Regulation of phospholipid biosynthesis during cholesterol influx and high density lipoprotein-mediated cholesterol efflux in macrophages

We have studied the rate of phospholipid synthesis and turnover in mouse peritoneal macrophages in reaction to cholesterol influx and high density lipoprotein (HDL)-mediated cholesterol efflux, using three different radioactive precursors, 32PO4(3-), [3H]choline, and [14C]oleic acid. The cells were loaded with cholesterol for up to 18 h with acetyl-low density lipoprotein (LDL), and phospholipid synthesis was measured at various time intervals and compared with nonloaded macrophages. In the first 2 h of cholesterol loading, a twofold increase in the rate of synthesis for sphingomyelin, phosphatidylcholine, phosphatidylserine-inositol, and phosphatidylethanolamine was observed. After this initial up-regulation, the rate of phospholipid synthesis continuously declined upon further cholesterol loading, while the turnover rate of cellular phospholipids was not affected under the same conditions. The lysosomal inhibitor chloroquine abolished the down-regulation, revealing a strong correlation between phospholipid synthesis and lysosomal enzyme activity which was presumably dependent on the release of cholesterol from the lysosome. The reduction in phospholipid synthesis induced by cholesterol loading is reversible by the addition of HDL3 to the cells. When HDL3 was added to the culture medium, a two- to threefold increase in phosphatidylcholine synthesis and a twofold increase in sphingomyelin formation was observed after 3 h. Ca2+ antagonists of the dihydropyridine type, which down-regulate HDL-receptor activity and promote the formation and cellular release of lamellar bodies derived from the lysosomal compartment (Schmitz, G., et al. 1988. Arteriosclerosis. 8: 46-56, and Robenek, H., and G. Schmitz. 1988. Arteriosclerosis. 8: 57-67), specifically enhance the synthesis of sphingomyelin in cholesterol-loaded macrophages. Inhibitors of acyl-CoA:cholesterol acyltransferase (Octimibate, progesterone) increase both the synthesis of sphingomyelin and phosphatidylcholine, and enhance HDL-receptor activity. The results indicate that cholesterol and phospholipid metabolism are coordinately regulated in macrophages. Moreover, the formation of phosphatidylcholine and sphingomyelin seems to be an important factor for the promotion of HDL-receptor-mediated cellular cholesterol efflux.     

Adiponectin prevents atherosclerosis by increasing cholesterol efflux from macrophages

Plasma high density lipoprotein (HDL)-cholesterol levels are inversely correlated to the risk of atherosclerotic cardiovascular diseases. Reverse cholesterol transport (RCT) is one of the major protective systems against atherosclerosis, in which HDL particles play a crucial role to carry cholesterol derived from peripheral tissues to the liver. Recently, ATP-binding cassette transporters (ABCA1, ABCG1) and scavenger receptor (SR-BI) have been identified as important membrane receptors to generate HDL by removing cholesterol from foam cells. Adiponectin (APN) secreted from adipocytes is one of the important molecules to inhibit the development of atherosclerosis. Epidemiological studies have revealed a positive correlation between plasma HDL-cholesterol and APN concentrations in humans, although its mechanism has not been clarified. Therefore, in the present study, we investigated the role of APN on RCT, in particular, cellular cholesterol efflux from human monocyte-derived and APN-knockout (APN-KO) mice macrophages. APN up-regulated the expression of ABCA1 in human macrophages, respectively. ApoA-1-mediated cholesterol efflux from macrophages was also increased by APN treatment. Furthermore, the mRNA expression of LXRα and PPARγ was increased by APN. In APN-KO mice, the expression of ABCA1, LXRα, PPARγ, and apoA-I-mediated cholesterol efflux was decreased compared with wild-type mice. In summary, APN might protect against atherosclerosis by increasing apoA-I-mediated cholesterol efflux from macrophages through ABCA1-dependent pathway by the activation of LXRα and PPARγ.     

