John F. Oram

Shotgun proteomics implicates protease inhibition and complement activation in the antiinflammatory properties of HDL.

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.

The Tangier disease gene product ABC1 controls the cellular apolipoprotein-mediated lipid removal pathway

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.

ABCA1 Is the cAMP-inducible Apolipoprotein Receptor That Mediates Cholesterol Secretion from Macrophages

Lipid-poor high density lipoprotein apolipoproteins remove cholesterol and phospholipids from cells by an active secretory pathway controlled by an ABC transporter called ABCA1. This pathway is induced by cholesterol and cAMP analogs in a cell-specific manner. Here we provide evidence that increased plasma membrane ABCA1 accounts for the enhanced apolipoprotein-mediated lipid secretion from macrophages induced by cAMP analogs. Treatment of RAW264 macrophages with 8-bromo-cAMP caused parallel increases in apoA-I-mediated cholesterol efflux, ABCA1 mRNA and protein levels, incorporation of ABCA1 into the plasma membrane, and binding of apoA-I to cell-surface ABCA1. All of these parameters declined to near base-line values within 6 h after removal of 8-bromo-cAMP, indicating that ABCA1 is highly unstable and is degraded rapidly in the absence of inducer. Thus, ABCA1 is likely to be the cAMP-inducible apolipoprotein receptor that promotes removal of cholesterol and phospholipids from macrophages.

Defective removal of cellular cholesterol and phospholipids by apolipoprotein A-I in Tangier Disease.

Tangier disease is a rare genetic disorder characterized by extremely low plasma levels of HDL and apo A-I, deposition of cholesteryl esters in tissues, and a high prevalence of cardiovascular disease. We examined the possibility that HDL apolipoprotein-mediated removal of cellular lipids may be defective in Tangier disease. With fibroblasts from normal subjects, purified apo A-I cleared cells of cholesteryl esters, depleted cellular free cholesterol pools available for esterification, and stimulated efflux of radiolabeled cholesterol, phosphatidylcholine, and sphingomyelin. With fibroblasts from two unrelated Tangier patients, however, apo A-I had little or no effect on any of these lipid transport processes. Intact HDL also was unable to clear cholesteryl esters from Tangier cells even though it promoted radiolabeled cholesterol efflux to levels 50-70% normal. Passive desorption of radiolabeled cholesterol or phospholipids into medium containing albumin or trypsinized HDL was normal for Tangier cells. Binding studies showed that the interaction of apo A-I with high-affinity binding sites on Tangier fibroblasts was abnormal. These results indicate that apo A-I has an impaired ability to remove cholesterol and phospholipid from Tangier fibroblasts, possibly because of a defective interaction of apo A-I with cell-surface binding sites. Failure of apo A-I to acquire cellular lipids may account for the rapid catabolism of nascent HDL particles and the low plasma HDL levels in Tangier disease.

ATP-Binding Cassette Cholesterol Transporters and Cardiovascular Disease

A hallmark of atherosclerotic cardiovascular disease (CVD) is the accumulation of cholesterol in arterial macrophages. Factors that modulate circulating and tissue cholesterol levels have major impacts on initiation, progression, and regression of CVD. Four members of the ATP-binding cassette (ABC) transporter family play important roles in this modulation. ABCA1 and ABCG1 export excess cellular cholesterol into the HDL pathway and reduce cholesterol accumulation in macrophages. ABCG5 and ABCG8 form heterodimers that limit absorption of dietary sterols in the intestine and promote cholesterol elimination from the body through hepatobiliary secretion. All 4 transporters are induced by the same sterol-sensing nuclear receptor system. ABCA1 expression and activity are also highly regulated posttranscriptionally by diverse processes. ABCA1 mutations can cause a severe HDL-deficiency syndrome characterized by cholesterol deposition in tissue macrophages and prevalent atherosclerosis. ABCG5 or ABCG8 mutations can cause sitosterolemia, in which patients accumulate cholesterol and plant sterols in the circulation and develop premature CVD. Disrupting Abca1 or Abcg1 in mice promotes accumulation of excess cholesterol in macrophages, and manipulating mouse macrophage ABCA1 expression affects atherogenesis. Overexpressing ABCG5 and ABCG8 in mice attenuates diet-induced atherosclerosis in association with reduced circulating and liver cholesterol. Metabolites elevated in individuals with the metabolic syndrome and diabetes destabilize ABCA1 protein and inhibit transcription of all 4 transporters. Thus, impaired ABC cholesterol transporters might contribute to the enhanced atherogenesis associated with common inflammatory and metabolic disorders. Their beneficial effects on cholesterol homeostasis have made these transporters important new therapeutic targets for preventing and reversing CVD.

