Cyclic AMP-specific phosphodiesterase 4 inhibitors promote ABCA1 expression and cholesterol efflux.

ATP cassette binding protein 1 (ABCA1) controls the apolipoprotein-mediated cholesterol efflux pathway and determines plasma HDL levels. Although cAMP is known to promote ABCA1 expression and cholesterol efflux from cells, it has not been determined whether cyclic nucleotide phosphodiesterase (PDE) isoforms regulate this pathway. We show that rolipram and cilomilast, inhibitors of cAMP-specific PDE4, increase apolipoprotein A-I (apoA-I)-mediated cholesterol efflux up to 80 and 140% in human THP-1 and mouse J774.A1 macrophages, respectively, concomitant with an elevation of cAMP levels. The EC(50) value was estimated to be 1 to 2 microM for both inhibitors. Rolipram and cilomilast also increase ABCA1 protein expression in THP-1 and J774.A1 macrophages. Thus, PDE4 inhibitors cause parallel increases in cAMP levels, ABCA1 expression and apoA-I-mediated cholesterol efflux. PDE4 inhibitors may provide a novel strategy for the treatment of cardiovascular disease by mobilizing cholesterol from atherosclerotic lesions.     

Importance of macrophage cholesterol content on the flux of cholesterol mass

Net flux of cholesterol represents the difference between efflux and influx and can result in net cell-cholesterol accumulation, net cell-cholesterol depletion, or no change in cellular cholesterol content. We measured radiolabeled cell-cholesterol efflux and cell-cholesterol mass using cholesterol-normal and -enriched J774 and elicited mouse peritoneal macrophage cells. Net cell-cholesterol effluxes were observed when cholesterol-enriched J774 cells were incubated with 3.5% apolipoprotein (apo) B depleted human serum, HDL₃, and apo A-I. Net cell-cholesterol influxes were observed when cholesterol-normal J774 cells were incubated with the same acceptors except apo A-I. When incubated with 2.5% individual sera, cholesterol mass efflux in free cholesterol (FC)-enriched J774 cells correlated with the HDL-cholesterol (HDL-C) concentrations (r² = 0.4; P=0.003), whereas cholesterol mass influx in cholesterol-normal J774 cells correlated with the LDL cholesterol (LDL-C) concentrations (r² = 0.6; P<0.0001) of the individual sera. A positive correlation was observed between measurements of [³H]cholesterol efflux and reductions in cholesterol mass (r² = 0.4; P=0.001) in FC-enriched J774 cells. In conclusion, isotopic efflux measurements from cholesterol-normal or cholesterol-enriched cells provide an accurate measurement of relative ability of an acceptor to remove labeled cholesterol under a specific set of experimental conditions, i.e., efflux potential. Moreover, isotopic efflux measurements can reflect changes in cellular cholesterol mass if the donor cells are enriched with cholesterol.     

Trypsin-sensitive and lipid-containing sites of the macrophage extracellular matrix bind apolipoprotein A-I and participate in ABCA1-dependent cholesterol efflux

A unique property of the extracellular matrix of J774 and THP-1 cells has been identified, which contributes to the ability of these cells to promote cholesterol efflux. We demonstrate high level apolipoprotein (apo) A-I binding to macrophage cells (THP-1 and J774) and to their extracellular matrix (ECM). However, high level apoA-I binding is not observed on fibroblasts, HepG2 cells, or U937 cells (a macrophage cell line that does not efflux cholesterol to apoA-I or bind apoA-I on their respective ECM). Binding to the ECM of THP-1 or J774 macrophages depends on the presence of apoA-I C-terminal helices and is markedly reduced with a mutant lacking residues 187-243 (apoA-I Delta(187-243)), suggesting that the hydrophobic C terminus forms a hydrophobic interaction with the ECM. ApoA-I binding is lost upon trypsin treatment or with Triton X-100, a preparation method that de-lipidates the ECM. However, binding is recovered with re-lipidation, and is preserved with ECM prepared using cytochalasin B, which conserves the endogenous phospholipid levels of the ECM. We also demonstrate that specific cholesterol efflux to apoA-I is much reduced in cells released from their native ECM, but fully restored when ECM-depleted cells are added back to ECM in the presence of apoA-I. The apoA-I-mediated efflux is deficient in plated or suspension U937 macrophages, but is restored to high levels when the suspension U937 cells are reconstituted with the ECM of J774 cells. The ECM-dependent activity was much reduced in the presence of glyburide, indicating participation of ABCA1 (ATP-binding cassette transporter 1) in the efflux mechanism. These studies establish a novel binding site for apoA-I on the macrophage ECM that may function together with ABCA1 in promoting cholesterol efflux.     

