Cholesterol accumulation regulates expression of macrophage proteins implicated in proteolysis and complement activation.

OBJECTIVE: Cholesterol accumulation by macrophages plays a key role in atherogenesis. To begin to develop a global picture of this process, we used proteomics and transcriptomics to analyze foam cells generated with acetyl-low-density lipoprotein, a classic ligand for scavenger receptors. METHODS AND RESULTS: Tandem mass spectrometry and stringent statistical analysis revealed that foam cells differentially expressed 15 of 542 proteins (2.8%) detected in macrophage-conditioned medium. Apolipoprotein E was one of the most upregulated proteins, confirming that proteins involved in lipid metabolism are important targets for regulation by sterol accumulation. However, levels of proteins linked to complement activation and lysosomal proteolysis also changed markedly. Transcriptional analysis demonstrated that 698 of 19,700 genes (3.5%) were regulated in foam cells, including many genes important in sterol metabolism. We also found that cholesterol accumulation regulated genes implicated in complement activation but failed to affect genes linked to proteolysis and macrophage polarization. Changes in protein levels in macrophage-conditioned medium were largely independent of changes in mRNA levels. CONCLUSIONS: Loading sterol into macrophages regulates levels of complement proteins and lysosomal proteases-key players in the immune system and plaque rupture. Posttranscriptional mechanisms are likely important for controlling levels of most of the proteins detected in macrophage medium.

Apolipoprotein A-I attenuates palmitate-mediated NF-κB activation by reducing Toll-like receptor-4 recruitment into lipid rafts.

While high-density lipoprotein (HDL) is known to protect against a wide range of inflammatory stimuli, its anti-inflammatory mechanisms are not well understood. Furthermore, HDL's protective effects against saturated dietary fats have not been previously described. In this study, we used endothelial cells to demonstrate that while palmitic acid activates NF-κB signaling, apolipoprotein A-I, (apoA-I), the major protein component of HDL, attenuates palmitate-induced NF-κB activation. Further, vascular NF-κB signaling (IL-6, MCP-1, TNF-α) and macrophage markers (CD68, CD11c) induced by 24 weeks of a diabetogenic diet containing cholesterol (DDC) is reduced in human apoA-I overexpressing transgenic C57BL/6 mice compared to age-matched WT controls. Moreover, WT mice on DDC compared to a chow diet display increased gene expression of lipid raft markers such as Caveolin-1 and Flotillin-1, and inflammatory Toll-like receptors (TLRs) (TLR2, TLR4) in the vasculature. However apoA-I transgenic mice on DDC show markedly reduced expression of these genes. Finally, we show that in endothelial cells TLR4 is recruited into lipid rafts in response to palmitate, and that apoA-I prevents palmitate-induced TLR4 trafficking into lipid rafts, thereby blocking NF-κB activation. Thus, apoA-I overexpression might be a useful therapeutic tool against vascular inflammation.

Obesity and weight loss result in increased adipose tissue ABCG1 expression in db/db mice.

The prevalence of obesity has reached epidemic proportions and is associated with several co-morbid conditions including diabetes, dyslipidemia, cancer, atherosclerosis and gallstones. Obesity is associated with low systemic inflammation and an accumulation of adipose tissue macrophages (ATMs) that are thought to modulate insulin resistance. ATMs may also modulate adipocyte metabolism and take up lipids released during adipocyte lipolysis and cell death. We suggest that high levels of free cholesterol residing in adipocytes are released during these processes and contribute to ATM activation and accumulation during obesity and caloric restriction. Db/db mice were studied for extent of adipose tissue inflammation under feeding conditions of ad libitum (AL) and caloric restriction (CR). The major finding was a marked elevation in epididymal adipose ABCG1 mRNA levels with obesity and CR (6-fold and 16-fold, respectively) over that seen for lean wild-type mice. ABCG1 protein was also elevated for CR as compared to AL adipose tissue. ABCG1 is likely produced by cholesterol loaded ATMs since this gene is not highly expressed in adipocytes and ABCG1 expression is sterol mediated. Our data supports the concept that metabolic changes in adipocytes due to demand lipolysis and cell death lead to cholesterol loading of ATMs. Based on finding cholesterol-loaded peritoneal leukocytes with elevated levels of ABCG1 in CR as compared to AL mice, we suggest that pathways for cholesterol trafficking out of adipose tissue involve ATM egress as well as ABCG1 mediated cholesterol efflux. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).

