Different cellular traffic of LDL-cholesterol and acetylated LDL-cholesterol leads to distinct reverse cholesterol transport pathways
2006; Elsevier BV; Volume: 48; Issue: 3 Linguagem: Inglês
10.1194/jlr.m600470-jlr200
ISSN1539-7262
AutoresMingdong Wang, Robert S. Kiss, Vivian Franklin, Heidi M. McBride, Stewart C. Whitman, Yves L. Marcel,
Tópico(s)Lipoproteins and Cardiovascular Health
ResumoEndocytosis of LDL and modified LDL represents regulated and unregulated cholesterol delivery to macrophages. To elucidate the mechanisms of cellular cholesterol transport and egress under both conditions, various primary macrophages were labeled and loaded with cholesterol or cholesteryl ester from LDL or acetylated low density lipoprotein (AcLDL), and the cellular cholesterol traffic pathways were examined. Confocal microscopy using fluorescently labeled 3,3′-dioctyldecyloxacarbocyanine perchlorate-labeled LDL and 1,1′-dioctyldecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate-labeled AcLDL demonstrated their discrete traffic pathways and accumulation in distinct endosomes. ABCA1-mediated cholesterol efflux to apolipoprotein A-I (apoA-I) was much greater for AcLDL-loaded macrophages compared with LDL. Treatment with the liver X receptor ligand 22-OH increased efflux to apoA-I in AcLDL-loaded but not LDL-loaded cells. In contrast, at a level equivalent to AcLDL, LDL-derived cholesterol was preferentially effluxed to HDL, in keeping with increased ABCG1. In vivo studies of reverse cholesterol transport (RCT) from cholesterol-labeled macrophages injected intraperitoneally demonstrated that LDL-derived cholesterol was more efficiently transported to the liver and secreted into bile than AcLDL-derived cholesterol. This indicates a greater efficiency of HDL than lipid-poor apoA-I in interstitial fluid in controlling in vivo RCT. These assays, taken together, emphasize the importance of mediators of diffusional cholesterol efflux in RCT. Endocytosis of LDL and modified LDL represents regulated and unregulated cholesterol delivery to macrophages. To elucidate the mechanisms of cellular cholesterol transport and egress under both conditions, various primary macrophages were labeled and loaded with cholesterol or cholesteryl ester from LDL or acetylated low density lipoprotein (AcLDL), and the cellular cholesterol traffic pathways were examined. Confocal microscopy using fluorescently labeled 3,3′-dioctyldecyloxacarbocyanine perchlorate-labeled LDL and 1,1′-dioctyldecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate-labeled AcLDL demonstrated their discrete traffic pathways and accumulation in distinct endosomes. ABCA1-mediated cholesterol efflux to apolipoprotein A-I (apoA-I) was much greater for AcLDL-loaded macrophages compared with LDL. Treatment with the liver X receptor ligand 22-OH increased efflux to apoA-I in AcLDL-loaded but not LDL-loaded cells. In contrast, at a level equivalent to AcLDL, LDL-derived cholesterol was preferentially effluxed to HDL, in keeping with increased ABCG1. In vivo studies of reverse cholesterol transport (RCT) from cholesterol-labeled macrophages injected intraperitoneally demonstrated that LDL-derived cholesterol was more efficiently transported to the liver and secreted into bile than AcLDL-derived cholesterol. This indicates a greater efficiency of HDL than lipid-poor apoA-I in interstitial fluid in controlling in vivo RCT. These assays, taken together, emphasize the importance of mediators of diffusional cholesterol efflux in RCT. The development of atherosclerosis is initiated by the formation of macrophage-derived foam cells (1Brown M.S. Goldstein J.L. Lipoprotein metabolism in the macrophage: implications for cholesterol deposition in atherosclerosis. Annu. Rev. Biochem. 