Moderate alcohol consumption increases cholesterol efflux mediated by ABCA1

Moderate alcohol consumption increases HDL cholesterol, which is involved in reverse cholesterol transport (RCT). The aim of this study was to investigate the effect of moderate alcohol consumption on cholesterol efflux, using J774 mouse macrophages and Fu5AH cells, and on other parameters in the RCT pathway. Twenty-three healthy men (45-65 years) participated in a randomized, partially diet-controlled, crossover trial. They consumed four glasses of whisky (40 g of alcohol) or water daily for 17 days. After 17 days of whisky consumption, serum capacity to induce ABCA1-dependent cholesterol efflux from J774 mouse macrophages was increased by 17.5% (P = 0.027) compared with water consumption. Plasma capacity to induce cholesterol efflux from Fu5AH cells increased by 4.6% (P = 0.002). Prebeta-HDL, apolipoprotein A-I (apoA-I), and lipoprotein A-I:A-II also increased by 31.6, 6.2, and 5.7% (P < 0.05), respectively, after whisky consumption compared with water consumption. Changes of cAMP-stimulated cholesterol efflux correlated (r = 0.65, P < 0.05) with changes of apoA-I but not with changes of prebeta-HDL (r = 0.30, P = 0.18). Cholesterol efflux capacities from serum of lean men were higher than those from overweight men. In conclusion, this study shows that moderate alcohol consumption increases the capacity of serum to induce cholesterol efflux from J774 mouse macrophages, which may be mediated by ABCA1.     

Interaction with proteoglycans enhances the sterol efflux produced by endogenous expression of macrophage apoE.

Endogenous expression of apolipoprotein (apo)E in macrophages facilitates cholesterol efflux in the presence and absence of extracellular sterol acceptors. A proteoglycan-associated pool of apoE has also been described. The relationship between a proteoglycan-associated pool of apoE and enhanced cholesterol efflux was investigated in these studies. Inhibition of proteoglycan expression reduced cholesterol efflux from apoE-expressing cells ( J774E(+)) in the presence and absence of HDL, but did not do so from nonexpressing cells ( J774E(-)). The effect of proteoglycan depletion on sterol efflux from J774E(+) cells was confirmed by measuring differences in cell sterol mass, secreted sterol mass, and sterol efflux rates. Furthermore, apoE-containing particles secreted from proteoglycan-depleted J774E(+) cells were denser than those secreted from J774E(+) cells with intact proteoglycan expression. Also, in J774E(+) cells with intact proteoglycans, apoE particles isolated from the cell surface proteoglycan layer were denser than secreted particles. The apoE-lipid particles isolated from the cell surface proteoglycan layer had a lower lipid-to-apoE and cholesterol-to-apoE ratio compared with secreted particles. In distinction, proteoglycan depletion of J774E(-) cells did not reduce sterol efflux produced by the exogenous addition of apoE. These observations indicate that one mechanism by which endogenous expression of apoE facilitates effective cholesterol efflux from macrophages is related to its retention at the cell surface in a proteoglycan-associated pool. Further, our data suggest that apoE arrives at the cell surface in a relatively lipid-poor state, and that a proximate source of lipid available to the proteoglycan-bound apoE at the cell surface resides in the plasma membrane.     