Acyl-CoA synthetase 1 is required for oleate and linoleate mediated inhibition of cholesterol efflux through ATP-binding cassette transporter A1 in macrophages.

Diabetes and insulin resistance increase the risk of cardiovascular disease caused by atherosclerosis through mechanisms that are poorly understood. Lipid-loaded macrophages are key contributors to all stages of atherosclerosis. We have recently shown that diabetes associated with increased plasma lipids reduces cholesterol efflux and levels of the reverse cholesterol transporter ABCA1 (ATP-binding cassette transporter A1) in mouse macrophages, which likely contributes to macrophage lipid accumulation in diabetes. Furthermore, we and others have shown that unsaturated fatty acids reduce ABCA1-mediated cholesterol efflux, and that this effect is mediated by the acyl-CoA derivatives of the fatty acids. We therefore investigated whether acyl-CoA synthetase 1 (ACSL1), a key enzyme mediating acyl-CoA synthesis in macrophages, could directly influence ABCA1 levels and cholesterol efflux in these cells. Mouse macrophages deficient in ACSL1 exhibited reduced sensitivity to oleate- and linoleate-mediated ABCA1 degradation, which resulted in increased ABCA1 levels and increased apolipoprotein A-I-dependent cholesterol efflux in the presence of these fatty acids, as compared with wildtype mouse macrophages. Conversely, overexpression of ACSL1 resulted in reduced ABCA1 levels and reduced cholesterol efflux in the presence of unsaturated fatty acids. Thus, the reduced ABCA1 and cholesterol efflux in macrophages subjected to conditions of diabetes and elevated fatty load may, at least in part, be mediated by ACSL1. These observations raise the possibility that ABCA1 levels could be increased by inhibition of acyl-CoA synthetase activity in vivo. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).

Serum amyloid A facilitates the binding of high-density lipoprotein from mice injected with lipopolysaccharide to vascular proteoglycans.

OBJECTIVE: Levels of serum amyloid A (SAA), an acute-phase protein carried on high-density lipoprotein (HDL), increase in inflammatory states and are associated with increased risk of cardiovascular disease. HDL colocalizes with vascular proteoglycans in atherosclerotic lesions. However, its major apolipoprotein, apolipoprotein A-I, has no proteoglycan-binding domains. Therefore, we investigated whether SAA, which has proteoglycan-binding domains, plays a role in HDL retention by proteoglycans. METHODS AND RESULTS: HDL from control mice and mice deficient in both SAA1.1 and SAA2.1 (SAA knockout mice) injected with bacterial lipopolysaccharide (LPS) was studied. SAA mRNA expression in the liver and plasma levels of SAA increased dramatically in C57BL/6 mice after LPS administration, although HDL cholesterol did not change. Fast protein liquid chromatography analysis showed most of the SAA to be in HDL. Mass spectrometric analysis indicated that HDL from LPS-injected control mice had high levels of SAA1.1/2.1 and reduced levels of apolipoprotein A-I. HDL from LPS-injected control mice demonstrated high-affinity binding to biglycan relative to normal mouse HDL. In contrast, HDL from LPS-injected SAA knockout mice showed very little binding to biglycan, consistent with SAA facilitating the binding of HDL to vascular proteoglycans. CONCLUSION: SAA enrichment of HDL under inflammatory conditions plays an important role in the binding of HDL to vascular proteoglycans.