1983; 52: 223-261Google Scholar). As dedicated scavenger and sentinel cells, macrophages actively take up and process apoptotic and necrotic cells (2Wong H. Hashimoto S. 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Acta. 2000; 1529: 164-174Crossref PubMed Scopus (119) Google Scholar). The cell can also export excess cholesterol to appropriate extracellular acceptors by transfer mechanisms through cholesterol gradients that involve mostly HDL, mediated via scavenger receptor class B type I (SR-BI) (4Ji Y. Jian B. Wang N. Sun Y. Moya M.D. Phillips M.C. Rothblat G.H. Swaney J.B. Tall A.R. Scavenger receptor BI promotes high density lipoprotein-mediated cellular cholesterol efflux. J. Biol. Chem. 1997; 272: 20982-20985Google Scholar) and the ABCG1/G4 transporter (5Wang N. Lan D. Chen W. Matsuura F. Tall A.R. ATP-binding cassette transporters G1 and G4 mediate cellular cholesterol efflux to high-density lipoproteins. Proc. Natl. Acad. Sci. USA. 2004; 101: 9774-9779Google Scholar, 6Vaughan A.M. Oram J.F. ABCG1 redistributes cell cholesterol to domains removable by high density lipoprotein but not by lipid-depleted apolipoproteins. J. Biol. 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Acetylated low-density lipoprotein is endocytosed through coated pits by rat peritoneal macrophages. Virchows Arch. B Cell Pathol. Incl. Mol. Pathol. 1986; 52: 1-13Crossref PubMed Scopus (27) Google Scholar). Whereas LDL is delivered to centrally located vesicles, βVLDL or AcLDL is observed in peripherally distributed vesicles, where its catabolism is slower (18Tabas I. Lim S Xu X.X. Maxfield F.R. Endocytosed beta-VLDL and LDL are delivered to different intracellular vesicles in mouse peritoneal macrophages. J. Cell Biol. 1990; 111: 929-940Google Scholar, 19Zha X.H. Tabas I. Leopold P.L. Jones N.L. Maxfield F.R. Evidence for prolonged cell-surface contact of acetyl-LDL before entry into macrophages. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 1421-1431Crossref PubMed Scopus (19) Google Scholar). 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Here, we demonstrate that LDL and AcLDL traffic to discrete endosomal compartments and that cholesterol derived from each of these lipoproteins enters different cellular pools and is effluxed in vitro through largely distinct pathways: LDL-derived cholesterol effluxes preferentially via the HDL-mediated pathway dependent in part on ABCG1, whereas AcLDL-derived cholesterol effluxes preferentially via the more specific lipid-poor apoA-I/ABCA1-dependent pathway. Interestingly, in vivo reverse cholesterol transport (RCT) to the liver and bile is shown to be more significant for the LDL-derived cholesterol, reflecting the importance of the available mediators of diffusional cholesterol efflux in RCT. ABCA1−/− mice were a kind gift from Dr. Edward M. Rubin (Department of Energy Joint Genome Institute, Berkeley, CA). SRA−/− mice were transferred from Dr. T. Kodama (University of Tokyo). C57Bl6, NPC1−/−, SR-BI−/−, LDLr−/−, and caveolin-1−/− mice were purchased from Jackson Laboratories and maintained and bred at the animal facility of the Ottawa Heart Institute. All protocols were approved by the University of Ottawa Animal Care Committee. The ACAT inhibitor Sandoz 58-035 was a gift from Novartis. Simvastatin was kindly given by Merck. Cholesterol-[1,2-3H], mevalono-lactone-Rs-[5-3H(N)], choline chloride-[methyl-3H], and cholesteryl oleate were obtained from Perkin-Elmer Life and Analytical Sciences. 22R-Hydroxy cholesterol and progesterone were purchased from Gibco. Methyl-β-cyclodextrin (mβ-CD) was obtained from Cerestar (Cargill, Inc., Minneapolis, MI). Recombinant human apoA-I was produced in our laboratory (27Bergeron J. Frank P.G. Emmanuel F. Latta M. Zhao Y.W. Sparks D.L. Rassart E. Denèfle P. Marcel Y.L. Characterization of human apolipoprotein A-I expressed in Escherichia coli. Biochim. Biophys. 