Tweaking the cholesterol efflux capacity of reconstituted HDL

Mechanisms to increase plasma high-density lipoprotein (HDL) or to promote egress of cholesterol from cholesterol-loaded cells (e.g., foam cells from atherosclerotic lesions) remain an important target to regress heart disease. Reconstituted HDL (rHDL) serves as a valuable vehicle to promote cellular cholesterol efflux in vitro and in vivo. rHDL were prepared with wild type apolipoprotein (apo) A-I and the rare variant, apoA-I Milano (M), and each apolipoprotein was reconstituted with phosphatidylcholine (PC) or sphingomyelin (SM). The four distinct rHDL generated were incubated with CHO cells, J774 macrophages, and BHK cells in cellular cholesterol efflux assays. In each cell type, apoA-I(M) SM-rHDL promoted the greatest cholesterol efflux. In BHK cells, the cholesterol efflux capacities of all four distinct rHDL were greatly enhanced by increased expression of ABCG1. Efflux to PC-containing rHDL was stimulated by transfection of a nonfunctional ABCA1 mutant (W590S), suggesting that binding to ABCA1 represents a competing interaction. This interpretation was confirmed by binding experiments. The data show that cholesterol efflux activity is dependent upon the apoA-I protein employed, as well as the phospholipid constituent of the rHDL. Future studies designed to optimize the efflux capacity of therapeutic rHDL may improve the value of this emerging intervention strategy.     

Structural modification of plasma HDL by phospholipids promotes efficient ABCA1-mediated cholesterol release

It has been suggested that ABCA1 interacts preferentially with lipid-poor apolipoprotein A-I (apoA-I). Here, we show that treatment of plasma with dimyristoyl phosphatidylcholine (DMPC) multilamellar vesicles generates prebeta(1)-apoA-I-containing lipoproteins (LpA-I)-like particles similar to those of native plasma. Isolated prebeta(1)-LpA-I-like particles inhibited the binding of (125)I-apoA-I to ABCA1 more efficiently than HDL(3) (IC(50) = 2.20 +/- 0.35 vs. 37.60 +/- 4.78 microg/ml). We next investigated the ability of DMPC-treated plasma to promote phospholipid and unesterified (free) cholesterol efflux from J774 macrophages stimulated or not with cAMP. At 2 mg DMPC/ml plasma, both phospholipid and free cholesterol efflux were increased ( approximately 50% and 40%, respectively) in cAMP-stimulated cells compared with unstimulated cells. Similarly, both phospholipid and free cholesterol efflux to either isolated native prebeta(1)-LpA-I and prebeta(1)-LpA-I-like particles were increased significantly in stimulated cells. Furthermore, glyburide significantly inhibited phospholipid and free cholesterol efflux to DMPC-treated plasma. Removal of apoA-I-containing lipoproteins from normolipidemic plasma drastically reduced free cholesterol efflux mediated by DMPC-treated plasma. Finally, treatment of Tangier disease plasma with DMPC affected the amount of neither prebeta(1)-LpA-I nor free cholesterol efflux. These results indicate that DMPC enrichment of normal plasma resulted in the redistribution of apoA-I from alpha-HDL to prebeta-HDL, allowing for more efficient ABCA1-mediated cellular lipid release. Increasing the plasma prebeta(1)-LpA-I level by either pharmacological agents or direct infusions might prevent foam cell formation and reduce atherosclerotic vascular disease.     

High-density lipoprotein particle uptake and selective uptake of high-density lipoprotein-associated cholesteryl esters by J774 macrophages