Enhancing apolipoprotein A-I-dependent cholesterol efflux elevates cholesterol export from macrophages in vivo

Eight proteins potentially involved in cholesterol efflux [ABCA1, ABCG1, CYP27A1, phospholipid transfer protein (PLTP), scavenger receptor type BI (SR-BI), caveolin-1, cholesteryl ester transfer protein, and apolipoprotein A-I (apoA-I)] were overexpressed alone or in combination in RAW 264.7 macrophages. When apoA-I was used as an acceptor, overexpression of the combination of ABCA1, CYP27A1, PLTP, and SR-BI (Combination I) enhanced the efflux by 4.3-fold. It was established that the stimulation of efflux was due to increased abundance of ABCA1 and increased apoA-I binding to non-ABCA1 sites on macrophages. This combination caused only a small increase of the efflux to isolated HDL. When HDL was used as an acceptor, overexpression of caveolin-1 or a combination of caveolin-1 and SR-BI (Combination II) was the most active, doubling the efflux to HDL, without affecting the efflux to apoA-I. When tested in the in vivo mouse model of cholesterol efflux, overexpression of ABCA1 and Combination I elevated cholesterol export from macrophages to plasma, liver, and feces, whereas overexpression of caveolin-1 or Combination II did not have an effect. We conclude that pathways of cholesterol efflux using apoA-I as an acceptor make a predominant contribution to cholesterol export from macrophages in vivo.     

Testosterone up-regulates scavenger receptor BI and stimulates cholesterol efflux from macrophages

By lowering high density lipoprotein (HDL) cholesterol, testosterone contributes to the gender difference in HDL cholesterol and has been accused to be pro-atherogenic. The mechanism by which testosterone influences HDL cholesterol is little understood. We therefore investigated the effect of testosterone on the gene expression of apolipoprotein A-I (apoA-I), hepatic lipase (HL), scavenger receptor B1 (SR-BI), and the ATP binding cassette transporter A1 (ABCA1), all of which are important regulators of HDL metabolism. In both cultivated HepG2 hepatocytes and primary human monocyte-derived macrophages, testosterone led to a dose-dependent up-regulation of SR-BI, which was assessed on both the mRNA and the protein levels. As a functional consequence, we observed an increased HDL(3)-induced cholesterol efflux from macrophages. At supraphysiological dosages, testosterone also increased the expression of HL in HepG2 cells. Testosterone had no effect on the expression of apoA-I in HepG2 cells and ABCA1 in either HepG2 cells or macrophages. These data suggest that testosterone, despite lowering HDL cholesterol, intensifies reverse cholesterol transport and thereby exerts an anti-atherogenic rather than a pro-atherogenic effect.     

ApoE-mediated cholesterol efflux from macrophages: separation of autocrine and paracrine effects

Macrophages in the vessel wall secrete high levels of apolipoprotein E (apoE). Cholesterol efflux from macrophages to apoE has been shown to decrease foam cell formation and prevent atherosclerosis. An apoE molecule can mediate cholesterol efflux from the macrophage that originally secreted it (autocrine effect) or from surrounding macrophages (paracrine effect). Traditional methodologies have not been able to separate these serial effects. The novel methodology presented here was developed to separate autocrine and paracrine effects by using a simple mathematical model to interpret the effects of dilution on apoE-mediated cholesterol efflux. Our results show that, at very dilute concentrations, the paracrine effect of apoE is not evident and the autocrine effect becomes the dominant mediator of efflux. However, at saturating concentrations, paracrine apoE causes 80–90% of the apoE-mediated cholesterol efflux, whereas autocrine apoE is responsible for the remaining 10–20%. These results suggest that the relative importance of autocrine and paracrine apoE depends on the size of the local distribution volume, a factor not considered in previous in vitro studies of apoE function. Furthermore, autocrine effects of apoE could be critical in the prevention of foam cell formation in vivo. This novel methodology may be applicable to other types of mixed autocrine/paracrine systems, such as signal transduction systems. autocrine/paracrine system; cholesterol acceptor; extracellular space; distribution volume     

Apolipoprotein A-I-stimulated apolipoprotein E secretion from human macrophages is independent of cholesterol efflux

