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Originally published In Press as doi:10.1074/jbc.M310672200 on November 24, 2003

J. Biol. Chem., Vol. 279, Issue 9, 8084-8092, February 27, 2004
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Suppression of Macrophage Eicosanoid Synthesis by Atherogenic Lipoproteins Is Profoundly Affected by Cholesterol-Fatty Acyl Esterification and the Niemann-Pick C Pathway of Lipid Trafficking*

Andrew R. Leventhal{ddagger}, Christina C. Leslie§, and Ira Tabas{ddagger}

From the {ddagger}Departments of Medicine and Anatomy and Cell Biology, Columbia University, New York, New York 10032 and the §Program in Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, Denver, Colorado 80206

Received for publication, September 26, 2003 , and in revised form, November 24, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Atheroma macrophages internalize large quantities of lipoprotein-derived lipids. While most emphasis has been placed on cholesterol, lipoprotein-derived fatty acids may also play important roles in lesional macrophage biology. Little is known, however, about the trafficking or metabolism of these fatty acids. In this study, we first show that the cholesterol-fatty acyl esterification reaction, catalyzed by acyl-CoA:cholesterol acyltransferase (ACAT), competes for the incorporation of lipoprotein-derived fatty acids into cellular phospholipids. Furthermore, conditions that inhibit trafficking of cholesterol from late endosomes/lysosomes to the endoplasmic reticulum (ER), such as the amphipathic amine U18666A and the Npc1+/- mutation, also inhibit incorporation of lipoprotein-derived fatty acids into phospholipids. The biological relevance of these findings was investigated by studying the suppression of agonist-induced prostaglandin E2 (PGE2) and leukotriene C4/D4/E4 production during lipoprotein uptake by macrophages, which has been postulated to involve enrichment of cellular phospholipids with non-arachidonic fatty acids (NAAFAs). We found that eicosanoid suppression was markedly enhanced when ACAT was inhibited and prevented when late endosomal/lysosomal lipid trafficking was blocked. Moreover, PGE2 suppression depended entirely on acetyl-LDL-derived NAAFAs, not on acetyl-LDL-cholesterol, and was not due to decreased cPLA2 activity per se. These data support the following model: lipoprotein-derived NAAFAs traffic via the NPC1 pathway from late endosomes/lysosomes to a critical pool of phospholipids. In competing reactions, these NAAFAs can be either esterified to cholesterol or incorporated into phospholipids, resulting in suppression of eicosanoid biosynthesis. In view of recent evidence suggesting dysfunctional cholesterol esterification in late lesional macrophages, these data predict that such cells would have highly suppressed eicosanoid synthesis, thus affecting eicosanoid-mediated cell signaling in advanced atherosclerosis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Macrophages are a prominent feature of atherosclerotic lesions and play critical roles in both lesion initiation and progression (1-3). During atherogenesis, macrophages internalize atherogenic lipoproteins in the arterial subendothelium via receptor-mediated endocytosis and phagocytosis (4, 5). In late endosomes/lysosomes or phagosomes, hydrolases release both cholesterol and fatty acids from these lipoproteins. Free cholesterol is exported from these degradative organelles by a vesicular transport process that requires the NPC11 protein and that is sensitive to a class of amphipathic amines, such as U18666A (6). An important fate of this exported cholesterol is its esterification to fatty acids in the ER by ACAT (4). This reaction is particularly critical during the early stages of atherogenesis, because early lesional macrophages accumulate large amounts of ACAT-derived cholesteryl esters ("foam cells") (5).

While the fate of lipoprotein-cholesterol has been widely studied, much less is known about the fate of lipoprotein-derived fatty acids. The majority of released fatty acids traffic to the plasma membrane for efflux. Of those retained, most are incorporated into phospholipids (7, 8). In contrast to the situation with lipoprotein-derived cholesterol, however, the mechanisms and consequences of lipoprotein-derived fatty acid transport and metabolism are poorly understood.

Our laboratory became interested in this topic in the context of our studies on lipid metabolism relevant to late lesional macrophages. In advanced atherosclerotic lesions, macrophages accumulate large amounts of unesterified, or "free," cholesterol (FC) (3), and recent in vivo data from our laboratory has provided evidence that this event is due to defective ACAT-mediated cholesterol-fatty acyl esterification (9). The consequences of FC accumulation are profound, including triggering of an ER stress pathway and apoptosis (3, 10). However, a pool of lipoprotein-derived fatty acids should also accumulate as a consequence of defective cholesterol-fatty acyl esterification, and we wondered what the consequences of this would be.

To explore this question, we took advantage of a model in which lipoprotein-derived fatty acids affect eicosanoid biosynthesis. In particular, investigators have observed that foam cell formation is associated with decreased eicosanoid synthesis, although the mechanism remains controversial (11, 12, 33). In this report, we explore the effects of altered lipid trafficking and cholesterol-fatty acyl esterification on lipoprotein-fatty acid incorporation into cellular phospholipids, cPLA2-induced fatty acid release, and eicosanoid synthesis in macrophages. We provide evidence for a model in which lipoprotein-derived non-arachidonic acid fatty acids (NAAFAs) traffic via the NPC1 pathway from late endosomes/lysosomes to a critical pool of phospholipids. In competing reactions, these NAAFAs can be either esterified to cholesterol or incorporated into phospholipids, resulting in suppression of eicosanoid biosynthesis.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Falcon tissue culture plastic ware was purchased from Fisher Scientific. Tissue culture reagents were from Invitrogen Life Technologies, Inc. Fetal Bovine Serum (FBS) was obtained from Hyclone Laboratories (Logan, UT). 3-[Decyldimethylsilyl]-N-[2-(4-methylphenyl)-1-phenylethyl]propanamide (compound 58035) (14), an inhibitor of acyl-CoA:cholesterol O-acyltransferase, was generously provided by Dr. John Heider, formerly of Sandoz, Inc. (East Hanover, NJ); a 10 mg/ml stock solution was prepared in dimethyl sulfoxide, and the final dimethyl sulfoxide concentration in both treated and control cells was 0.05%. U18666A (3-{beta}-[2-diethylaminoethoxy]androst-5-en-17-one hydrochloride) was from Biomol (Plymouth Meeting, PA). Cholesteryl-[1-14C]oleate was purchased from Amersham Biosciences, and phosphatidylcholine-1-stearoyl-2-[3H]arachidonyl was from New England Nuclear. Dioleoylglycerol and sn-2 arachidonyl PC were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). Human GST-tagged recombinant cPLA2{alpha} was produced in Sf9 cells using a baculovirus transfection system and was purified by anti-GST affinity chromatography. All other chemicals and reagents were obtained from Sigma Chemical Co.