High-density lipoprotein (HDL) cholesteryl esters are taken up by fibroblasts via HDL particle uptake and via selective uptake, i.e., cholesteryl ester uptake independent of HDL particle uptake. In the present study we investigated HDL selective uptake and HDL particle uptake by J774 macrophages. HDL3 (d = 1.125-1.21 g/ml) was labeled with intracellularly trapped tracers: 125I-labeled N-methyltyramine-cellobiose-apo A-I (125I-NMTC-apo A-I) to trace apolipoprotein A-I (apo A-I) and [3H]cholesteryl oleyl ether to trace cholesteryl esters. J774 macrophages, incubated at 37 degrees C in medium containing doubly labeled HDL3, took up 125I-NMTC-apo A-I, indicating HDL3 particle uptake (102.7 ng HDL3 protein/mg cell protein per 4 h at 20 micrograms/ml HDL3 protein). Apparent HDL3 uptake according to the uptake of [3H]cholesteryl oleyl ether (470.4 ng HDL3 protein/mg cell protein per 4 h at 20 micrograms/ml HDL3 protein) was in significant excess on 125I-NMTC-apo A-I uptake, i.e., J774 macrophages demonstrated selective uptake of HDL3 cholesteryl esters. To investigate regulation of HDL3 uptake, cell cholesterol was modified by preincubation with low-density lipoprotein (LDL) or acetylated LDL (acetyl-LDL). Afterwards, uptake of doubly labeled HDL3, LDL (apo B,E) receptor activity or cholesterol mass were determined. Preincubation with LDL or acetyl-LDL increased cell cholesterol up to approx. 3.5-fold over basal levels. Increased cell cholesterol had no effect on HDL3 particle uptake. In contrast, LDL- and acetyl-LDL-loading decreased selective uptake (apparent uptake 606 vs. 366 ng HDL3 protein/mg cell protein per 4 h in unloaded versus acetyl-LDL-loaded cells at 20 micrograms HDL3 protein/ml). In parallel with decreased selective uptake, specific 125I-LDL degradation was down-regulated. Using heparin as well as excess unlabeled LDL, it was shown that HDL3 uptake is independent of LDL (apo B,E) receptors. In summary, J774 macrophages take up HDL3 particles. In addition, J774 cells also selectively take up HDL3-associated cholesteryl esters. HDL3 selective uptake, but not HDL3 particle uptake, can be regulated.     

Regulation of high density lipoprotein receptors in cultured macrophages: role of acyl-CoA:cholesterol acyltransferase

The interaction of human serum high density lipoproteins (HDL) with mouse peritoneal macrophages and human blood monocytes was studied. Saturation curves for binding of apolipoprotein E-free [125I]HDL3 showed at least two components: non-specific binding and specific binding that saturated at approximately 40 micrograms HDL protein/ml. Scatchard analysis of specific binding of apo E-free [125I]-HDL3 to cultured macrophages yielded linear plots indicative of a single class of specific binding sites. Pretreatment of [125I]HDL3 with various apolipoprotein antibodies (anti apo A-I, anti apo A-II, anti apo C-II, anti apo C-III and anti apo E) and preincubation of the cells with anti-idiotype antibodies against apo A-I and apo A-II prior to the HDL binding studies revealed apolipoprotein A-I as the ligand involved in specific binding of HDL. Cellular cholesterol accumulation via incubation with acetylated LDL led to an increase in HDL binding sites as well as an increase in the activity of the cytoplasmic cholesterol esterifying enzyme acyl-CoA:cholesterol acyltransferase (ACAT). Incubation of the cholesterol-loaded cells in the presence of various ACAT inhibitors (Sandoz 58.035, Octimibate-Nattermann, progesterone) revealed a time- and dose-dependent amplification in HDL binding and HDL-mediated cholesterol efflux. It is concluded that the homeostasis of cellular cholesterol in macrophages is regulated in part by the number of HDL binding sites and that ACAT inhibitors enhance HDL-mediated cholesterol efflux from peripheral cells.     

A unique protease-sensitive high density lipoprotein particle containing the apolipoprotein A-I(Milano) dimer effectively promotes ATP-binding Cassette A1-mediated cell cholesterol efflux