Apolipoprotein A-I (apoA-I)-mediated cholesterol efflux involves the binding of apoA-I to the plasma membrane via its C terminus and requires cellular ATP-binding cassette transporter (ABCA1) activity. ApoA-I also stimulates secretion of apolipoprotein E (apoE) from macrophage foam cells, although the mechanism of this process is not understood. In this study, we demonstrate that apoA-I stimulates secretion of apoE independently of both ABCA1-mediated cholesterol efflux and of lipid binding by its C terminus. Pulse-chase experiments using (35)S-labeled cellular apoE demonstrate that macrophage apoE exists in both relatively mobile (E(m)) and stable (E(s)) pools, that apoA-I diverts apoE from degradation to secretion, and that only a small proportion of apoA-I-mobilized apoE is derived from the cell surface. The structural requirements for induction of apoE secretion and cholesterol efflux are clearly dissociated, as C-terminal deletions in recombinant apoA-I reduce cholesterol efflux but increase apoE secretion, and deletion of central helices 5 and 6 decreases apoE secretion without perturbing cholesterol efflux. Moreover, a range of 11- and 22-mer alpha-helical peptides representing amphipathic alpha-helical segments of apoA-I stimulate apoE secretion whereas only the C-terminal alpha-helix (domains 220-241) stimulates cholesterol efflux. Other alpha-helix-containing apolipoproteins (apoA-II, apoA-IV, apoE2, apoE3, apoE4) also stimulate apoE secretion, implying a positive feedback autocrine loop for apoE secretion, although apoE4 is less effective. Finally, apoA-I stimulates apoE secretion normally from macrophages of two unrelated subjects with genetically confirmed Tangier Disease (mutations C733R and c.5220-5222delTCT; and mutations A1046D and c.4629-4630insA), despite severely inhibited cholesterol efflux. We conclude that apoA-I stimulates secretion of apoE independently of cholesterol efflux, and that this represents a novel, ABCA-1-independent, positive feedback pathway for stimulation of potentially anti-atherogenic apoE secretion by alpha-helix-containing molecules including apoA-I and apoE.     

Acid sphingomyelinase-deficient macrophages have defective cholesterol trafficking and efflux

Cholesterol efflux from macrophage foam cells, a key step in reverse cholesterol transport, requires trafficking of cholesterol from intracellular sites to the plasma membrane. Sphingomyelin is a cholesterol-binding molecule that transiently exists with cholesterol in endosomes and lysosomes but is rapidly hydrolyzed by lysosomal sphingomyelinase (L-SMase), a product of the acid sphingomyelinase (ASM) gene. We therefore hypothesized that sphingomyelin hydrolysis by L-SMase enables cholesterol efflux by preventing cholesterol sequestration by sphingomyelin. Macrophages from wild-type and ASM knockout mice were incubated with [(3)H]cholesteryl ester-labeled acetyl-LDL and then exposed to apolipoprotein A-I or high density lipoprotein. In both cases, [(3)H]cholesterol efflux was decreased substantially in the ASM knockout macrophages. Similar results were shown for ASM knockout macrophages labeled long-term with [(3)H]cholesterol added directly to medium, but not for those labeled for a short period, suggesting defective efflux from intracellular stores but not from the plasma membrane. Cholesterol trafficking to acyl-coenzyme A:cholesterol acyltransferase (ACAT) was also defective in ASM knockout macrophages. Using filipin to probe cholesterol in macrophages incubated with acetyl-LDL, we found there was modest staining in the plasma membrane of wild-type macrophages but bright, perinuclear fluorescence in ASM knockout macrophages. Last, when wild-type macrophages were incubated with excess sphingomyelin to "saturate" L-SMase, [(3)H]cholesterol efflux was decreased. Thus, sphingomyelin accumulation due to L-SMase deficiency leads to defective cholesterol trafficking and efflux, which we propose is due to sequestration of cholesterol by sphingomyelin and possibly other mechanisms. This model may explain the low plasma high density lipoprotein found in ASM-deficient humans and may implicate L-SMase deficiency and/or sphingomyelin enrichment of lipoproteins as novel atherosclerosis risk factors.     