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Uptake by J774 macrophages of very-low-density lipoproteins isolated from apoE-deficient mice is mediated by a distinct receptor and stimulated by lipoprotein lipase. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 498-504Google Scholar). Fetal liver-derived macrophages were harvested from pregnant mice before delivery (31Boltz-Nitulescu G. Wiltschke C. Holzinger C. Fellinger A. Scheiner O. Gessl A. Forster O. Differentiation of rat bone marrow cells into macrophages under the influence of mouse L929 cell supernatant. J. Leukoc. Biol. 1987; 41: 83-91Google Scholar). Bone marrow-derived macrophages (BMDMs) were flushed from mouse femurs. Macrophages were generated by incubating fetal liver cells (2 × 106 cells/ml) or bone marrow cells (106 cells/ml) with DMEM and 10% FBS complemented with 15% L929-conditioned medium for 7 days. Unless indicated in the legends, labeling conditions were as follows. Macrophages were washed three times with plain DMEM and then labeled with LDL or AcLDL (50 μg protein/ml) that had been preincubated with 5 μCi of [3H]cholesterol in 1% FBS of DMEM for 24 h. The cells were equilibrated with 2 mg/ml BSA overnight. For labeling with [3H]mevalonate (10 μCi/ml), [3H]acetate (20 μCi/ml), [3H]cholesteryl oleate (5 μCi/ml), or [3H]choline (5 μCi/ml), the cells were incubated for 40 h in DMEM with 1% FBS. Incorporation of [3H]cholesteryl oleate into LDL and AcLDL was carried out as described previously (32Vassiliou G. Benoist F. Lau P. Kavaslar G.N. McPherson R. The low density lipoprotein receptor-related protein contributes to selective uptake of high density lipoprotein cholesteryl esters by SW872 liposarcoma cells and primary human adipocytes. J. Biol. Chem. 2001; 276: 48823-48830Google Scholar). Efflux to apoA-I (50 μg in 2 mg/ml BSA medium) was monitored for 3–5 h. Efflux to BSA (2 mg/ml BSA of DMEM) was allowed to proceed for 16 h. Efflux to mβ-CD (10 mM in 2 mg/ml BSA of DMEM) was carried out for 15 min at 37°C or 4°C. Cellular lipids were extracted (33Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification. Can. J. Biochem. 1959; 37: 911-917Google Scholar) and separated by TLC using hexane-diethylether-acetic acid (105:45:1.5) as running solvent on Sil-G TLC plates (EMD Chemicals, Darmstadt, Germany). Lipid bands were detected by exposure to iodine vapors and scraped off the TLC plate, and radioactivity was measured with a scintillation counter. For total cholesterol determination, cells were washed with cold PBS, and cholesterol was extracted by isopropanol and measured by colorimetric assay (Wako Chemicals, Richmond, VA). Cellular proteins were solubilized in RIPA buffer [20 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, 1% deoxycholic acid, 1% Triton X-100, and cocktail protease inhibitors (Roche)], electrophoresed on a 6% SDS-polyacrylamide gel, and transferred to nitrocellulose at 125 V for 4 h. ABCA1 and ABCG1 antibodies (Novus Biologicals; 1:500 dilution) were used for detection, with an anti-rabbit secondary antibody conjugated with horseradish peroxidase (Amersham Biosciences) for detection. Macrophages from ABCA1−/−, ABCA1−/+, or ABCA1+/+ mice were labeled with cholesterol delivered by LDL or AcLDL for 24 h. Cells are removed with 5 mM EDTA PBS and injected intraperitoneally into C57BL6 mice (34Zhang Y. Zanotti I. Reilly M.P. Glick J.M. Rothblat G.H. Rader D.J. Overexpression of apolipoprotein A-I promotes reverse transport of cholesterol from macrophages to feces in vivo. Circulation. 