Harvesting, Culturing, and Incubations of Mouse Peritoneal Macrophages—The mice used in this study were wild-type C57BL6/J (Jackson Laboratories), C57BL6/J in which a floxed macrophage CTP:phosphocholine cytidylyltransferase {alpha} gene was silenced by LysMCre (15), and Nctr-npc1N heterozygous/C57BL6/J. The npc1N mutation was detected by PCR analysis of tail DNA using PCR primers mp25-8f (GGTGCTGGACAGCCAAGTA) and mp25-INTR3 (GATGGTCTGTTCTCCCATG) as described by Loftus et al. (16). Macrophages were harvested from the peritoneum of the mice 3 days after the intraperitoneal injection of 40 µg of concanavalin A in 0.5 ml of phosphate-buffered saline and then cultured as described previously (17). On the day of the experiment, the cells were incubated for 5 h in 1% FBS in Dulbecco's modified Eagle's medium (w/v) containing 100 µg of acetyl-LDL/ml and 10 µg of compound 58035/ml, either in combination or separately as described previously (18).

Preparation of Membranes and Cytosol—The details of this procedure are described elsewhere (19). Briefly, murine peritoneal macrophages were plated to confluency on a 6-well dish. After treatment, the monolayers were washed twice with 1 ml of ice-cold phosphate-buffered saline. Cells were scraped in 200 µl of 10 mM HEPES, 10% glycerol, 0.34 M sucrose, 1 mM EGTA, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinin, pH 7.4. Cell suspensions were then sonicated at 4 °C using twenty 0.5-s bursts at setting no. 1 on a Branson 450 sonicator equipped with a tapered microtip. The homogenate was centrifuged at 2000 x g for 5 min at 4 °C to remove whole cells, and then the supernatant fraction was centrifuged at 100,000 x g for 1 h at 4 °C. The pellet resulting from this centrifugation was used as a source of cellular membranes, and the supernatant fraction was used as a source of cytosol. The cytosol was used immediately for the cPLA2 activity assay. For immunoblotting, the cytosol was stored at -20 °C and thawed once.

Lipoproteins—LDL (d, 1.020-1.063 g/ml) from fresh human plasma were isolated by preparative ultracentrifugation (20), and acetyl-LDL was prepared by reaction with acetic anhydride as described previously (21). Acetyl-LDL was reconstituted with defined neutral lipids as described by Krieger (27). Briefly, 1.9 mg of acetyl-LDL and 25 mg of potato starch were frozen and lyophilized in a 15-ml glass tube. The endogenous core lipids were then extracted three times with 5 ml of heptane, and then 6 mg of the desired neutral lipid core (dissolved in 200 µl of heptane) was added to the tube. For cholesteryl-[14C]oleate, 2500 cpm/nmol neutral lipid was added. This mixture was dried, and the sample was resuspended in 1 ml of 10 mM Tricine, pH 8.4. This suspension was incubated for 10-24 h. The solubilized reconstituted lipoprotein was separated from the bulk of the starch and excess lipid by low-speed centrifugation (2000 rpm). The specimen was further clarified by two additional centrifugations at 10,000 rpm for 10 min and stored under argon at 4 °C.

Arachidonic Acid Release and Eicosanoid Production—Confluent mouse peritoneal macrophages were grown on 24-well plates and incubated overnight in Dulbecco's modified Eagle's medium containing 10% FBS, 100 units/ml penicillin G, 100 µg/ml streptomycin sulfate, 0.29 mg/ml glutamine, and 0.5 µCi/ml [3H]arachidonic acid. Cells were washed three times with phosphate-buffered saline, incubated with lipoproteins, and then stimulated with media containing 0.2% BSA and 20 µM A23187 [GenBank] . After 5 min, the media were withdrawn and centrifuged for 5 min at 14,000 rpm in a microcentrifuge to remove cellular debris, and the radioactivity was quantified by liquid scintillation counting. The cells were dissolved in 1 ml of 0.1 N NaOH at room temperature for 5 h. A 100-µl aliquot of the cell lysate was counted, and the percent efflux was calculated as [(media cpm)/(cell + media cpm)] x 100. Background release from unstimulated cells (2-3%) was subtracted from each experimental point. The amount of PGE2 and LT-C4/D4/E4 in the media was determined using displacement enzyme-linked immunosorbent assay kits from Amersham Biosciences, Inc.

cPLA2 Immunoblotting—Laemmli electrophoresis sample buffer (5x) was added to 20 µg of cytosol. This mixture was boiled for 10 min, and the proteins were separated by SDS-polyacrylamide gel electrophoresis. After electrotransfer to a 0.22-µm nitrocellulose membrane, the blot was incubated overnight at 4 °C with rabbit anti-cPLA2 serum (1:2500) (23). The protein bands were detected with horseradish peroxidase-conjugated secondary antibodies (Bio-Rad) and ECL (Amersham Biosciences). The membrane was then reprobed with an anti-{beta}-actin monoclonal antibody to control for possible differences in loading.

cPLA2 Activity Assay—cPLA2 activity in macrophage cytosol fractions was assayed using a liposomal substrate as described by de Carvalho et al. (19). Briefly, 30 µM unlabeled sn-2 arachidonyl PC, and 30 µM dioleoylglycerol, and phosphatidylcholine-1-stearoyl-2-[3H]arachidonyl (100,000 cpm/assay) were dried under nitrogen and then resuspended in 150 mM NaCl, 1 mg/ml BSA, with or without 1 mM CaCl2. The assay tubes were incubated at 37 °C in a shaking water bath for 5 min, and the reaction was started by the addition of 20 µg cytosol (50 µl final assay volume). The mixture was incubated for an additional 10 min, and then the reaction was stopped by the addition of 2.5 ml of Dole reagent (isopropyl alcohol/heptane/1 N H2SO4; 300:75:15) plus 20 µg of oleic acid carrier. To extract the lipids, 1.5 ml of heptane and 1 ml of water were added, followed by vortexing and centrifugation. The upper phase was passed over a 3-ml silica column (Supelco Cupelclean LC-Si SPE), and the released [3H]arachidonic acid was eluted with 2 ml of chloroform. The chloroform was evaporated, and the radioactivity was counted using liquid scintillation. For the experiments using cPLA2{alpha}, unlabeled sn-2 linoleoyl phosphatidylcholine was added to create liposomes with varying sn-2 arachidonoyl percentages. These liposomes were then incubated with cPLA2 for 1 min, and the release of [3H]AA was assayed as above. The cpm data were converted to mass data based upon the specific activity of [3H]AA in each liposome preparation.

Fatty Acid Analysis—Macrophage lipids were extracted with 0.5 ml of hexane/isopropyl alcohol (3:2, v/v) for two 30-min periods. The phospholipids were isolated by thin-layer chromatography on soft-layer adsorbosil using a solvent system of hexane/ethyl ether/glacial acidic acid (80:20:1, v/v/v). The phospholipid spot was scraped, and methyl ester derivatives of the phospholipid fatty acids were prepared by adding 1.5 ml of 7% acetyl chloride/dry methanol to the silica. After boiling for 60 min, the released fatty acyl methyl esters were extracted with 100 µl of hexane and 5 ml of 6% K2CO3. After vortexing and centrifugation, the fatty acyl methyl esters in the upper phase were analyzed by gas-liquid chromatography as described previously (24).