High-density lipoprotein suppresses the type I interferon response, a family of potent antiviral immunoregulators, in macrophages challenged with lipopolysaccharide.

BACKGROUND: High-density lipoprotein (HDL) protects the artery wall by removing cholesterol from lipid-laden macrophages. However, recent evidence suggests that HDL might also inhibit atherogenesis by combating inflammation. METHODS AND RESULTS: To identify potential antiinflammatory mechanisms, we challenged macrophages with lipopolysaccharide, an inflammatory microbial ligand for Toll-like receptor 4. HDL inhibited the expression of 30 (277 of 911) of the genes normally induced by lipopolysaccharide, microarray analysis revealed. One of its major targets was the type I interferon response pathway, a family of potent viral immunoregulators controlled by Toll-like receptor 4 and the TRAM/TRIF signaling pathway. Unexpectedly, the ability of HDL to inhibit gene expression was independent of macrophage cholesterol stores. Immunofluorescent studies suggested that HDL promoted TRAM translocation to intracellular compartments, which impaired subsequent signaling by Toll-like receptor 4 and TRIF. To examine the potential in vivo relevance of the pathway, we used mice deficient in apolipoprotein A-I, the major protein of HDL. After infection with Salmonella typhimurium, a Gram-negative bacterium that expresses lipopolysaccharide, apolipoprotein A-I-deficient mice had 6-fold higher plasma levels of interferon-ß, a key regulator of the type I interferon response, than did wild-type mice. CONCLUSIONS: HDL inhibits a subset of lipopolysaccharide-stimulated macrophage genes that regulate the type I interferon response, and its action is independent of sterol metabolism. These findings raise the possibility that regulation of macrophage genes by HDL might link innate immunity and cardioprotection.

Modifying apolipoprotein A-I by malondialdehyde, but not by an array of other reactive carbonyls, blocks cholesterol efflux by the ABCA1 pathway.

Dysfunctional high density lipoprotein (HDL) is implicated in the pathogenesis of cardiovascular disease, but the underlying pathways remain poorly understood. One potential mechanism involves covalent modification by reactive carbonyls of apolipoprotein A-I (apoA-I), the major HDL protein. We therefore determined whether carbonyls resulting from lipid peroxidation (malondialdehyde (MDA) and hydroxynonenal) or carbohydrate oxidation (glycolaldehyde, glyoxal, and methylglyoxal) covalently modify lipid-free apoA-I and inhibit its ability to promote cellular cholesterol efflux by the ABCA1 pathway. MDA markedly impaired the ABCA1 activity of apoA-I. In striking contrast, none of the other four carbonyls were effective. Liquid chromatography-electrospray ionization-tandem mass spectrometry of MDA-modified apoA-I revealed that Lys residues at specific sites had been modified. The chief adducts were MDA-Lys and a Lys-MDA-Lys cross-link. Lys residues in the C terminus of apoA-I were targeted for cross-linking in high yield, and this process may hinder the interaction of apoA-I with lipids and ABCA1, two key steps in reverse cholesterol transport. Moreover, levels of MDA-protein adducts were elevated in HDL isolated from human atherosclerotic lesions, suggesting that lipid peroxidation might render HDL dysfunctional in vivo. Taken together, our observations indicate that MDA damages apoA-I by a pathway that generates lysine adducts at specific sites on the protein. Such damage may facilitate the formation of macrophage foam cells by impairing cholesterol efflux by the ABCA1 pathway.

A macrophage sterol-responsive network linked to atherogenesis.