Avian phospholipid transfer protein causes HDL conversion without affecting cholesterol efflux from macrophages

Circulatory phospholipid transfer protein (PLTP) has two major functions: 1) transfer of phospholipids towards HDL particles; and 2) modulation of HDL size and composition via the HDL conversion process. In the laying hen (Gallus gallus), the massive oocyte-targeted lipid flow is achieved through the concerted actions of lipases, lipid transfer proteins, and relatives of the LDL receptor family. The aim of the study was to gain insights into the structure and functions of chicken PLTP. The results demonstrate that PLTP is highly conserved from chicken to mammals, as (i) chicken PLTP is associated with plasma HDL; (ii) it clearly possesses phospholipid transfer activity; (iii) it is inactivated at + 58 °C; and (iv) it mediates conversion of avian and human HDL into small preβ-mobile HDL and large fused α-mobile HDL particles. Our data show that HDL from different chicken models is similar in chemical and physical properties to that of man based on PLTP activity, cholesterol efflux, and HDL conversion assays. In contrast to mammals, PLTP-facilitated HDL remodeling did not enhance cholesterol efflux efficiency of chicken HDL particles.     

Lycopene regulation of cholesterol synthesis and efflux in human macrophages

Hypercholesterolemia is one of the most important risk factors for atherosclerosis, and tomato lycopene has been suggested to have beneficial effects against such a disease, although the exact molecular mechanism is unknown. We tested the hypothesis that lycopene may exert its antiatherogenic role through changes in cholesterol metabolism. Incubation of THP-1 cells with lycopene (0.5–2 μM) dose-dependently reduced intracellular total cholesterol. Such an effect was associated with a decrease in reduction of 3-hydroxy-3-methylglutaryl coenzyme A reductase expression and with an increase in ABCA1 and caveolin-1 (cav-1) expressions. In addition, lycopene enhanced RhoA levels in the cytosolic fraction, activating peroxisome proliferator-activated receptor gamma (PPARγ) and liver X receptor alpha expressions. Concomitant addition of lycopene and the PPARγ inhibitor GW9662 or lycopene and mevalonate blocked the carotenoid-induced increase in ABCA1 and cav-1 expressions. These results imply a potential role of lycopene in attenuating foam cell formation and, therefore, in preventing atherosclerosis by a cascade mechanism involving inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase, RhoA inactivation and subsequent increase in PPARγ and liver X receptor alpha activities and enhancement of ABCA1 and cav-1 expressions.     

Cell surface-expressed moesin-like HDL/apoA-I binding protein promotes cholesterol efflux from human macrophages

HDL and its major component, apolipoprotein A-I (apoA-I), play a central role in reverse cholesterol transport. We recently reported the involvement of a glycosylphosphatidylinositol anchor (GPI anchor) in the binding of HDL and apoA-I on human macrophages, and purified an 80 kDa HDL/apoA-I binding protein. In the present study, we characterized the GPI-anchored HDL/apoA-I binding protein from macrophages. The HDL/apoA-I binding protein was purified from macrophages and digested with endopeptidase, and the resultant fragments were sequenced. Cholesterol efflux, flow cytometry, immunoblotting, and immunohistochemical analyses were performed to characterize the HDL/apoA-I binding protein. Two parts of seven amino acid sequences completely matched those of moesin. Flow cytometry, immunoblotting, and immunohistochemistry using anti-moesin antibody showed that the HDL/apoA-I binding protein was N-glycosylated and expressed on the cell surface. It was termed moesin-like protein. Treatment of macrophages with anti-moesin antibody blocked the binding of HDL/apoA-I and suppressed cholesterol efflux. The moesin-like protein was exclusively expressed on macrophages and was upregulated by cholesterol loading and cell differentiation. Our results indicate that the moesin-like HDL/apoA-I binding protein is specifically expressed on the surface of human macrophages and promotes cholesterol efflux from macrophages.-Matsuyama, A, N. Sakai, H. Hiraoka, K-i. Hirano, and S. Yamashita. Cell surface-expressed moesin-like HDL/apoA-I binding protein promotes cholesterol efflux from human macrophages.     