Whole Cell Cholesterol Esterification Assay—Macrophages were incubated for 5 h in Dulbecco's modified Eagle's medium, 1% FBS containing 0.1 mM [14C]oleate complexed with albumin and 100 µg/ml AcLDL. Then, the cells were washed two times with ice-cold PBS, and cellular lipids were extracted twice with 0.5 ml of hexane/isopropyl alcohol (3:2, v/v) for 30 min at room temperature. Whole cell cholesterol esterification activity was assayed by determining the cellular content of cholesteryl-[14C]oleate by thin-layer chromatography (25). The lipid-extracted cellular material was dissolved in 1 ml of 0.1 N NaOH, and aliquots were assayed for protein by the method of Lowry et al. (26).

Trafficking of Lipoprotein-derived Fatty Acids to Phospholipids— Mouse peritoneal macrophages were grown to confluency on 24-well plates. The cells were incubated for 5 h in Dulbecco's modified Eagle's medium, 1% FBS containing 100 µg/ml reconstituted acetyl-LDL containing a neutral lipid core of cholesteryl-[14C]oleate. Various pharmacological agents were added as described under "Results." At the end of the 5-h incubation, cellular lipids were extracted in hexane/isopropyl alcohol (3:2) as described above. The phospholipids were isolated by TLC using 80:20:1 (v/v) hexane/diethyl ether/glacial acetic acid. The phospholipid spots were scraped, and [14C]oleate incorporation was determined by liquid scintillation counting.

Statistics—Values are given as means ± S.E. (n = 3); absent error bars in the bar graphs signify S.E. values smaller than the graphic symbols.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Incorporation of Lipoprotein-derived Fatty Acids into Cellular Phospholipids Is Enhanced by ACAT Inhibition and Is Dependent on a U18666A-senstitive, NPC1-dependent Trafficking Pathway—The incorporation of lipoprotein-derived fatty acids into cellular phospholipids can have important biological consequences, yet little is known about the how such events are regulated. Our first goal was to determine how cholesterol-fatty acyl esterification, a potentially competing reaction for fatty acid incorporation into phospholipids, affects this process. To evaluate the incorporation of lipoprotein-derived fatty acids into cellular phospholipids, the neutral lipid core of acetyl-LDL was reconstituted with cholesteryl-[14C]oleate using the procedure of Krieger (27). These lipoproteins were then incubated with mouse peritoneal macrophages for 5 h. Cellular lipids were extracted, and phospholipids were isolated by thin-layer chromatography to determine formation of phospholipid-[14C]oleate. To determine how cholesterol-fatty acyl esterification might affect this process, the ACAT inhibitor 58035 was included in some of the incubations. As shown by the black bars in Fig. 1A, inhibition of esterification led to a substantial 2.6-fold increase in the incorporation of lipoprotein-derived fatty acids into cellular phospholipids. These data suggest cholesterol-fatty acyl esterification uses a pool of lipoprotein-derived fatty acids that would otherwise be incorporated into cellular phospholipids. Thus, when ACAT is active, lipoprotein-derived fatty acid incorporation into phospholipids is limited by the competing cholesterol esterification reaction.



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  		FIG. 1.
Effect of inhibition of ACAT and of late endosomal/lysosomal trafficking on the incorporation of lipoprotein-derived fatty acids into cellular phospholipids. A, macrophages were incubated for 5 h in media containing 100 µg/ml acetyl-LDL reconstituted with cholesteryl-[14C]oleate in the absence or presence of 10 µg/ml 58035 or 1 µM U18666A. B, macrophages from Npc1+/+ (black bars) and Npc1+/- (gray bars) mice were incubated for 5 h with 100 µg/ml acetyl-LDL reconstituted with cholesteryl-[14C]oleate in the absence or presence of 10 µg/ml 58035. Cellular phospholipids were isolated and assayed for [14C]oleate incorporation.

 
Previous work from our laboratory has shown that incubation of macrophages with acetyl-LDL plus 58035 induces de novo phospholipid biosynthesis via activation of the rate-limiting enzyme, CTP:phosphocholine cytidylyltransferase {alpha} (CT{alpha}) (18, 28). Thus, the increased incorporation of fatty acids into phospholipids displayed in Fig. 1A might result from an overall increase in de novo phospholipid biosynthesis. To test this possibility, CT{alpha}-deficient macrophages, which have markedly diminished de novo phospholipid biosynthesis in response to acetyl-LDL and 58035 (15), were incubated with cholesteryl-[14C]oleate-labeled acetyl-LDL and 58035 and compared with wild-type macrophages in their incorporation of lipoprotein fatty acids into cellular phospholipids. We found that the CT{alpha}-deficient macrophages showed the same increase in phospholipid [14C]oleate incorporation as wild-type macrophages (1.6-fold increase in macrophages lacking CT{alpha} versus 1.7-fold for macrophages with functional CT{alpha}). These data suggest that lipoprotein-derived fatty acids, when not utilized for the competing cholesterol esterification reaction, are incorporated into cellular phospholipids via transesterification rather than by an increase in de novo phospholipid biosynthesis.

Recent evidence suggests that lipoprotein-derived cholesterol-fatty acyl esters are hydrolyzed in an endosomal compartment and then transferred to late endosomes/lysosomes, where the liberated cholesterol is trafficked to peripheral cellular sites via an NPC1-dependent pathway (29). To determine if the trafficking of acetyl-LDL-derived [14C]oleate to cellular phospholipids shares properties with the trafficking of lipoprotein-derived cholesterol, we determined the susceptibility of phospholipid-[14C]oleate formation to the cholesterol trafficking inhibitor, U18666A. U18666A is an amphipathic amine that blocks the NPC1-dependent pathway of late endosomal/lysosomal cholesterol transport in cells (30). As shown by the gray bars in Fig. 1A, U18666A markedly diminished the incorporation of lipoprotein-derived [14C]oleate into phospholipids, an effect particularly apparent in the ACAT-inhibited macrophages. These data imply that lipoprotein-derived fatty acids traffic via an U18666A-inhibitable pathway from late endosomes/lysosomes to cellular phospholipids.

To further support a role for late endosomal/lysosomal trafficking, we employed macrophages with a heterozygous mutation in Npc1, which have a partial defect in intracellular cholesterol trafficking (10, 31). These Npc1+/- mice, unlike Npc1-/- mice, have a normal lifespan and thus are more amenable for the isolation of peritoneal macrophages. Because of the partial defect in cholesterol trafficking, we predicted that these macrophages would have a partial defect in the trafficking of lipoprotein-derived fatty acids. To test this idea, peritoneal macrophages from Npc1+/+ and Npc1± mice were assayed for their ability to incorporate acetyl-LDL-derived [14C]oleate into phospholipids. As predicted, [14C]oleate incorporation into phospholipids was partially suppressed in Npc1+/- macrophages, which was most apparent in ACAT-inhibited cells (Fig. 1B). Thus, the NPC1 pathway is not only involved in late endosomal/lysosomal cholesterol trafficking (32) but also in the trafficking of lipoprotein-derived fatty acids.