Carriers of the apolipoprotein A-I(Milano) (A-I(M)) variant present with severe reductions of plasma HDL levels, not associated with premature coronary heart disease (CHD). Sera from 14 A-I(M) carriers and matched controls were compared for their ability to promote ABCA1-driven cholesterol efflux from J774 macrophages and human fibroblasts. When both cell types are stimulated to express ABCA1, the efflux of cholesterol through this pathway is greater with A-I(M) than control sera (3.4 +/- 1.0% versus 2.3 +/- 1.0% in macrophages; 5.2 +/- 2.4% versus 1.9 +/- 0.1% in fibroblasts). A-I(M) and control sera are instead equally effective in removing cholesterol from unstimulated cells and from fibroblasts not expressing ABCA1. The A-I(M) sera contain normal amounts of apoA-I-containing prebeta-HDL and varying concentrations of a unique small HDL particle containing a single molecule of the A-I(M) dimer; chymase treatment of serum degrades both particles and abolishes ABCA1-mediated cholesterol efflux. The serum content of chymase-sensitive HDL correlates strongly and significantly with ABCA1-mediated cholesterol efflux (r = 0.542, p = 0.004). The enhanced capacity of A-I(M) serum for ABCA1 cholesterol efflux is thus explained by the combined occurrence in serum of normal amounts of apoA-I-containing prebeta-HDL, together with a unique protease-sensitive, small HDL particle containing the A-I(M) dimer, both effective in removing cell cholesterol via ABCA1.     

Sterol efflux to apolipoprotein A-I originates from caveolin-rich microdomains and potentiates PDGF-dependent protein kinase activity

The kinetics of sterol efflux from human aortic smooth muscle cells equilibrated with a [(3)H]benzophenone-modified photoactivable free cholesterol analogue ((3)H-FCBP) did not differ significantly from those labeled with free cholesterol ((3)H-FC). Trypsin digestion of caveolin cross-linked by photoactivation of FCBP led to association of radiolabel in a single low molecular weight fraction, indicating relative structural homogeneity of caveolin-bound sterol. These findings were used to investigate the organization of sterols in caveolae and the ability of these domains to transfer sterols to apolipoprotein A-I (apo A-I), the major protein of human plasma high-density lipoproteins (HDL). During long-term (4-5 h) incubation with apo A-I, caveolin-associated (3)H-FC and (3)H-FCBP decreased, in parallel with an increase in apo A-I-associated sterol. Assay of caveolin-associated labeled sterols indicated that caveolae were a major source of sterol lost from the cells during HDL formation. Short-term changes of sterol distribution in caveolae were assayed using platelet-derived growth factor (PDGF). PDGF was without effect on FC efflux in the absence of apo A-I, but when apo A-I was present, PDGF increased FC efflux approximately 3-fold beyond the efflux rate catalyzed by apo A-I alone. At the same time, caveolin-associated FC decreased, and PDGF-dependent protein kinase activity was stimulated. Parallel results were obtained with (3)H-FCBP-equilibrated cells, in which apo A-I potentiated a PDGF-mediated reduction of radiolabel cross-linked to caveolin following photoactivation. These results suggest that sterols within caveolae are mobile and selectively transferred to apo A-I. They also suggest a novel role for sterol efflux in amplifying PDGF-mediated signal transduction.     

Potential role of ABCA7 in cellular lipid efflux to apoA-I

ABCA7 is homologous to ABCA1 and has recently been shown in cell culture to bind apolipoprotein A-I (apoA-I) and to promote the efflux of phospholipids. However, it is not known if ABCA7 promotes lipid efflux in vivo. When expressed in HEK293 cells, both human and mouse ABCA7 promoted phospholipid efflux to apoA-I but no detectable cholesterol efflux. However, genetic knockdown of ABCA7 in mouse peritoneal macrophages did not affect phospholipid or cholesterol efflux to apoA-I. Moreover, in ABCA1-knockout macrophages, there was no detectable apoA-I-stimulated phospholipid efflux, inconsistent with a residual role of ABCA7. In contrast to plasma membrane localization of ABCA7 in transfected embryonic kidney cells, immunofluorescence microscopy of endogenous ABCA7 in macrophages showed a predominantly intracellular localization of the protein. Strikingly, immunofluorescence studies of adult mouse kidney revealed an apical brush border membrane localization of ABCA7 in the proximal tubule, suggesting that ABCA7 may come in contact with apoA-I in the glomerular filtrate. Although ABCA7 does not contribute to apolipoprotein-mediated lipid efflux in resting macrophages, its cell surface location in the kidney suggests that it could serve such a role in tissue microenvironments.     