A kinetic model to evaluate cholesterol efflux from THP-1 macrophages to apolipoprotein A-1

The kinetics (0 to 3 h) of cholesterol efflux to delipidated apolipoprotein A-1 were investigated, and the experimental data were best fitted to a mathematical model that involves two independent pathways of cholesterol efflux. The first pathway with a rate constant of 4.6 h(-1) is fast but removes only 3-5% of total cholesterol. After preconditioning apoA-1, it was found that this pathway remains, and hence it is a property of the cholesterol-loaded cells rather than due to modification on the apolipoprotein. This fast initial efflux does not seem to contribute to cholesterol efflux at later stages (>1 h) where a second pathway predominates. However, the fast initial efflux pool can be restored if apoA-1 is withdrawn. The second slower pathway (k(membrane--media) = 0.79 h(-1)) is associated with cholesterol ester hydrolysis whose rate constant could be experimentally verified (k(cal) = 0.43, k(exp) = 0.38 +/- 0.05). The model suggests that two different plasma membrane domains are involved in the two pathways. Loading of the cells with an oxysterol, 7-ketocholesterol (7K), inhibits efflux from both pathways. The model predicts that 7K decreases the initial efflux by decreasing the available cholesterol (by possibly affecting lipid packing), while all rate constants in the second pathway are decreased. In conclusion, the kinetic model suggests that cholesterol efflux to apoA-1 is a two-step process. In the first step, some of the plasma membrane cholesterol contributes to a fast initial efflux (possibly from lipid rafts) and leads to a second pathway that mobilizes intracellular cholesterol mobilization.     

Selective uptake of HDL cholesteryl esters and cholesterol efflux from mouse peritoneal macrophages independent of SR-BI

Scavenger receptor class B type I (SR-BI) mediates the selective uptake of HDL cholesteryl esters (CEs) and facilitates the efflux of unesterified cholesterol. SR-BI expression in macrophages presumably plays a role in atherosclerosis. The role of SR-BI for selective CE uptake and cholesterol efflux in macrophages was explored. Macrophages and HDL originated from wild-type (WT) or SR-BI knockout (KO; homozygous) mice. For uptake, macrophages were incubated in medium containing 125I-/3H-labeled HDL. For lipid removal, [3H]cholesterol efflux was analyzed using HDL as acceptor. Selective uptake of HDL CE ([3H]cholesteryl oleyl ether - 125I-tyramine cellobiose) was similar in WT and SR-BI KO macrophages. Radiolabeled SR-BI KO-HDL yielded a lower rate of selective uptake compared with WT-HDL in WT and SR-BI KO macrophages. Cholesterol efflux was similar in WT and SR-BI KO cells using HDL as acceptor. SR-BI KO-HDL more efficiently promoted cholesterol removal compared with WT-HDL from both types of macrophages. Macrophages selectively take up HDL CE independently of SR-BI. Additionally, in macrophages, there is substantial cholesterol efflux that is not mediated by SR-BI. Therefore, SR-BI-independent mechanisms mediate selective CE uptake and cholesterol removal. SR-BI KO-HDL is an inferior donor for selective CE uptake compared with WT-HDL, whereas SR-BI KO-HDL more efficiently promotes cholesterol efflux.     

Oxidation of high density lipoproteins: characterization and effects on cholesterol efflux from J774 macrophages