Suppression of A23187 [GenBank] -induced Eicosanoid Secretion Is Enhanced by ACAT Inhibition and is Dependent on a U18666A-senstitive, NPC1-dependent Trafficking Pathway—We hypothesized that the effects of cholesterol-fatty acyl esterification and late endosomal/lysosomal trafficking on lipoprotein-derived fatty acid incorporation into cellular phospholipid would have important biological effects on cells that internalize lipoproteins. To test this idea, we first used a model in which incubation of macrophages with foam cell-inducing lipoproteins like acetyl-LDL causes a defect in agonist-induced AA release and PGE2 production (11, 12, 33). Despite the commonality of this observation, there has been confusion about mechanism. Field and coworkers (33) concluded that cholesterol-loading of macrophages leads to a defect in cPLA2 activation. Pollaud et al. (12) hypothesized that stimulation of ACAT was necessary for the suppression in PGE2 synthesis by sequestering AA. Arai et al. (11) demonstrated an inverse correlation with percent AA composition of cellular phospholipids, which is consistent with a mechanism in which enrichment of cellular phospholipids with lipoprotein-derived non-AA fatty acids suppresses PGE2 synthesis.

To address these uncertainties in the context of our new findings on lipoprotein-fatty acid trafficking and metabolism, we first assayed PGE2 secretion in macrophages incubated with acetyl-LDL in the absence or presence of 58035 and U18666A. In particular, we hypothesized that if lipoprotein-derived fatty acid incorporation into cellular phospholipids was important in suppressing PGE2 production, then ACAT inhibition should enhance the suppression and U18666A should prevent the suppression. Thus, macrophages were incubated for 5 h with 100 µg/ml acetyl-LDL alone or in addition to 10 µg/ml 58035, 1 µM U18666A, or both compounds. PGE2 secretion was then stimulated for 5 min with 20 µM A23187 [GenBank] , a calcium ionophore that stimulates cPLA2-mediated free arachidonic acid release and PGE2 secretion. As shown in Fig. 2A, incubation with acetyl-LDL alone caused a ~50% decrease in A23187 [GenBank] -induced PGE2 release, consistent with the previous reports mentioned above. Remarkably, inclusion of the ACAT inhibitor almost completely abolished PGE2 secretion, and U18666A fully restored PGE2 release even in the presence of the ACAT inhibitor. Inclusion of the ACAT inhibitor alone did not diminish PGE2 synthesis (data not shown). The effect of the Npc1+/- mutation is shown in Fig. 2B. Consistent with the data in Fig. 1B, the suppression of PGE2 production in macrophages incubated with acetyl-LDL plus the ACAT inhibitor is partially yet substantially prevented in Npc1+/- macrophages. These data support the hypothesis the suppression of PGE2 production in macrophages by atherogenic lipoproteins involves late endosomal/lysosomal trafficking of lipoprotein-derived fatty acids to cellular phospholipids and incorporation into these phospholipids by a reaction that is competed by cholesterol-fatty acyl esterification.



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  		FIG. 2.
Effect of inhibition of ACAT and of late endosomal/lysosomal trafficking on A23187 [GenBank] -induced PGE2 production. A, macrophages were incubated for 5 h in media alone or containing 100 µg/ml acetyl-LDL in the absence or presence of 10 µg/ml 58035 or 1 µM U18666A. B, macrophages from Npc1+/+ and Npc1+/- mice were incubated for 5 h in media alone or media containing 100 µg/ml acetyl-LDL plus 10 µg/ml 58035. The cells were then incubated for 5 min with 20 µM A23187 [GenBank] , and the amount of PGE2 in the media was quantified by ELISA.

 
To broaden the scope of these findings, we explored the effects of acetyl-LDL loading and inhibition of cholesterol-fatty acyl esterification and lipid trafficking on the production of another macrophage eicosanoid, leukotriene C4/D4/E4 (LT-C4/D4/E4). As shown in Fig. 3, macrophages produced large quantities of LT-C4/D4/E4 in response to A23187 [GenBank] , and this was partially suppressed by incubation with acetyl-LDL. Most importantly, this suppression was markedly enhanced when cholesterol-fatty acyl esterification was inhibited by 58035 and completely prevented when late endosomal/lysosomal lipid trafficking was inhibited by U18666A. Thus, the model proposed above is applicable to the synthesis of both of the major classes of macrophage eicosanoids.



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  		FIG. 3.
Effect of inhibition of ACAT and of late endosomal/lysosomal trafficking on A23187 [GenBank] -induced LT-C4/D4/E4 production. Macrophages were incubated for 5 h in media alone or containing 100 µg/ml acetyl-LDL in the absence or presence of 10 µg/ml 58035 or 1 µM U18666A. The cells were then incubated for 5 min with 20 µM A23187 [GenBank] or buffer control, and the amount of LT-C4/D4/E4 in the media was quantified by ELISA.

 
The Effects of Cholesterol-Fatty Acyl Esterification and Late Endosomal Lipid Trafficking on Lipoprotein-mediated Eicosanoid Suppression Occur at the Level of AA Release from Phospholipids but Does Not Involve Direct Inhibition of cPLA2 Activity—The commonality of the results with both prostaglandins and leukotrienes suggested that cholesterol-fatty acyl esterification and late endosomal/lysosomal lipid trafficking were affecting eicosanoid production proximal to the late stage biosynthetic reactions of any individual class of eicosanoids. In this context, we considered a model in which these processes were occurring at the level of cPLA2-mediated AA release from cellular phospholipids. To directly test this point, we assayed AA release by measuring A23187 [GenBank] -induced efflux of cellular AA to BSA-containing medium. Macrophage AA pools were labeled by overnight incubation with media containing 0.5 µCi of [3H]arachidonic acid, and the cells were then either left untreated or incubated for 5 h in media containing 100 µg/ml acetyl-LDL in the absence or presence of 58035 or U18666A. cPLA2-mediated hydrolysis of AA from phospholipids was stimulated by a 5-min incubation in media containing 20 µM A23187 [GenBank] and 0.2% BSA, and the [3H]arachidonic acid content of the media and cells was determined. The results are shown in Fig. 4A. Similar to the case with PGE2 release, acetyl-LDL alone caused a decrease in AA release of ~50%. Inclusion of the ACAT inhibitor further inhibited AA release to ~80%, while inclusion of U18666A completely abolished the suppression of AA release. Thus, the effects of blocking cholesterol esterification and late endosomal/lysosomal lipid trafficking on eicosanoid production are paralleled by effects on AA release, further indicating that the effects are proximal to the eicosanoid synthesizing enzymes.