Helical apolipoproteins of high-density lipoprotein enhance phagocytosis by stabilizing ATP-binding cassette transporter A7

We previously reported that the endogenous ATP-binding cassette transporter (ABC)A7 strongly associates with phagocytic function rather than biogenesis of high-density lipoprotein (HDL), being regulated by sterol-regulatory element binding protein (SREBP)2. Phagocytic activity was found enhanced by apolipoprotein (apo)A-I and apoA-II more than twice the maximum in J774 and mouse peritoneal macrophages. Therefore we investigated the molecular basis of this reaction in association with the function of ABCA7. Similar to ABCA1, ABCA7 was degraded, likely by calpain, and apoA-I and apoA-II stabilize ABCA7 against degradation. Cell surface biotinylation experiments demonstrated that endogenous ABCA7 predominantly resides on the cell surface and that the apolipoproteins increase the surface ABCA7. The increase of phagocytosis by apolipoproteins was retained in the J774 cells treated with ABCA1 siRNA and in the peritoneal macrophages from ABCA1-knockout mice, but it was abolished in the J774 cells treated with ABCA7 siRNA and in the peritoneal macrophages from ABCA7-knockout mice. Phagocytosis was decreased in the cells in the peritoneal cavity of the ABCA7-knockout mouse compared with the wild-type control. We thus concluded that extracellular helical apolipoproteins augment ABCA7-associated phagocytosis by stabilizing ABCA7. The results demonstrated direct enhancement of the host defense system by HDL components.     

The correlation of ATP-binding cassette 1 mRNA levels with cholesterol efflux from various cell lines

Studies show that lipid-free apoA-I stimulates release of cholesterol and phospholipid from fibroblasts and macrophages. ATP-binding cassette 1 (ABC1) is implicated in this release and has been identified as the genetic defect in Tangier disease, evidence that ABC1 is critical to the biogenesis of high density lipoprotein. We quantified levels of ABC1 mRNA, protein, and cholesterol efflux from J774 mouse macrophages +/- exposure to a cAMP analog. Up-regulating ABC1 mRNA correlated to increased cholesterol efflux in a dose- and time-dependent manner. mRNA levels rose after 15 min of exposure while protein levels rose after 1 h, with increased efflux 2-4 h post-treatment. In contrast to cells from wild-type mice, peritoneal macrophages from the Abc1 -/- mouse showed a lower level of basal efflux and no increase with cAMP treatment. The stimulation of efflux exhibits specificity for apoA-I, high density lipoprotein, and other apolipoproteins as cholesterol acceptors, but not for small unilamellar vesicles, bile acid micelles, or cyclodextrin. We have studied a number of cell types and found that while other cell lines express ABC1 constitutively, only J774 and elicited mouse macrophages show a substantial increase of mRNA and efflux with cAMP treatment. ApoA-I-stimulated efflux was detected from the majority of cell lines examined, independent of treatment.     

An analysis of the role of a retroendocytosis pathway in ABCA1-mediated cholesterol efflux from macrophages

The ATP binding cassette transporter A-1 (ABCA1) is critical for apolipoprotein-mediated cholesterol efflux, an important mechanism employed by macrophages to avoid becoming lipid-laden foam cells, the hallmark of early atherosclerotic lesions. It has been proposed that lipid-free apolipoprotein A-I (apoA-I) enters the cell and is resecreted as a lipidated particle via a retroendocytosis pathway during ABCA1-mediated cholesterol efflux from macrophages. To determine the functional importance of such a pathway, confocal microscopy was used to characterize the internalization of a fully functional apoA-I cysteine mutant containing a thiol-reactive fluorescent probe in cultured macrophages. ApoA-I was also endogenously labeled with (35)S-methionine to quantify cellular uptake and to determine the metabolic fate of the internalized protein. It was found that apoA-I was specifically taken inside macrophages and that a small amount of intact apoA-I was resecreted from the cells. However, a majority of the label that reappeared in the media was degraded. We estimate that the mass of apoA-I retroendocytosed is not sufficient to account for the HDL produced by the cholesterol efflux reaction. Furthermore, we have demonstrated that lipid-free apoA-I-mediated cholesterol efflux from macrophages can be pharmacologically uncoupled from apoA-I internalization into cells. On the basis these findings, we present a model in which the ABCA1-mediated lipid transfer process occurs primarily at the membrane surface in macrophages, but still accounts for the observed specific internalization of apoA-I.     