Oxidative modification of high density lipoproteins (HDL) may alter their capacity to mediate cellular cholesterol efflux. We studied the kinetics of copper-mediated oxidation of HDL and cholesterol efflux mediated by unmodified and oxidized HDL (oxHDL). Oxidation was measured by increases in absorbance at 234 nm (ΔA234), production of thiobarbituric acid reactive substances (TBARS) and loss of trinitrobenzene sulfonic acid reactivity. Oxidation was dependent on copper concentration and showed a lag phase and propagation phase. Efflux of cholesterol from J774 macrophages measured by appearance of cellular [³H]cholesterol in the medium was lower by 16% after 4 h and 36% after 24 h with oxHDL compared to HDL. OxHDL-mediated efflux was also lower by 27% to 36% at lipoprotein concentrations of 10 to 200 μg protein/ml. Cholesterol efflux correlated negatively with TBARS production (r= −0.97, P < 0.003) and ΔA234 (r = −0.77, P < 0.080). There was no difference in efflux mediated by apoproteins prepared from HDL and oxHDL. Efflux measured by change in cholesterol mass in medium was 78% lower with oxHDL. Inhibition of oxidation with butylated hydroxytoluene maintained the capacity of HDL to stimulate efflux. These results suggest that oxidation of HDL may impair its protective role against atherosclerosis.     

Regulation of macrophage cholesterol efflux through hydroxymethylglutaryl-CoA reductase inhibition: a role for RhoA in ABCA1-mediated cholesterol efflux

The cholesterol biosynthetic pathway produces numerous signaling molecules. Oxysterols through liver X receptor (LXR) activation regulate cholesterol efflux, whereas the non-sterol mevalonate metabolite, geranylgeranyl pyrophosphate (GGPP), was recently demonstrated to inhibit ABCA1 expression directly, through antagonism of LXR and indirectly through enhanced RhoA geranylgeranylation. We used HMG-CoA reductase inhibitors (statins) to test the hypothesis that reduced synthesis of mevalonate metabolites would enhance cholesterol efflux and attenuate foam cell formation. Preincubation of THP-1 macrophages with atorvastatin, dose dependently (1-10 microm) stimulated cholesterol efflux to apolipoprotein AI (apoAI, 10-60%, p < 0.05) and high density lipoprotein (HDL(3)) (2-50%, p < 0.05), despite a significant decrease in cholesterol synthesis (2-90%). Atorvastatin also increased ABCA1 and ABCG1 mRNA abundance (30 and 35%, p < 0.05). Addition of mevalonate, GGPP or farnesyl pyrophosphate completely blocked the statin-induced increase in ABCA1 expression and apoAI-mediated cholesterol efflux. A role for RhoA was established, because two inhibitors of Rho protein activity, a geranylgeranyl transferase inhibitor and C3 exoenzyme, increased cholesterol efflux to apoAI (20-35%, p < 0.05), and macrophage expression of dominant-negative RhoA enhanced cholesterol efflux to apoAI (20%, p < 0.05). In addition, atorvastatin increased the RhoA levels in the cytosol fraction and decreased the membrane localization of RhoA. Atorvastatin treatment activated peroxisome proliferator activated receptor gamma and increased LXR-mediated gene expression suggesting that atorvastatin induces cholesterol efflux through a molecular cascade involving inhibition of RhoA signaling, leading to increased peroxisome proliferator activated receptor gamma activity, enhanced LXR activation, increased ABCA1 expression, and cholesterol efflux. Finally, statin treatment inhibited cholesteryl ester accumulation in macrophages challenged with atherogenic hypertriglyceridemic very low density lipoproteins indicating that statins can regulate foam cell formation.     

15-Lipoxygenase-mediated modification of high-density lipoproteins impairs SR-BI- and ABCA1-dependent cholesterol efflux from macrophages

Elevated plasma levels of high-density lipoprotein cholesterol (HDL-C) are atheroprotective and HDL-dependent reverse cholesterol transport has been related to this effect. HDL particles may, however, undergo modifications that affect their biological activities. Lipoxygenases (LOs) belong to a family of lipid peroxidizing enzymes; among them, reticulocyte-type 15-lipoxygenase (15-LO-1) appears to play a pathophysiological role in atherosclerosis, as its expression is increased in atherosclerotic plaques and it has been shown to oxidize low-density lipoproteins to an atherogenic form. In this work we investigated the impact of in vitro 15-lipoxygenase-catalyzed modification of HDL3 on their ability to act as cholesterol acceptor and found that 15-LO-modified HDL3 were less effective in mediating cholesterol efflux from lipid-laden J774 cells. A reduced binding of 15-LO-modified HDL3 to scavenger receptor class B, type I (SR-BI), due to HDL apoproteins cross-linking, explained, at least in part, the observed reduction of cholesterol efflux. In addition, ATP-binding cassette transporter A1 (ABCA1)-mediated cholesterol efflux was also reduced, as a consequence of pre-beta-particles loss after HDL3 modification. These results suggest that 15-lipoxygenase might induce structural alterations of HDL3 particles that impair their capability of triggering reverse cholesterol transport.     