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  		FIG. 4.
Effect of inhibition of ACAT and of late endosomal/lysosomal trafficking on A23187 [GenBank] -induced AA release from cellular lipids. A, macrophages prelabeled for 24 h with [3H]AA were incubated for 5 h in media alone or containing 100 µg/ml acetyl-LDL in the absence or presence of 10 µg/ml 58035 or 1 µM U18666A. The cells were then incubated for 5 min in media containing 0.2% BSA and 20 µM A23187 [GenBank] , and the cells and media were assayed for free [3H]AA. The data are expressed as percent [3H]AA cpm in the media. B, cytosol (20 µg of protein) harvested from macrophages incubated for 5 h in media alone or containing 100 µg/ml acetyl-LDL was assayed for cPLA2 activity.

 
The defect in phospholipid-AA release could be due to alterations in the phospholipids themselves or to direct suppression of cPLA2 protein or activity. We therefore assayed cPLA2 protein and activity in cytosolic fractions from untreated and lipoprotein-treated macrophages. We found that that cPLA2 protein levels by immunoblot analysis (data not shown) and, most importantly, cPLA2 activity in untreated and acetyl-LDL-loaded macrophages were very similar (Fig. 4B). Furthermore, calcium-dependent binding of cPLA2 to cellular membranes, an important step in phospholipase activity in stimulated cells, was also not altered by acetyl-LDL loading (data not shown). These data support a model in which cholesterol-fatty acyl esterification and late endosomal/lysosomal lipid trafficking affect lipoprotein-mediated eicosanoid suppression by altering the phospholipid substrates for cPLA2.

Lipoprotein-mediated PGE2 Suppression in ACAT-inhibited Macrophages Depends on Lipoprotein-derived Non-arachidonic Acid Fatty Acids, Not Cholesterol—A plausible explanation for how cholesterol-fatty acyl esterification affects lipoprotein-mediated eicosanoid suppression is by alterations in enrichment of cellular cPLA2-substrate phospholipids with NAAFAs. An alternative explanation, particularly in the context of the late endosomal/lysosomal trafficking data, is that excess cellular free cholesterol inhibits cPLA2-mediated AA release. Indeed, FC loading of macrophages has profound effects on the function of the ER (10), which is a major site of cPLA2-mediated hydrolysis of AA-containing phospholipids (34).

To identify the component of acetyl-LDL responsible for the suppression of PGE2 production in ACAT-inhibited macrophages, we reconstituted acetyl-LDL with one of four neutral lipid cores: cholesteryl linoleate (rAcLDL-Chol-18:2), cholesteryl arachidonate (rAcLDL-Chol-20:4), trilinolein (rAcLDL-Tri-18:2) or triarachidonate (rAcLDL-Tri-20:4). Macrophages were incubated for 5 h with 100 µg/ml of the various rAcLDLs in the presence of 10 µg/ml 58035, and the cells were then stimulated for 5 min with 20 µM A23187 [GenBank] and assayed for PGE2 production. As shown in the first three bars in Fig. 5, incubation with rAcLDL-Chol-18:2 led to a ~50% decrease in PGE2 production, while rAcLDL-Chol-20:4 caused no suppression. This difference was observed despite the similar ability of both particles to deliver cholesterol to cells as determined by a [14C]oleate whole-cell cholesterol esterification assay: cholesteryl-[14C]oleate formation (cpm/µg cell protein ± S.E.) was 106.2 ± 12.3 for rAcLDL-Chol-18:2 and 131.0 ± 4.2 for rAcLDL-Chol-20:4. Moreover, rAcLDL-tri-18:2, a particle essentially free of cholesterol, was able to completely abolish PGE2 secretion, while rAcLDL-tri-20:4 had relatively little effect (last two bars in Fig. 5).2 These data show that the suppression of PGE2 synthesis in lipoprotein-loaded, ACAT-inhibited macrophages is independent of cholesterol but requires lipoprotein-derived NAAFAs.



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  		FIG. 5.
Effect of varying lipoprotein neutral lipid core composition on PGE2 secretion in lipoprotein-loaded, ACAT-inhibited macrophages. Macrophages were incubated for 5 h in media alone or media containing 100 µg/ml of reconstituted acetyl-LDL plus 10 µg/ml 58035. The acetyl-LDL with reconstituted with cholesteryl linoleate (rAcLDL-Chol-18:2), cholesteryl arachidonate (rAcLDL-Chol-20:4), trilinolein (rAcLDL-tri-18:2), and triarachidonate (rAcLDL-tri-20:4). The cells were then incubated for 5 min with 20 µM A23187 [GenBank] , and the amount of PGE2 in the media was quantified by ELISA.

 

To test the principle that enrichment of phospholipids with NAAFAs leads to less cPLA2-mediated release of AA, we first measured the change in phospholipid composition in control macrophages and macrophages incubated with acetyl-LDL plus 58035 and then determined whether this degree of change could effect cPLA2-mediated AA release in an in vitro system. As shown in Fig. 6A, the percent AA in cellular phospholipids was decreased from 34 to 23% by incubation of macrophages with acetyl-LDL plus the ACAT inhibitor. Using an in vitro assay, a similar change in percent AA in liposomal phospholipids led to an ~2-fold decrease in cPLA2-mediated AA release (Fig. 6B). Although the actual change in percent AA in the subpopulation of cellular phospholipids that is the substrate pool of cPLA2 would be difficult to determine, these data demonstrate that enrichment of phospholipids with NAAFAs can lead to decreased cPLA2-mediated AA release.



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  		FIG. 6.
Effect of varying phospholipid AA:NAAFA ratio on cPLA2-mediated AA hydrolysis. A, macrophages were incubated for 5 h in media alone or media containing 100 µg/ml of acetyl-LDL plus 10 µg/ml 58035. Cellular phospholipids were isolated and assayed for the mass of individual fatty acid species. The data are expressed as percent AA in cellular phospholipids. B, purified recombinant cPLA2 (100 ng) was added to phosphatidylcholine liposomes with varying percentages of sn-2 arachidonoyl versus sn-2 linoleoyl, and the release of AA was determined.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The data in this report support the following model of lipoprotein-derived fatty acid trafficking and metabolism in the context of prostanoid biosynthesis (Fig. 7): in non-loaded macrophages (Fig. 7A), endogenous phospholipids containing AA in the sn-2 position supply ample substrate for cPLA2-induced eicosanoid synthesis. With lipoprotein loading and full ACAT activity (Fig. 7B), most of the lipoprotein-derived fatty acids and cholesterol that are delivered to the ER are esterified by ACAT, but a portion of the fatty acids are incorporated into phospholipids. In the case of human LDL, which is the source of acetyl-LDL used in this study, most of these fatty acids are NAAFAs, and so the AA:NAAFA ratio in these phospholipids is decreased. This leads to a decreased release of AA upon activation of cPLA2 and thus a lower production of eicosanoids. When ACAT activity is compromised (Fig. 7C), more of the lipoprotein-derived NAAFAs are available for incorporation into phospholipids, thus exacerbating the suppression of eicosanoid production. If lipoprotein-derived NAAFAs are prevented from trafficking to the site of phospholipid biosynthesis (Fig. 7D), cellular phospholipids remain relatively enriched in AA, and agonist-induced eicosanoid production remains high.



