 50 mmol/mol HDL) versus large HDL2 (15 to 20 mmol/mol HDL; PϽ0.01). Small HDL3c was equally enriched in LpA-I particles relative to LpA-I:A-II. The sphingosine-1-phosphate/sphingomyelin ratio correlated positively with the capacities of HDL subspecies to attenuate apoptosis in endothelial cells (rϭ0.73, PϽ0.001) and to retard LDL oxidation (rϭ0.58, PϽ0.01). Conclusions-An elevated sphingosine-1-phosphate/sphingomyelin ratio is an integral feature of small dense HDL3, reflecting enrichment in sphingosine-1-phosphate, a key antiapoptotic molecule, and depletion of sphingomyelin, a structural lipid with negative impact on surface fluidity and LCAT activity. These findings further distinguish the structure and antiatherogenic activities of small, dense HDL. Key Words: sphingolipids Ⅲ phospholipid molecular species Ⅲ cholesteryl esters Ⅲ HDL particle remodeling L ow circulating levels of high density lipoprotein (HDL)-cholesterol (HDL-C) constitute a major independent and predictive cardiovascular (CV) risk factor; in contrast, elevated HDL-C concentrations may be atheroprotective. 1 Indeed, HDL particles possess multiple antiatherogenic properties, including cellular cholesterol efflux capacity as well as antiapoptotic, antioxidative, antiinflammatory, and vasodilatory activities. 1 Such diversity in biological function is intimately related to the marked heterogeneity of HDL particles, which present as a continuum of subpopulations distinct in physicochemical properties, structure, and intravascular metabolism.The biological activities and atheroprotective function of HDL are inseparably linked to the physicochemical properties of both lipid and protein moieties, and equally to particle structure. Indeed, small, dense, lipid-poor HDL particles possess elevated capacities to accept cellular cholesterol, 2 to inhibit cellular expression of adhesion molecules, 3 and to protect LDL from oxidation 4 as compared with large, light, lipid-rich HDL. The intravascular metabolism of lipid components of HDL is regulated by cholesteryl ester transfer protein (CETP), lecithin:cholesterol acyltransferase (LCAT), phospholipid tr...

Plasma high-density lipoproteins (HDLs) protect endothelial cells against apoptosis induced by oxidized low-density lipoprotein (oxLDL). The specific component(s) of HDLs implicated in such cytoprotection remain(s) to be identified. Human microvascular endothelial cells (HMEC-1) were incubated with mildly oxLDL in the presence or absence of each of five physicochemically distinct HDL subpopulations fractionated from normolipidemic human plasma (n= 7) by isopycnic density gradient ultracentrifugation. All HDL subfractions protected HMEC-1 against oxLDL-induced primary apoptosis as revealed by nucleic acid staining, annexin V binding, quantitative DNA fragmentation, inhibition of caspase-3 activity and reduction of cytoplasmic release of cytochrome c and apoptosis-inducing factor. Small, dense HDL 3c displayed twofold superior intrinsic cytoprotective activity (as determined by mitochondrial dehydrogenase activity) relative to large, light HDL 2b on a per particle basis (P < 0.05). Equally, all HDL subfractions attenuated intracellular generation of reactive oxygen species (ROS); such anti-oxidative activity diminished from HDL 3c to HDL 2b. The HDL protein moiety, in which apolipoprotein A-I (apoA-I) predominated, accounted for ∼70% of HDL anti-apoptotic activity. Furthermore, HDL reconstituted with apoA-I, cholesterol and phospholipid potently protected HMEC-1 from apoptosis. By contrast, modification of the content of sphingosine-1-phosphate in HDL did not significantly alter cytoprotection. We conclude that small, dense, lipid-poor HDL 3 potently protects endothelial cells from primary apoptosis and intracellular ROS generation induced by mildly oxLDL, and that apoA-I is pivotal to such protection.