Human atherosclerotic intima and blood of patients with established coronary artery disease contain high density lipoprotein damaged by reactive nitrogen species

High density lipoprotein (HDL) is the major carrier of lipid hydroperoxides in plasma, but it is not yet established whether HDL proteins are damaged by reactive nitrogen species in the circulation or artery wall. One pathway that generates such species involves myeloperoxidase (MPO), a major constituent of artery wall macrophages. Another pathway involves peroxynitrite, a potent oxidant generated in the reaction of nitric oxide with superoxide. Both MPO and peroxynitrite produce 3-nitrotyrosine in vitro. To investigate the involvement of reactive nitrogen species in atherogenesis, we quantified 3-nitrotyrosine levels in HDL in vivo. The mean level of 3-nitrotyrosine in HDL isolated from human aortic atherosclerotic intima was 6-fold higher (619 ± 178 μmol/mol Tyr) than that in circulating HDL (104 ± 11 μmol/mol Tyr; p < 0.01). Immunohistochemical studies demonstrated striking colocalization of MPO with epitopes reactive with an antibody to 3-nitrotyrosine. However, there was no significant correlation between the levels of 3-chlorotyrosine, a specific product of MPO, and those of 3-nitrotyrosine in lesion HDL. We also detected 3-nitrotyrosine in circulating HDL, and linear regression analysis demonstrated a strong correlation between the levels of 3-chlorotyrosine and levels of 3-nitrotyrosine. These observations suggest that MPO promotes the formation of 3-chlorotyrosine and 3-nitrotyrosine in circulating HDL but that other pathways also produce 3-nitrotyrosine in atherosclerotic tissue. Levels of HDL isolated from plasma of patients with established coronary artery disease contained twice as much 3-nitrotyrosine as HDL from plasma of healthy subjects, suggesting that nitrated HDL might be a marker for clinically significant vascular disease. The detection of 3-nitrotyrosine in HDL raises the possibility that reactive nitrogen species derived from nitric oxide might promote atherogenesis. Thus, nitrated HDL might represent a previously unsuspected link between nitrosative stress, atherosclerosis, and inflammation.

ABCA1-mediated transport of cellular cholesterol and phospholipids to HDL apolipoproteins.

Lipid-poor apolipoproteins remove cellular cholesterol and phospholipids by an active transport pathway controlled by an ATP binding cassette transporter called ABCA1 (formerly ABC1). Mutations in ABCA1 cause Tangier disease, a severe HDL deficiency syndrome characterized by a rapid turnover of plasma apolipoprotein A-I, accumulation of sterol in tissue macrophages, and prevalent atherosclerosis. This implies that lipidation of apolipoprotein A-I by the ABCA1 pathway is required for generating HDL particles and clearing sterol from macrophages. Thus, the ABCA1 pathway has become an important therapeutic target for mobilizing excess cholesterol from tissue macrophages and protecting against atherosclerosis.

ABCG1 redistributes cell cholesterol to domains removable by high density lipoprotein but not by lipid-depleted apolipoproteins.