The efflux of lysosomal cholesterol from cells

To gain insight into the transport of sterol from lysosomes to the plasma membrane, we studied the efflux of lysosomal free cholesterol from intact Fu5AH rat hepatoma cells to high density lipoprotein (HDL) and other extracellular acceptors that promote sterol desorption from the plasma membrane. The procedures involved pulsing cells at 15 degrees C with low density lipoprotein that had been reconstituted with [3H]cholesteryl oleate and then incubating the cells at 37 degrees C in the presence of a sterol acceptor, while monitoring both the hydrolysis of [3H]cholesteryl oleate in lysosomes and the efflux of the resulting [3H]free cholesterol to the acceptor. After warming cells to 37 degrees C, rapid hydrolysis of [3H]cholesteryl oleate began after 10-20 min, and the lysosomally generated [3H]free cholesterol became available for efflux after an additional delay of 40-50 min. The kinetics of hydrolysis and the delay between hydrolysis and efflux were unchanged over a wide range of HDL3 concentrations (10-1000 micrograms of protein/ml), and with acceptors that do not interact with HDL-specific cell surface binding sites (phospholipid vesicles, dimethyl suberimidate cross-linked HDL). In addition, the delivery of lysosomal cholesterol to the plasma membrane was unaffected when cellular cholesterol content was elevated 2.6-fold above the normal control level, or when the activity of cellular acyl-coenzyme A/cholesterol acyltransferase (ACAT) was stimulated with exogenous oleic acid. We conclude that in the Fu5AH cell, a maximum of 40-50 min is required for the transport of cholesterol from lysosomes to the plasma membrane and that this transport is not regulated in response to either specific extracellular acceptors or the content of sterol in cells. The lack of effect of increased ACAT activity implies that the pathway for this transport does not involve passage of sterol through the rough endoplasmic reticulum, the subcellular location of ACAT.     

Purified human paraoxonase-1 interacts with plasma membrane lipid rafts and mediates cholesterol efflux from macrophages

Paraoxonase-1 (PON1) is a high-density lipoprotein (HDL)-associated serum enzyme thought to make a major contribution to the antioxidant and anti-inflammatory capacities of HDLs. However, the role of PON1 in the modulation of cholesterol efflux is poorly understood. The aim of our study was to investigate the involvement of PON1 in the regulation of cholesterol efflux, especially the mechanism by which it modulates HDL-mediated cholesterol transport. The enrichment of HDL(3) with human PON1 enhanced, in a dose-dependent manner, cholesterol efflux from THP-1 macrophage-like cells and ABCA1-enriched J774 macrophages. Moreover, an additive effect was observed when ABCA1-enriched J774 macrophages were incubated with both PON1 and apo-AI. Interestingly, PON1 alone was able to mediate cholesterol efflux from J774 macrophages and to upregulate ABCA1 expression on J774 macrophages. Immunofluorescence measurement showed an increase in PON1 levels in the cytoplasm of J774 macrophages overexpressing ABCA1. PON1 used an apo-AI-like mechanism to modulate cholesterol efflux from rapid and slow efflux pools derived from the lipid raft and nonraft domains of the plasma membrane, respectively. This was supported by the fact that ABCA1 protein was incrementally expressed by J774 macrophages within the first few hours of incubation with cholesterol-loaded J774 macrophages and that cyclodextrin significantly inhibited the capacity of PON1 to modulate cholesterol efflux from macrophages. This finding suggested that PON1 plays an important role in the antiatherogenic properties of HDLs and may exert its protective function outside the lipoprotein environment.