2003; 108: 661-663Google Scholar). Gallbladders, livers, and feces were harvested 24 h later. Tissues and feces were treated with 0.5 N NaOH, and lipid radioactivity was counted. Lipoproteins were labeled with 3,3′-dioctyldecyloxacarbocyanine perchlorate (DiO) and 1,1′-dioctyldecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate (DiD) (Molecular Probes) as reported previously (18Tabas I. Lim S Xu X.X. Maxfield F.R. Endocytosed beta-VLDL and LDL are delivered to different intracellular vesicles in mouse peritoneal macrophages. J. Cell Biol. 1990; 111: 929-940Google Scholar, 32Vassiliou G. Benoist F. Lau P. Kavaslar G.N. McPherson R. The low density lipoprotein receptor-related protein contributes to selective uptake of high density lipoprotein cholesteryl esters by SW872 liposarcoma cells and primary human adipocytes. J. Biol. Chem. 2001; 276: 48823-48830Google Scholar). Dansyl-cholestanol was synthesized according to the method of Wiegand et al. (35Wiegand V. Chang T.Y. Strauss III, J.F. Fahrenholz F. Gimpl G. Transport of plasma membrane-derived cholesterol and the function of Niemann-Pick C1 protein. FASEB J. 2003; 17: 782-784Google Scholar). Macrophages were loaded with LDL or AcLDL that was preequilibrated with dansyl-cholestanol. An Olympus FV1000 confocal microscope, complete with a 100× objective (numerical aperture 1.4), a 488 argon-ion laser, and a 633 helium/neon laser, was used for DiO and DiD fluorescence microscopy. For dansyl-cholestanol imaging, an Olympus IX70 inverted fluorescence microscope outfitted with a monochrometer from Till Photonics was used. Images were captured using the Imago charge-coupled device camera and processed using TILLvisION software. Student's t-test was applied to evaluate significant differences. BMDMs were labeled and loaded with [3H]cholesterol delivered by either AcLDL or LDL. AcLDL-loaded macrophages effluxed cholesterol to apoA-I at a rate nearly 7-fold higher than that of the macrophages loaded with LDL (Fig. 1A ). Furthermore, apoA-I-mediated cholesterol efflux from the cells loaded with AcLDL was totally dependent on ABCA1 expression, whereas cells loaded with LDL displayed ∼25% residual apoA-I-specific cholesterol efflux in ABCA1−/− macrophages (Fig. 1A). In contrast, macrophages loaded with LDL effluxed cholesterol to HDL and BSA at a significantly greater rate than those loaded with AcLDL (Fig. 1B, C). This difference was not related to the preferential plasma membrane cholesterol labeling by LDL cholesterol, because mβ-CD extraction at 4°C, which measures the proportion of cholesterol present at the cell surface, showed equivalent plasma membrane labeling by LDL (9.9%) and AcLDL (10.2%) (Fig. 1D). However, short-term cholesterol efflux to mβ-CD at 37°C, which measures cholesterol present at the plasma membrane and the recycling compartment (36Zheng H. Kiss R.S. Franklin V. Wang M.D. Haidar B. Marcel Y.L. ApoA-I lipidation in primary mouse hepatocytes: separate controls for phospholipid and cholesterol transfers. J. Biol. Chem. 2005; 280: 21612-21621Google Scholar), was significantly greater from cells loaded with LDL (44%) compared with AcLDL (22%), regardless of ABCA1 expression level (Fig. 1D). Therefore cholesterol derived from LDL preferentially loaded the recycling compartment, a conclusion supported by the greater efflux to HDL and to mβ-CD at 37°C. This difference was not related to the use of specific differentiated murine macrophages, because the same efflux specificity existed in all tested murine macrophages (Fig. 1E). The same specificity was also observed for efflux to immunoprecipitated apoA-I (Fig. 1F). To further document the differential trafficking of LDL- and AcLDL-derived cholesterol and its independence of the labeling efficiency of cell surface compartments, macrophages were labeled to an equivalent level with [3H]cholesteryl oleate and preincorporated into LDL or AcLDL