Cholesteryl ester accumulation by macrophages is a critical early event in atherogenesis. To test the hypothesis that sterol loading promotes foam cell formation and vascular disease by perturbing a network of interacting proteins, we used a global approach to identify proteins that are differentially expressed when macrophages are loaded with cholesterol in vivo. Our analysis revealed a sterol-responsive network that is highly enriched in proteins with known physical interactions, established roles in vesicular transport, and demonstrated atherosclerotic phenotypes in mice. Pharmacologic intervention with a statin or rosiglitazone and use of mice deficient in LDL receptor or apolipoprotein E implicated the network in atherosclerosis. Biochemical fractionation revealed that most of the sterol-responsive proteins resided in microvesicles, providing a physical basis for the network's functional and biochemical properties. These observations identify a highly integrated network of proteins whose expression is influenced by environmental, genetic, and pharmacological factors implicated in atherogenesis.

Oxidation of apolipoprotein A-I by myeloperoxidase impairs the initial interactions with ABCA1 required for signaling and cholesterol export.

A key cardioprotective effect of high-density lipoprotein involves the interaction of its major protein, apolipoprotein A-I (apoA-I) with ATP-binding cassette transporter A1 (ABCA1), a macrophage cholesterol exporter. ApoA-I is thought to remove cholesterol from macrophages by a cascade of events. First it binds directly to ABCA1, activating signaling pathways, and then it binds to and solubilizes lipid domains generated by ABCA1. HDL isolated from human atherosclerotic lesions and blood of subjects with established coronary artery disease contains elevated levels of 3-chlorotyrosine and 3-nitrotyrosine, two characteristic products of myeloperoxidase (MPO), a heme protein secreted by macrophages. Here we show that chlorination (but not nitration) of apoA-I by the MPO pathway impairs its ability to interact directly with ABCA1, to activate the Janus kinase 2 signaling pathway, and to promote efflux of cellular cholesterol. In contrast, oxidation of apoA-I has little effect on its ability to stabilize ABCA1 protein or to solubilize phospholipids. Our results indicate that chlorination of apoA-I by the MPO pathway selectively inhibits two critical early events in cholesterol efflux: (1) the binding of apoA-I to ABCA1 and (2) the activation of a key signaling pathway. Therefore, oxidation of apoA-I in the artery wall by MPO-generated chlorinating intermediates may contribute to atherogenesis by impairing cholesterol efflux from macrophages.

Myeloperoxidase: an oxidative pathway for generating dysfunctional high-density lipoprotein.

Accumulation of low-density lipoprotein (LDL)-derived cholesterol by artery wall macrophages triggers atherosclerosis, the leading cause of cardiovascular disease. Conversely, high-density lipoprotein (HDL) retards atherosclerosis by promoting cholesterol efflux from macrophages by the membrane-associated ATP-binding cassette transporter A1 (ABCA1) pathway. HDL has been proposed to lose its cardioprotective effects in subjects with atherosclerosis, but the underlying mechanisms are poorly understood. One potential pathway involves oxidative damage by myeloperoxidase (MPO), a heme enzyme secreted by human artery wall macrophages. We used mass spectrometry to demonstrate that HDL isolated from patients with established cardiovascular disease contains elevated levels of 3-chlorotyrosine and 3-nitrotyrosine, two characteristic products of MPO. When apolipoprotein A-I (apoA-I), the major HDL protein, was oxidized by MPO, its ability to promote cellular cholesterol efflux by ABCA1 was impaired. Moreover, oxidized apoA-I was unable to activate lecithin:cholesterol acyltransferase (LCAT), which rapidly converts free cholesterol to cholesteryl ester, a critical step in HDL maturation. Biochemical studies implicated tyrosine chlorination and methionine oxygenation in the loss of ABCA1 and LCAT activity by oxidized apoA-I. Oxidation of specific residues in apoA-I inhibited two key steps in cholesterol efflux from macrophages, raising the possibility that MPO initiates a pathway for generating dysfunctional HDL in humans.

Diabetes reduces the cholesterol exporter ABCA1 in mouse macrophages and kidneys.

