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HDL lowers the risk for atherosclerotic cardiovascular disease by promoting cholesterol efflux from macrophage foam cells. However, other antiatherosclerotic properties of HDL are poorly understood. To test the hypothesis that the lipoprotein carries proteins that might have novel cardioprotective activities, we used shotgun proteomics to investigate the composition of HDL isolated from healthy subjects and subjects with coronary artery disease (CAD). Unexpectedly, our analytical strategy identified multiple complement-regulatory proteins and a diverse array of distinct serpins with serine-type endopeptidase inhibitor activity. Many acute-phase response proteins were also detected, supporting the proposal that HDL is of central importance in inflammation. Mass spectrometry and biochemical analyses demonstrated that HDL3 from subjects with CAD was selectively enriched in apoE, raising the possibility that HDL carries a unique cargo of proteins in humans with clinically significant cardiovascular disease. Collectively, our observations suggest that HDL plays previously unsuspected roles in regulating the complement system and protecting tissue from proteolysis and that the protein cargo of HDL contributes to its antiinflammatory and antiatherogenic properties.

Clearance of excess cholesterol from cells by HDL is facilitated by the interaction of HDL apolipoproteins with cell-surface binding sites or receptors, a process that may be important in preventing atherosclerosis. In this study, synthetic peptides containing 18-mer amphipathic helices of the class found in HDL apolipoproteins (class A) were tested for their abilities to remove cholesterol and phospholipid from cultured sterol-laden fibroblasts and macrophages and to interact with cell-surface HDL binding sites. Lipid-free peptides containing two identical tandem repeats of class A amphipathic helices promoted cholesterol and phospholipid efflux from cells and depleted cellular cholesterol accessible for esterification by acyl CoA/cholesterol acyltransferase, similar to what was observed for purified apolipoprotein A-I. Peptide-mediated removal of plasma membrane cholesterol and depletion of acyl CoA/cholesterol acyltransferase-accessible cholesterol appeared to occur by separate mechanisms, as the latter process was less dependent on extracellular phospholipid. The dimeric amphipathic helical peptides also competed for high-affinity HDL binding sites on cholesterol-loaded fibroblasts and displayed saturable high-affinity binding to the cell surface. In contrast, peptides with a single helix had little or no ability to remove cellular cholesterol and phospholipid, or to interact with HDL binding sites, suggesting that cooperativity between two or more helical repeats is required for these activities. Thus, synthetic peptides comprising dimers of a structural motif common to exchangeable apolipoproteins can mimic apolipoprotein A-I in both binding to putative cell-surface receptors and clearing cholesterol from cells.

The ABC1 transporter was identified as the defect in Tangier disease by a combined strategy of gene expression microarray analysis, genetic mapping, and biochemical studies. Patients with Tangier disease have a defect in cellular cholesterol removal, which results in near zero plasma levels of HDL and in massive tissue deposition of cholesteryl esters. Blocking the expression or activity of ABC1 reduces apolipoprotein-mediated lipid efflux from cultured cells, and increasing expression of ABC1 enhances it. ABC1 expression is induced by cholesterol loading and cAMP treatment and is reduced upon subsequent cholesterol removal by apolipoproteins. The protein is incorporated into the plasma membrane in proportion to its level of expression. Different mutations were detected in the ABC1 gene of 3 unrelated patients. Thus, ABC1 has the properties of a key protein in the cellular lipid removal pathway, as emphasized by the consequences of its defect in patients with Tangier disease.