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  		FIG. 7.
Model of how lipoproteins, cholesterol esterification, and late endosomal/lysosomal trafficking affect eicosanoid synthesis in macrophages. A, when cPLA2 activity is stimulated in the absence of prior lipoprotein loading, cellular phospholipids (PLs) containing AA in the sn-2 position contribute to robust prostaglandin and leukotriene (PG/LT) synthesis. B, with lipoprotein loading, some of the cellular phospholipids are remodeled with lipoprotein-derived NAAFAs in the sn-2 position, thus limiting the amount of eicosanoids synthesized in response to agonists. This remodeling is limited because a substantial portion of the lipoprotein-derived NAAFAs are esterified to cholesterol by ACAT. C, when ACAT is inhibited, a greater portion of lipoprotein-derived NAAFAs are incorporated into phospholipids, because the NAAFAs are not utilized in the cholesterol esterification reaction. Thus, eicosanoid synthesis is maximally suppressed. D, when late endosomal/lysosomal lipid trafficking is inhibited, lipoprotein-derived NAAFAs cannot traffic to the site of phospholipid synthesis, presumably the ER. Thus, phospholipids are not remodeled with NAAFAs, and eicosanoid synthesis is not suppressed, even in the face of ACAT inhibition.

 
There are three important implications of these findings. First, this study provides new information about the trafficking and metabolism of lipoprotein-derived fatty acids. In particular, we show that the fatty acids destined for incorporation into a cPLA2 substrate pool of phospholipids use a late endosomal/lysosomal trafficking pathway that appears to be the same as that used by lipoprotein-derived cholesterol. The role of NPC1 in lipoprotein-derived fatty acid transport is of particular interest. NPC1 is a glycoprotein that resides in the membranes of late endosomes/lysosomes (35). Initial interest in NPC1 stemmed from the observation that cells lacking this protein accumulate large amounts of cholesterol within their endosomal-lysosomal system (32, 36). For this reason, and because the protein contains a putative sterol-sensing domain (35), the cholesterol transport function of NPC1 has been widely studied. However, mammalian cells with the Npc1-/- mutation accumulate a variety of lipids, notably gangliosides (37-40), and our findings provide evidence that NPC1 can play a role in the incorporation of lipoprotein-derived fatty acids into cellular phospholipids, at least in the case of lipoprotein-loaded macrophages.

Moreover, we show that the ACAT-mediated cholesterol-fatty acyl esterification reaction competes for the pool of lipoprotein-derived fatty acids that otherwise would be incorporated into cellular phospholipids. In an analogous study, we showed that the ACAT reaction also competes for an important pool of cholesterol, namely, that used in sterol-mediated down-regulation of the LDL receptor and hydroxymethylglutarylcoenzyme A reductase (41). Of interest, both of the reactions competed for by ACAT-fatty acid incorporation into phospholipids and cholesterol-mediated regulation occur in the ER (42, 43). Thus, the focality of these reactions may be important in their utilization of common substrate pools.

Second, the study helps resolve a controversy in the literature related to the mechanism by which PGE2 synthesis is suppressed during macrophage foam cell formation. The idea that there is direct inhibition of cPLA2 activity, as proposed by Mathur et al. (33), is clearly not supported by the data in this report. Another study proposed that acetyl-LDL caused suppression of PGE2 synthesis by stimulating ACAT activity and thus sequestering cellular AA into cholesteryl esters (12). The ACAT inhibitor data presented here indicate that cholesterol esterification is not necessary for this effect. Finally, other investigators showed an inverse correlation between PGE2 production and lipoprotein-derived NAAFA content of macrophage phospholipids (11), but definitive proof of a direct causal relationship was not provided. In this report, the results of experiments using reconstituted acetyl-LDL (Fig. 5), liposomes of various NAAFA:AA ratios (Fig. 6), and manipulations of lipoprotein-derived fatty acid trafficking and metabolism (Figs. 1, 2, 3, 4) provide direct evidence for the hypothesis that incorporation of lipoprotein-derived NAAFAs into cellular phospholipids directly causes the suppression of cPLA2-mediated AA release, leading to decreased production of both PGE2 and LT-C4/D4/E4. Of interest, previous work has shown that incubation of other cell types with lipoproteins that are enriched in AA can actually enhance eicosanoid biosynthesis (44, 45). These data are consistent with the overall model outlined in this paper (Fig. 7) and are consistent with our finding that AA-rich lipoproteins failed to suppress eicosanoid production (Fig. 5). However, unlike the studies cited above, AA-rich lipoproteins in A23187 [GenBank] -stimulated macrophages did not enhance PGE2 production above the medium-alone control (Fig. 5).

Regarding the mechanism of suppression of cPLA2-mediated AA release, there may be two related processes working in concert. In the simplest scenario, the sn-2 arachidonoyl substrate pool for cPLA2 would be rate-limiting when the enzyme encounters phospholipids that are relatively enriched in NAAFAs versus AA. An additional possible mechanism is based on previous work showing the translocation of cPLA2 from the cytosol to ER/Golgi membranes, which is the essential first step in cPLA2 action, is insensitive to the identity of the acyl chain in the sn-2 position of membranes phospholipids (46). Fatty acid release by cPLA2, however, is strongly dependent on the identity of the sn-2 fatty acid, with a 20-40-fold preference of arachidonyl over stearoyl (47). Thus, it is possible that membranes enriched in NAAFAs trap limiting amounts of cPLA2 on non-substrate phospholipids.

Third, the findings in this report may have direct relevance to eicosanoid signaling in advanced atherosclerotic lesions. In light of the evidence for lipoprotein loading and dysfunctional cholesterol-fatty acyl esterification in advanced lesional macrophages (9), we propose that these macrophages might have highly suppressed eicosanoid production. The net effect of this suppression would depend upon the relative pathobiological roles of macrophage-derived PGE2, LT-C4/D4/E4, and perhaps other AA-derived bioactive molecules. On the one hand, macrophage-derived leukotrienes may be atherogenic (48), although little is known about the specific effects of these molecules on atherosclerotic processes (49). On the other hand, the effects of PGE2 on atherosclerotic processes have been widely studied, and the findings suggest an overall protective role. For example, PGE2 has been shown to inhibit T-cell proliferation (50); selectively suppress the expression of several pro-atherogenic chemokines in activated macrophage via interaction with the EP4 receptor (51); enhance platelet aggregation at low concentrations through the EP3 receptor but inhibit platelet aggregation at high concentrations through activation of the prostacyclin receptor (52); and inhibit IL-1-induced SMC proliferation (53). Moreover, another AA-derived product in macrophages, 15-hydroxyeicosatetraenoic acid (15-HETE), is a ligand for PPAR-{gamma}, and thus would also be expected confer an overall protective effect on atherosclerosis (54, 55). Therefore, if suppression of PGE2 and possibly 15-HETE are dominant effects in advanced lesional macrophages, lesion development and lesional complications might be exacerbated. Of interest in this regard, macrophages with defective cholesterol esterification also accumulate FC, which induces cholesterol efflux defects, ER dysfunction, apoptosis, and lesional necrosis (9, 10, 31, 56, 57). What is remarkable about these detrimental consequences of macrophage ACAT dysfunction is that all are ameliorated by interrupting the NPC1-dependent late endosomal/lysosomal trafficking pathway (data here and in Refs. 9, 10, 31, 58). Indeed, late lesional complications are significantly prevented in Npc1+/- mice (9), suggesting that partial interruption of the NPC1-dependent late endosomal/lysosomal trafficking pathway may be the basis of a novel plaque-stabilizing therapeutic strategy.