ATP binding cassette transporter G1 (ABCG1) mediates the transport of cholesterol from cells to high density lipoprotein (HDL) but not to lipid-depleted apolipoprotein A-I. Here we show that human ABCG1 overexpressed in baby hamster kidney cells in the absence of lipoproteins traffics to the plasma membrane and redistributes membrane cholesterol to cell-surface domains accessible to treatment with the enzyme cholesterol oxidase. Cholesterol removed by HDL was largely derived from these domains in ABCG1 transfectants but not in cells lacking ABCG1. Overexpression of ABCG1 also increased cholesterol esterification, which was decreased by the addition of HDL, suggesting that a proportion of the cell-surface cholesterol not removed by HDL is transported to the intracellular esterifying enzyme acyl-CoA:cholesterol acyltransferase. A 638-amino acid ABCG1, which lacked the 40 N-terminal amino acids of the predicted full-length protein, was fully functional and of a similar size to ABCG1 expressed by cholesterol-loaded human monocyte-derived macrophages. Mutating an essential glycine residue in the Walker A motif abolished ABCG1-dependent cholesterol efflux and esterification and prevented localization of ABCG1 to the cell surface, indicating that the ATP binding domain in ABCG1 is essential for both lipid transport activity and protein trafficking. These studies show that ABCG1 redistributes cholesterol to cell-surface domains where it becomes accessible for removal by HDL, consistent with a direct role of ABCG1 in cellular cholesterol transport.

ABCA1 and ABCG1 or ABCG4 act sequentially to remove cellular cholesterol and generate cholesterol-rich HDL

Recent developments in lipid metabolism have shown the importance of ATP binding cassette transporters (ABCs) in controlling cellular and total body lipid homeostasis. ABCA1 mediates the transport of cholesterol and phospholipids from cells to lipid-poor apolipoprotein A-I (apoA-I), whereas ABCG1 and ABCG4 mediate the transport of cholesterol from cells to lipidated lipoproteins. ABCA1, ABCG1, and ABCG4 are all expressed in cholesterol-loaded macrophages, and macrophages from ABCA1 and ABCG1 knockout mice accumulate cholesteryl esters. Here, we show that the lipidated particles generated by incubating cells overexpressing ABCA1 with apoA-I are efficient acceptors for cholesterol released from cells overexpressing either ABCG1 or ABCG4. The cholesterol released to the particles was derived from a cholesterol oxidase-accessible plasma membrane pool in both ABCG1 and ABCG4 cells, which is the same pool of cholesterol shown previously to be removed by high density lipoproteins. ABCA1 cells incubated with apoA-I generated two major populations of cholesterol- and phospholipid-rich lipoprotein particles that were converted by ABCG1 or ABCG4 cells to one major particle population that was highly enriched in cholesterol. These results suggest that ABCG1 and ABCG4 act in concert with ABCA1 to maximize the removal of excess cholesterol from cells and to generate cholesterol-rich lipoprotein particles.

Regulation of High Density Lipoprotein Receptor Activity in Cultured Human Skin Fibroblasts and Human Arterial Smooth Muscle Cells

Cultured human skin fibroblasts and human arterial smooth muscle cells possess high-affinity binding sites specific for high density lipoproteins (HDL). Results from the present study demonstrate that binding of HDL to these sites is up-regulated in response to cholesterol loading of cells. When fibroblasts or smooth muscle cells were preincubated with nonlipoprotein cholesterol, cellular binding of 125I-HDL3 was enhanced severalfold. This enhancement was sustained in the presence of cholesterol but was readily reversed when cells were exposed to cholesterol-free medium. The stimulatory effect of cholesterol treatment was prevented by cycloheximide, suggesting the involvement of protein synthesis. Kinetic analysis of HDL3 binding showed that prior exposure to cholesterol led to an induction of high-affinity binding sites on the cell surface. In the up-regulated state, the apparent dissociation constant (Kd) of these sites was approximately 2 micrograms protein/ml. Competition studies indicated that the HDL binding sites recognized either HDL3 or HDL2 but interacted weakly with low density lipoprotein (LDL). Exposure of cells to lipoprotein cholesterol in the form of LDL also enhanced HDL binding by a process related to delivery of sterol into cells via the LDL receptor pathway. Enhancement of HDL binding to fibroblasts by either nonlipoprotein cholesterol or LDL was associated with an increased cell cholesterol content, a suppressed rate of cholesterol synthesis, decreased LDL receptor activity, and an enhanced rate of cholesterol ester formation. A comparison of HDL3 binding with the effects of HDL3 on cholesterol transport from cells revealed similar saturation profiles, implying a link between the two processes. Thus, cultured human fibroblasts and human arterial smooth muscle cells appear to possess specific receptors for HDL that may function to facilitate cholesterol removal from cells.