Depletion of pre-beta-high density lipoprotein by human chymase impairs ATP-binding cassette transporter A1- but not scavenger receptor class B type I-mediated lipid efflux to high density lipoprotein

The ATP-binding cassette transporter A1 (ABCA1) mediates the efflux of cellular unesterified cholesterol and phospholipid to lipid-poor apolipoprotein A-I. Chymase, a protease secreted by mast cells, selectively cleaves pre-beta-migrating particles from high density lipoprotein (HDL)(3) and reduces the efflux of cholesterol from macrophages. To evaluate whether this effect is the result of reduction of ABCA1-dependent or -independent pathways of cholesterol efflux, in this study we examined the efflux of cholesterol to preparations of chymase-treated HDL(3) in two types of cell: 1) in J774 murine macrophages endogenously expressing low levels of scavenger receptor class B, type I (SR-BI), and high levels of ABCA1 upon treatment with cAMP; and 2) in Fu5AH rat hepatoma cells endogenously expressing high levels of the SR-BI and low levels of ABCA1. Treatment of HDL(3) with the human chymase resulted in rapid depletion of pre-beta-HDL and a concomitant decrease in the efflux of cholesterol and phospholipid (2-fold and 3-fold, respectively) from the ABCA1-expressing J774 cells. In contrast, efflux of free cholesterol from Fu5AH to chymase-treated and to untreated HDL(3) was similar. Incubation of HDL(3) with phospholipid transfer protein led to an increase in pre-beta-HDL contents as well as in ABCA1-mediated cholesterol efflux. A decreased cholesterol efflux to untreated HDL(3) but not to chymase-treated HDL(3) was observed in ABCA1-expressing J774 with probucol, an inhibitor of cholesterol efflux to lipid-poor apoA-I. Similar results were obtained using brefeldin and gliburide, two inhibitors of ABCA1-mediated efflux. These results indicate that chymase treatment of HDL(3) specifically impairs the ABCA1-dependent pathway without influencing either aqueous or SR-BI-facilitated diffusion and that this effect is caused by depletion of lipid-poor pre-beta-migrating particles in HDL(3). Our results are compatible with the view that HDL(3) promotes ABCA1-mediated lipid efflux entirely through its lipid-poor fraction with pre-beta mobility.     

Reduction in ABCG1 in Type 2 diabetic mice increases macrophage foam cell formation

Atherosclerosis development is accelerated severalfold in patients with Type 2 diabetes. In the initial stages of disease, monocytes transmigrate into the subendothelial space and differentiate into foam cells. Scavenger receptors and ATP binding cassette (ABC) Transporters play an important role in foam cell formation as they regulate the influx and efflux of oxidized lipids. Here, we show that peritoneal macrophages isolated from Type 2 diabetic db/db mice have decreased expression of the ABC transporter ABCG1 and increased expression of the scavenger receptor CD36. We found a 2-fold increase in accumulation of esterified cholesterol in diabetic db/db macrophages compared with wild-type control macrophages. Diabetic db/db macrophages also had impaired cholesterol efflux to high density lipoprotein but not to lipid-free apo A-I, suggesting that the increased esterified cholesterol in diabetic db/db macrophages was due to a selective loss of ABCG1-mediated efflux to high density lipoprotein. Additionally, we were able to confirm down-regulation of ABCG1 using C57BL/6J peritoneal macrophages cultured in elevated glucose in vitro (25 mM glucose for 7 days), suggesting that ABCG1 expression in diabetic macrophages is regulated by chronic exposure to elevated glucose. Diabetic KK(ay) mice were also studied and were found to have decreased ABCG1 expression without an increase in CD36. These observations demonstrate that ABCG1 plays a major role in macrophage cholesterol efflux and that decreased ABCG1 function can facilitate foam cell formation in Type 2 diabetic mice.     