   FOOTNOTES
 
* This study was supported by National Institutes of Health Grants HL-56984 and HL-57560 (to I. T.) and HL-34303 and HL-61378 (to C. C. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Dept. of Medicine, Columbia University, 630 West 168th Street, New York, NY 10032. Tel.: 212-305-9430; Fax: 212-305-4834; E-mail: iat1{at}columbia.edu.

1 The abbreviations used are: NPC, Niemann-Pick C; AA, arachidonic acid; ACAT, acyl-CoA:cholesterol acyltransferase; cPLA2, cytosolic phospholipase A2; ER, endoplasmic reticulum; FC, free cholesterol; LDL, low-density lipoprotein; LT, leukotriene; NAAFA, non-arachidonic fatty acid; PGE2, prostaglandin E2; rAcLDL, reconstituted acetyl-LDL; FBS, fetal bovine serum; BSA, bovine serum albumin; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. Back

2 The enhanced inhibition of PGE2 production seen with rAcLDL-Tri-18:2 in comparison with rAcLDL-Chol-18:2 was not associated with an increased incorporation of rAcLDL-Tri-18:2-derived fatty acids into cellular phospholipids (data not shown). Therefore, it is possible that fatty acids derived from lipoprotein-triglycerides might incorporate preferentially into a subset of phospholipids critically important in eicosanoid synthesis. Indeed, others have observed that the mode of delivery of fatty acids to cells can influence fatty acid trafficking and metabolism (59). Back