Accumulation of cholesterol in arterial macrophages may contribute to diabetes-accelerated atherosclerotic cardiovascular disease. The ATP-binding cassette transporter ABCA1 is a cardioprotective membrane protein that mediates cholesterol export from macrophages. Factors elevated in diabetes, such as reactive carbonyls and free fatty acids, destabilize ABCA1 protein in cultured macrophages, raising the possibility that impaired ABCA1 plays an atherogenic role in diabetes. We therefore examined the modulation of ABCA1 in two mouse models of diabetes. We isolated peritoneal macrophages, livers, kidneys, and brains from type 1 non-obese diabetic (NOD) mice and mice made diabetic by viral-induced autoimmune destruction of pancreatic beta-cells, and we measured ABCA1 protein and mRNA levels and cholesterol contents. ABCA1 protein levels and cholesterol export activity were reduced by 40-44% (P<0.01) in peritoneal macrophages and protein levels by 48% (P<0.001) in kidneys in diabetic NOD mice compared with nondiabetic animals, even though ABCA1 mRNA levels were not significantly different. A similar selective reduction in ABCA1 protein was found in peritoneal macrophages (33%, P<0.05) and kidneys (35%, P<0.05) from the viral-induced diabetic mice. In liver and brain, however, diabetes had no effect or slightly increased ABCA1 protein and mRNA levels. The reduced ABCA1 in macrophages and kidneys was associated with increased cholesterol content. Impaired ABCA1-mediated cholesterol export could therefore contribute to the increased atherosclerosis and nephropathy associated with diabetes.

Tumor necrosis factor-alpha and lymphotoxin-alpha increase macrophage ABCA1 by gene expression and protein stabilization via different receptors.

OBJECTIVE: The tumor necrosis factor superfamily may exert cardioprotective or atherogenic effects, depending on the state of lesion progression. Tumor necrosis factor-alpha (TNF) induces macrophage ATP-binding cassette transporter A1 (ABCA1), a cardioprotective transmembrane protein that exports cellular cholesterol to apolipoprotein A-I. Here we examined the role of TNF receptors (TNFRs) in ABCA1 induction and tested the effects of lymphotoxin-alpha (LT), another TNF family member, on macrophage ABCA1 levels. METHODS: Primary macrophages taken from mice deficient in TNF receptors were used to determine ABCA1 expression and cholesterol efflux activity in response to treatment with exogenous TNF or LT. RESULTS: We studied TNFR2(-/-) and TNFR1(-/-)/R2(-/-) mice and found that both receptors are necessary for maximal induction of ABCA1 by TNF. Peritoneal macrophages from TNFR1(-/-)/R2(-/-) mice had no change in ABCA1 mRNA levels when treated with TNF while cells from TNFR2(-/-) mice had ABCA1 mRNA levels that were half that of wild-type (WT) cells. In contrast, incubating TNFR1(-/-)/R2(-/-) mice with LT increased ABCA1 by stabilizing the protein, which was not observed in WT mice and this was associated with downstream signaling through the LTbeta receptor. CONCLUSION: TNF requires both of its receptors to maximally induce ABCA1. Despite previous studies suggesting that LT has proatherogenic properties, we found that LT increases ABCA1 protein in TNFR1(-/-)/R2(-/-) but not WT macrophages and may supplement TNF in enhancing ABCA1-dependent cholesterol export from early atherosclerotic lesions.

The macrophage cholesterol exporter ABCA1 functions as an anti-inflammatory receptor.

ATP-binding cassette transporter A1 (ABCA1) is a cell membrane protein that exports excess cholesterol from cells to apolipoprotein (apo) A-I, the major protein in high density lipoproteins. Genetic studies have shown that ABCA1 protects against cardiovascular disease. The interaction of apoA-I with ABCA1 promotes cholesterol removal and activates signaling molecules, such as Janus kinase 2 (JAK2), that optimize the lipid export activity of ABCA1. Here we show that the ABCA1-mediated activation of JAK2 also activates STAT3, which is independent of the lipid transport function of ABCA1. ABCA1 contains two candidate STAT3 docking sites that are required for the apoA-I/ABCA1/JAK2 activation of STAT3. The interaction of apoA-I with ABCA1-expressing macrophages suppressed the ability of lysopolysaccaride to induce the inflammatory cytokines interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha, which was reversed by silencing STAT3 or ABCA1. Thus, the apoA-I/ABCA1 pathway in macrophages functions as an anti-inflammatory receptor through activation of JAK2/STAT3. These findings implicate ABCA1 as a direct molecular link between the cardioprotective effects of cholesterol export from arterial macrophages and suppressed inflammation.