Influence of ApoA-I structure on the ABCA1-mediated efflux of cellular lipids

The influence of apolipoprotein (apo) A-I structure on ABCA1-mediated efflux of cellular unesterified (free) cholesterol (FC) and phospholipid (PL) is not well understood. To address this issue, we used a series of apoA-I mutants to examine the contributions of various domains in the molecule to ABCA1-mediated FC and PL efflux from mouse J774 macrophages and human skin fibroblasts. Irrespective of the cell type, deletion or disruption of the C-terminal lipid-binding domain of apoA-I drastically reduced the FC and PL efflux ( approximately 90%), indicating that the C-terminal amphipathic alpha-helix is required for high affinity microsolubilization of FC and PL. Deletion in the N-terminal region of apoA-I also reduced the lipid efflux ( approximately 30%) and increased the K(m) about 2-fold compared with wild type apoA-I, whereas deletion of the central domain (Delta123-166) had no effect on either K(m) or V(max). These results indicate that ABCA1-mediated lipid efflux is relatively insensitive to the organization of the apoA-I N-terminal helix-bundle domain. Alterations in apoA-I structure caused parallel changes in its ability to bind to a PL bilayer and to induce efflux of FC and PL. Overall, these results are consistent with a two-step model for ABCA1-mediated lipid efflux. In the first step, apoA-I binds to ABCA1 and hydrophobic alpha-helices in the C-terminal domain of apoA-I insert into the region of the perturbed PL bilayer created by the PL transport activity of ABCA1, thereby allowing the second step of lipidation of apoA-I and formation of nascent high density lipoprotein particles to occur.     

Phospholipid transfer protein enhances removal of cellular cholesterol and phospholipids by high-density lipoprotein apolipoproteins

High-density lipoprotein (HDL) apolipoproteins remove excess cholesterol from cells by an active transport pathway that may protect against atherosclerosis. Here we show that treatment of cholesterol-loaded human skin fibroblasts with phospholipid transfer protein (PLTP) increased HDL binding to cells and enhanced cholesterol and phospholipid efflux by this pathway. PLTP did not stimulate lipid efflux in the presence of albumin, purified apolipoprotein A-I, and phospholipid vesicles, suggesting specificity for HDL particles. PLTP restored the lipid efflux activity of mildly trypsinized HDL, presumably by regenerating active apolipoproteins. PLTP-stimulated lipid efflux was absent in Tangier disease fibroblasts, induced by cholesterol loading, and inhibited by brefeldin A treatment, indicating selectivity for the apolipoprotein-mediated lipid removal pathway. The lipid efflux-stimulating effect of PLTP was not attributable to generation of preβ HDL particles in solution but instead required cellular interactions. These interactions increased cholesterol efflux to minor HDL particles with electrophoretic mobility between α and preβ. These findings suggest that PLTP promotes cell-surface binding and remodeling of HDL so as to improve its ability to remove cholesterol and phospholipids by the apolipoprotein-mediated pathway, a process that may play an important role in enhancing flux of excess cholesterol from tissues and retarding atherogenesis.     

Expression of functional RANK on mature rat and human osteoclasts

The present study was aimed at investigating effects of hypochlorite (HOCl) modification of high density lipoproteins subclass 3 (HDL3) on their ability for cellular cholesterol removal from permanent J774 macrophages. Our findings indicate that HOCl (added as reagent or generated enzymatically by the myeloperoxidase/H2O2/Cl- system) damages apolipoprotein A-I, the major protein component of HDL3. Fatty acid analysis of native and HOCl-modified HDL3 revealed that unsaturated fatty acids in both major lipid subclasses (phospholipids and cholesteryl esters) are targets for HOCl attack. HOCl modification resulted in impaired HDL3-mediated cholesterol efflux from J774 cells, regardless of whether reagent or enzymatically generated HOCl was used to modify the lipoprotein. Decreased cholesterol efflux was also observed after HOCl modification of reconstituted HDL particles. Impairment of cholesterol efflux from macrophages was noticed at low and physiologically occurring HOCl concentrations.