   ACKNOWLEDGMENTS
 
We thank Inge Hansen for conducting the fatty acid mass assays.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Ross, R. (1995) Annu. Rev. Physiol. 57, 791-804[CrossRef][Medline] [Order article via Infotrieve]
  2. Libby, P. and Clinton, S. K. (1993) Curr. Opin. Lipidol. 4, 355-363[CrossRef]
  3. Tabas, I. (2002) J. Clin. Invest. 110, 905-911[CrossRef][Medline] [Order article via Infotrieve]
  4. Brown, M. S., and Goldstein, J. L. (1983) Annu. Rev. Biochem. 52, 223-261[CrossRef][Medline] [Order article via Infotrieve]
  5. Tabas, I. (2000) Biochim. Biophys. Acta 1529, 164-174[Medline] [Order article via Infotrieve]
  6. Liscum, L., and Munn, N. J. (1999) Biochim. Biophys. Acta 1438, 19-37[Medline] [Order article via Infotrieve]
  7. Groener, J. E., Bax, W., and Poorthuis, B. J. (1996) J. Lipid Res. 37, 2271-2279[Abstract]
  8. Li, Q., Fuhrman, B., and Aviram, M. (1995) Isr. J. Med. Sci. 31, 474-478[Medline] [Order article via Infotrieve]
  9. Feng, B., Zhang, D., Kuriakose, G., Devlin, C. M., Kockx, M., and Tabas, I. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 10423-10428[Abstract/Free Full Text]
  10. Feng, B., Yao, P. M., Li, Y., Devlin, C. M., Zhang, D., Harding, H. P., Sweeney, M., Rong, J. X., Kuriakose, G., Fisher, E. A., Marks, A. R., Ron, D., and Tabas, I. (2003) Nat. Cell Biol. 5, 781-792[CrossRef][Medline] [Order article via Infotrieve]
  11. Arai, H., Nagano, Y., Narumiya, S., and Kita, T. (1992) J. Biochem. (Tokyo) 112, 482-487[Abstract/Free Full Text]
  12. Pollaud, C., Krause, S., Lepert, J. C., Orfila, C., Seguelas, M., Festal, D., Decerprit, J., and Pipy, B. (1995) Biochim. Biophys. Acta 1259, 211-219[Medline] [Order article via Infotrieve]
  13. Deleted in proof
  14. Ross, A. C., Go, K. J., Heider, J. G., and Rothblat, G. H. (1984) J. Biol. Chem. 259, 815-819[Abstract/Free Full Text]
  15. Zhang, D., Tang, W., Yao, P. M., Yang, C., Xie, B., Jackowski, S., and Tabas, I. (2000) J. Biol. Chem. 275, 35368-35376[Abstract/Free Full Text]
  16. Loftus, S. K., Morris, J. A., Carstea, E. D., Gu, J. Z., Cummings, C., Brown, A., Ellison, J., Ohno, K., Rosenfeld, M. A., Tagle, D. A., Pentchev, P. G., and Pavan, W. J. (1997) Science 277, 232-235[Abstract/Free Full Text]
  17. Tang, W., Walsh, A., and Tabas, I. (1999) Biochim. Biophys. Acta 1437, 301-316[Medline] [Order article via Infotrieve]
  18. Shiratori, Y., Okwu, A. K., and Tabas, I. (1994) J. Biol. Chem. 269, 11337-11348[Abstract/Free Full Text]
  19. de Carvalho, M. G., Garritano, J., and Leslie, C. C. (1995) J. Biol. Chem. 270, 20439-20446[Abstract/Free Full Text]
  20. Havel, R. J., Eder, H., and Bragdon, J. (1955) J. Clin. Invest. 34, 1345-1353[Medline] [Order article via Infotrieve]
  21. Basu, S. K., Goldstein, J. L., Anderson, R. G. W., and Brown, M. S. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 3178-3182[Abstract/Free Full Text]
  22. Deleted in proof
  23. Leslie, C. C. (1991) J. Biol. Chem. 266, 11366-11371[Abstract/Free Full Text]
  24. Kuwae, T., Schmid, P. C., Johnson, S. B., and Schmid, H. H. (1990) J. Biol. Chem. 265, 5002-5007[Abstract/Free Full Text]
  25. Tabas, I., Boykow, G. C., and Tall, A. R. (1987) J. Clin. Invest. 79, 418-426[Medline] [Order article via Infotrieve]
  26. Lowry, O. H., Rosenbrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275[Free Full Text]
  27. Krieger, M. (1986) Methods Enzymol. 128, 608-613[Medline] [Order article via Infotrieve]
  28. Shiratori, Y., Houweling, M., Zha, X., and Tabas, I. (1995) J. Biol. Chem. 270, 29894-29903[Abstract/Free Full Text]
  29. Sugii, S., Reid, P. C., Ohgami, N., Du, H., and Chang, T. Y. (2003) J. Biol. Chem. 278, 27180-27189[Abstract/Free Full Text]
  30. Liscum, L. (1990) Biochim. Biophys. Acta 1045, 40-48[Medline] [Order article via Infotrieve]
  31. Feng, B., and Tabas, I. (2002) J. Biol. Chem. 277, 43271-43280[Abstract/Free Full Text]
  32. Liscum, L., and Klansek, J. J. (1998) Curr. Opin. Lipidol. 9, 131-135[CrossRef][Medline] [Order article via Infotrieve]
  33. Mathur, S. N., Albright, E., and Field, F. J. (1989) J. Lipid Res. 30, 1385-1395[Abstract]
  34. Gijon, M. A., and Leslie, C. C. (1999) J. Leukoc. Biol. 65, 330-336[Abstract]
  35. Carstea, E. D., Morris, J. A., Coleman, K. G., Loftus, S. K., Zhang, D., Cummings, C., Gu, J., Rosenfeld, M. A., Pavan, W. J., Krizman, D. B., Nagle, J., Polymeropoulos, M. H., Sturley, S. L., Ioannou, Y. A., Higgins, M. E., Comly, M., Cooney, A., Brown, A., Kaneski, C. R., Blanchette-Mackie, E. J., Dwyer, N. K., Neufeld, E. B., Chang, T. Y., Liscum, L., Tagle, D. A., et al. (1997) Science 277, 228-231[Abstract/Free Full Text]
  36. Pentchev, P. G., Vanier, M. T., Suzuki, K., and Patterson, M. C. (1995) in Metabolic Basis of Inherited Disease (Scriver, C. R., Beaudet, A. L., Sly, W. S., and Valle, D., eds) pp. 2625-2639, McGraw-Hill, New York
  37. Sugimoto, Y., Ninomiya, H., Ohsaki, Y., Higaki, K., Davies, J. P., Ioannou, Y. A., and Ohno, K. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 12391-12396[Abstract/Free Full Text]
  38. Vanier, M. T. (1999) Neurochem. Res. 24, 481-489[CrossRef][Medline] [Order article via Infotrieve]
  39. Harzer, K., and Kustermann-Kuhn, B. (2001) Clin. Chim. Acta 305, 65-73[CrossRef][Medline] [Order article via Infotrieve]
  40. Harzer, K., Massenkeil, G., and Frohlich, E. (2003) FEBS Lett. 537, 177-181[CrossRef][Medline] [Order article via Infotrieve]
  41. Tabas, I., Weiland, D. A., and Tall, A. R. (1986) J. Biol. Chem. 261, 3147-3155[Abstract/Free Full Text]
  42. Starodub, O., Jolly, C. A., Atshaves, B. P., Roths, J. B., Murphy, E. J., Kier, A. B., and Schroeder, F. (2000) Am. J. Physiol. Cell. Physiol. 279, C1259-C1269[Abstract/Free Full Text]
  43. Horton, J. D., Goldstein, J. L., and Brown, M. S. (2002) Cold Spring Harb. Symp. Quant. Biol. 67, 491-498[CrossRef][Medline] [Order article via Infotrieve]
  44. Pomerantz, K. B., Tall, A. R., Feinmark, S. J., and Cannon, P. J. (1984) Circ. Res. 54, 554-565[Abstract/Free Full Text]
  45. Salbach, P. B., Specht, E., von Hodenberg, E., Kossmann, J., Janssen-Timmen, U., Schneider, W. J., Hugger, P., King, W. C., Glomset, J. A., and Habenicht, A. J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 2439-2443[Abstract/Free Full Text]
  46. Clark, J. D., Lin, L. L., Kriz, R. W., Ramesha, C. S., Sultzman, L. A., Lin, A. Y., Milona, N., and Knopf, J. L. (1991) Cell 65, 1043-1051[CrossRef][Medline] [Order article via Infotrieve]
  47. Diez, E., Louis-Flamberg, P., Hall, R. H., and Mayer, R. J. (1992) J. Biol. Chem. 267, 18342-18348[Abstract/Free Full Text]
  48. Mehrabian, M., Allayee, H., Wong, J., Shi, W., Wang, X. P., Shaposhnik, Z., Funk, C. D., Lusis, A. J., and Shih, W. (2002) Circ. Res. 91, 120-126[Abstract/Free Full Text]
  49. Tabas, I. (2002) in Biochemistry of Lipids, Lipoproteins and Membranes (Vance, D. E., and Vance, J., eds) pp. 573-597, Elsevier, Amsterdam
  50. Goodwin, J. S., Bankhurst, A. D., and Messner, R. P. (1977) J. Exp. Med. 146, 1719-1734[Abstract/Free Full Text]
  51. Takayama, K., Garcia-Cardena, G., Sukhova, G. K., Comander, J., Gimbrone, M. A., Jr., and Libby, P. (2002) J. Biol. Chem. 277, 44147-44154[Abstract/Free Full Text]
  52. Fabre, J. E., Nguyen, M., Athirakul, K., Coggins, K., McNeish, J. D., Austin, S., Parise, L. K., FitzGerald, G. A., Coffman, T. M., and Koller, B. H. (2001) J. Clin. Investig. 107, 603-610[Medline] [Order article via Infotrieve]
  53. Libby, P., Warner, S. J., and Friedman, G. B. (1988) J. Clin. Investig. 81, 487-498[Medline] [Order article via Infotrieve]
  54. Huang, J. T., Welch, J. S., Ricote, M., Binder, C. J., Willson, T. M., Kelly, C., Witztum, J. L., Funk, C. D., Conrad, D., and Glass, C. K. (1999) Nature 400, 378-382[CrossRef][Medline] [Order article via Infotrieve]
  55. Duval, C., Chinetti, G., Trottein, F., Fruchart, J. C., and Staels, B. (2002) Trends Mol. Med. 8, 422-430[CrossRef][Medline] [Order article via Infotrieve]
  56. Warner, G. J., Stoudt, G., Bamberger, M., Johnson, W. J., and Rothblat, G. H. (1995) J. Biol. Chem. 270, 5772-5778[Abstract/Free Full Text]
  57. Yao, P. M., and Tabas, I. (2000) J. Biol. Chem. 275, 23807-23813[Abstract/Free Full Text]
  58. Kellner-Weibel, G., Jerome, W. G., Small, D. M., Warner, G. J., Stoltenborg, J. K., Kearney, M. A., Corjay, M. H., Phillips, M. C., and Rothblat, G. H. (1998) Arterioscler. Thromb. Vasc. Biol. 18, 423-431[Abstract/Free Full Text]
  59. Teruya, J., Cluette-Brown, J., Szczepiorkowski, Z. M., and Laposata, M. (1995) J. Lipid Res. 36, 266-276[Abstract]

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