to the same specific activity (Fig. 2A ). Under these conditions, cholesterol efflux to apoA-I from macrophages loaded with AcLDL remained significantly greater (P < 0.004) than that from cells loaded with LDL by ∼3-fold (Fig. 2B). When cells were equally loaded with cholesteryl ester-labeled lipoproteins, the proportion of accumulated ACAT-generated cholesteryl esters was ∼10-fold greater with AcLDL compared with LDL (Fig. 2C). Similar to [3H]cholesterol labeling of lipoproteins, cholesterol derived from [3H]cholesteryl oleate was equally accessible to mβ-CD extraction at 4°C whether delivered by LDL or AcLDL (Fig. 2D). However, mβ-CD extraction at 37°C was much higher for LDL-loaded macrophages, similar to the previous results (Fig. 1D) and confirming a preferential labeling by LDL of the recycling compartment. It is well known that AcLDL is a potent ACAT activator (37Linton M.F. Fazio S. Macrophages, inflammation, and atherosclerosis. Int. J. Obes. Relat. Metab. Disord. 2003; 27 (Suppl. 3): 35-40Crossref PubMed Scopus (233) Google Scholar, 38Moore K.J. Freeman M.W. Scavenger receptors in atherosclerosis: beyond lipid uptake. Arterioscler. Thromb. Vasc. Biol. 2006; 26: 1702-1711Crossref PubMed Scopus (432) Google Scholar), and our own data showed that treatment with the same amount of lipoprotein (either LDL or AcLDL) in macrophages causes significantly more cholesterol esterification by labeling with [3H]oleate in AcLDL-treated cells (data not shown). To understand whether the increased apoA-I-mediated efflux was attributable simply to increased cholesteryl ester formation, because cholesterol efflux to apoA-I is closely related to the level of cellular cholesteryl ester (see supplementary Fig. II) and in keeping with previous studies (39Rigamonti E. Helin L. Lestavel S. Mutka A.L. Lepore M. Fontaine C. Bouhlel M.A. Bultel S. Fruchart J.C. Ikonen E. Liver X receptor activation controls intracellular cholesterol trafficking and esterification in human macrophages. Circ. Res. 2005; 97 (et al.): 682-689Google Scholar), macrophages were loaded for 5 or 24 h with AcLDL or LDL to achieve similar levels of radioactive labeling (Fig. 2E) and cholesteryl ester formation (Fig. 2G). Under these conditions, a clear difference in the amount of apoA-I-mediated cholesterol efflux was evident (4-fold increase for AcLDL-loaded cells) (Fig. 2F). Increased efflux to apoA-I might reflect preferential cholesterol mobilization from the ACAT-accessible pool; however, ACAT inhibition during the labeling period increased apoA-I-mediated cholesterol efflux from the macrophages labeled with AcLDL but not LDL (Fig. 2H). Increased efflux to apoA-I might reflect preferential cholesterol mobilization from the ACAT-accessible pool (cholesteryl ester droplets). However, administration of the ACAT inhibitor Sandoz 58-035 during the labeling period completely suppressed the cholesterol reesterification cycle (data not shown) and increased apoA-I-mediated cholesterol efflux from the macrophages labeled with AcLDL but not LDL (Fig. 2H). These results demonstrate that the observed differences of LDL- and AcLDL-loaded macrophages are not simply attributable to increased loading or preferential targeting to an ACAT-accessible pool; rather, they further demonstrate that lipoprotein-derived cholesterol, which internalizes through distinct mechanisms (see below), remains in two functionally distinct pools. Next, we showed that the specificity of the pathways is independent of the net uptake of lipoprotein cholesterol. Increasing LDL concentration resulted in a linear and nonsaturating increase in uptake, which at 250 μg/ml doubled total cellular cholesterol and increased cholesteryl ester level to ∼5% (Fig. 3A , B), indicating that a non-receptor-independent