The cell cholesterol exporter ABCA1 as a protector from cardiovascular disease and diabetes.

ATP-binding cassette transporter A1 (ABCA1) is an integral cell membrane protein that exports cholesterol from cells and suppresses macrophage inflammation. ABCA1 exports cholesterol by a multistep pathway that involves forming cell-surface lipid domains, solubilizing these lipids by apolipoproteins, binding of apolipoproteins to ABCA1, and activating signaling processes. Thus, ABCA1 behaves both as a lipid exporter and a signaling receptor. ABCA1 transcription is highly induced by sterols, and its expression and activity are regulated post-transcriptionally by diverse processes. ABCA1 mutations can reduce plasma HDL levels, accelerate cardiovascular disease, and increase the risk for type 2 diabetes. Genetic manipulations of ABCA1 expression in mice also affect plasma HDL levels, inflammation, atherogenesis, and pancreatic beta cell function. Metabolites elevated in individuals with the metabolic syndrome and diabetes destabilize ABCA1 protein and decrease cholesterol export from macrophages, raising the possibility that an impaired ABCA1 pathway contributes to the enhanced atherogenesis associated with common inflammatory and metabolic disorders. The ABCA1 pathway has therefore become a promising new therapeutic target for treating cardiovascular disease and diabetes.

Effects of acceptor composition and mechanism of ABCG1-mediated cellular free cholesterol efflux.

Among the known mechanisms of reverse cholesterol transport (RCT), ATP binding cassette transporter G1 (ABCG1)-mediated free cholesterol (FC) transport is the most recent and least studied. Here, we have characterized the efficiencies of different acceptors using baby hamster kidney (BHK) cells transfected with human ABCG1 cDNA, which is inducible upon treatment with mifepristone. When normalized on particle number and particle surface area, the acceptor efficiency for FC efflux was as follows: small unilamellar vesicles (SUV)>LDL>reconstituted HDL>HDL(2) = HDL(3). Based on phospholipid content, the order was reversed. ABCG1 also mediated phospholipid efflux to human serum and HDL(3). ABCG1-mediated FC efflux correlated significantly with a number of HDL subfractions and components in serum collected from 25 normolipidemic individuals: apolipoprotein A-II (apoA-II) (r(2) = 0.7), apolipoprotein A-I (apoA-I) (r(2) = 0.5), HDL-C (r(2) = 0.4), HDL-PL (r(2) = 0.4), alpha-2 HDL (r(2) = 0.4), and prebeta HDL (r(2) = 0.2). ABCG1 did not enhance influx of FC or cholesteryl oleyl ether (COE) when cells were incubated with radiolabeled HDL(3). ABCG1 expression did not increase the association of HDL(3) with cells. Compared with control cells, ABCG1 expression significantly increased the FC pool available for efflux and the rate constant for efflux. In conclusion, composition and particle size determine the acceptor efficiency for ABCG1-mediated efflux. ABCG1 increases cell membrane FC pools and changes its rate of desorption into the aqueous phase without enhancing the association with the acceptor.

ABCA1 mutants reveal an interdependency between lipid export function, apoA-I binding activity, and Janus kinase 2 activation.

ABCA1 exports cholesterol and phospholipids from cells by a multistep pathway that involves forming cell surface lipid domains, solubilizing these lipids by apolipoproteins, binding of apolipoproteins to ABCA1, and activating signaling processes. Here we used a mutational analysis approach to evaluate the relationship between these events. We prepared seven naturally occurring mutants and one artificial missense mutant of ABCA1 with varying degrees of impaired function, expressed them to similar levels as wild-type ABCA1 on the cell surface of BHK cells, and measured ABCA1-dependent lipid export, apolipoprotein A-I (apoA-I) binding, and signaling activities. Linear regression analyses showed that cholesterol and phospholipid efflux and cellular apoA-I binding correlated significantly with the ability of ABCA1 to form cell surface lipid domains. Lipid export and cellular apoA-I binding activities and formation of lipid domains also correlated with the amount of apoA-I that could be cross-linked to ABCA1. Moreover, each of these lipid export and apoA-I binding activities correlated with apoA-I-induced Janus kinase 2 (JAK2) activation. Thus, these missense mutations in ABCA1 impair lipid export, apoA-I binding, and apoA-I-stimulated JAK2 activities to similar extents, indicating that these processes are highly interactive components of a pathway that functions to export lipids from cells.

An amphipathic helical region of the N-terminal barrel of phospholipid transfer protein is critical for ABCA1-dependent cholesterol efflux.

Phospholipid lipid transfer protein (PLTP) mimics high-density lipoprotein apolipoproteins in removing cholesterol and phospholipids from cells through the ATP-binding cassette transporter A1 (ABCA1). Because amphipathic alpha-helices are the structural determinants for ABCA1 interactions, we examined the ability of synthetic peptides corresponding to helices in PLTP to remove cellular cholesterol by the ABCA1 pathway. Of the seven helices tested, only one containing PLTP residues 144-163 (p144), located at the tip of the N-terminal barrel, promoted ABCA1-dependent cholesterol efflux and stabilized ABCA1 protein. Mutating methionine 159 (Met-159) in this helix in PLTP to aspartate (M159D) or glutamate (M159E) nearly abolished the ability of PLTP to remove cellular cholesterol and dramatically reduced PLTP binding to phospholipid vesicles and its phospholipid transfer activity. These mutations impaired PLTP binding to ABCA1-generated lipid domains and PLTP-mediated stabilization of ABCA1 but increased PLTP binding to ABCA1. PLTP interactions with ABCA1 also mimicked apolipoproteins in activating Janus kinase 2; however, the M159D/E mutants were also able to activate this kinase. Structural analyses showed that the M159D/E mutations had only minor effects on PLTP conformation. These findings indicate that PLTP helix 144-163 is critical for removing lipid domains formed by ABCA1, stabilizing ABCA1 protein, interacting with phospholipids, and promoting phospholipid transfer. Direct interactions with ABCA1 and activation of signaling pathways likely involve other structural determinants of PLTP.

Myeloperoxidase and inflammatory proteins: pathways for generating dysfunctional high-density lipoprotein in humans.

High-density lipoprotein (HDL) inhibits atherosclerosis by removing cholesterol from artery wall macrophages. Additionally, HDL is anti-inflammatory in animal studies, suggesting that this property might also be important for its cardioprotective effects. Recent studies in subjects with established cardiovascular disease (CVD) demonstrate that myeloperoxidase targets HDL for oxidation and blocks the lipoprotein's ability to remove excess cholesterol from cells, raising the possibility that the enzyme provides a specific mechanism for generating dysfunctional HDL in humans. Shotgun proteomic analysis of HDL identified multiple complement regulatory proteins, protease inhibitors, and acute-phase response proteins, supporting a central role for HDL in inflammation. Mass spectrometry and biochemical analyses demonstrated that HDL(3) from CVD subjects was selectively enriched in apolipoprotein E, suggesting that it carries a unique cargo of proteins in humans with clinically significant CVD. Thus, oxidative modifications to HDL and changes in its protein composition might be useful biomarkers-and perhaps mediators-of CVD.