mechanism contributed to macrophage LDL uptake, as reported by others (13Kruth H.S. Huang W Ishii I. Zhang W.Y. Macrophage foam cell formation with native low density lipoprotein. J. Biol. Chem. 2002; 277: 34573-34580Google Scholar, 14Kruth H.S. Jones N.L. Huang W. Zhao B. Ishii I. Chang J. Combs C.A. Malide D. Zhang W.Y. Macropinocytosis is the endocytic pathway that mediates macrophage foam cell formation with native low density lipoprotein. J. Biol. Chem. 2005; 280: 2352-2360Google Scholar). In contrast, increasing AcLDL concentration up to 50 μg/ml rapidly increased cellular cholesterol, which plateaued at a 3-fold increase, and was accompanied by an increase in cholesteryl ester to ∼40% (Fig. 3C, D) as well as a greater upregulation of ABCA1 (Fig. 3E). Under conditions that achieve equivalent cholesterol loading with LDL and AcLDL, we measured the total cellular cholesterol level before the addition of apoA-I and then carried out an efflux assay (Fig. 3G, H). Thus, AcLDL at 6.25 μg/ml and LDL at 150 μg/ml loaded the cells to the same level, 30.2 and 29 μg of total cholesterol mass, respectively (SD < 15%), but AcLDL elicited twice as much ABCA1-mediated efflux. The same difference in efflux was maintained with 12.5 μg/ml AcLDL and 250 μg/ml LDL, which increased cellular cholesterol to 38.7 and 35.6 μg/mg cellular protein, respectively. Because ABCG1 mediates efflux to HDL (5Wang N. Lan D. Chen W. Matsuura F. Tall A.R. ATP-binding cassette transporters G1 and G4 mediate cellular cholesterol efflux to high-density lipoproteins. Proc. Natl. Acad. Sci. USA. 2004; 101: 9774-9779Google Scholar, 40Kennedy M.A. Barrera G.C. Nakamura K. Baldan A. Tarr P. Fishbein M.C. Frank J. Francone O.L. Edwards P.A. ABCG1 has a critical role in mediating cholesterol efflux to HDL and preventing cellular lipid accumulation. Cell Metab. 2005; 1: 121-131Google Scholar) and LDL-derived cholesterol is effluxed to HDL at a higher rate than AcLDL-derived cholesterol (Fig. 1B), we also measured ABCG1 protein level in LDL- and AcLDL-treated cells (Fig. 3F). Clearly, ABCG1 protein expression was increased in LDL-treated cells, but not as much as in AcLDL-treated cells, suggesting that the robust cholesterol efflux to HDL in LDL-treated cells was not related directly only to ABCG1 expression but also to a specific targeting of cholesterol, and possibly of the transporter, to the recycling compartment. Alternatively, this discrepancy may reflect the contribution of another, yet uncharacterized, mediator of the diffusional pathway (41Duong M. Collins H.L. Jin W. Zanotti I. Favari E. Rothblat G.H. Relative contributions of ABCA1 and SR-BI to cholesterol efflux to serum from fibroblasts and macrophages. Arterioscler. Thromb. Vasc. Biol. 2006; 26: 541-547Google Scholar). To determine the specificity of LDL cholesterol internalization via the LDLr and AcLDL via SRA in macrophages, we first labeled BMDMs from LDLr−/− and wild-type mice with [3H]cholesteryl oleate-labeled LDL. Both BMDMs were pretreated with 3% lipoprotein deficient serum to upregulate LDLr by depleting cellular cholesterol. Under this condition, ∼70% of LDL cholesterol internalization could be inhibited by the addition of cold LDL, indicating its dependence on LDLr (Fig. 4A ), whereas the fraction of LDL cholesterol uptake, which could not be inhibited by cold LDL, may be mediated by macropinocytosis (13Kruth H.S. Huang W Ishii I. Zhang W.Y. Macrophage foam cell formation with native low density lipoprotein. J. Biol. Chem. 2002; 277: 34573-34580Google Scholar, 14Kruth H.S. Jones N.L. Huang W. Zhao B. Ishii I. Chang J. Combs C.A. Malide D. Zhang W.Y. Macropinocytosis is the endocytic pathway that mediates macrophage foam cell formation with native low density lipoprotein. J. Biol. Chem. 2005; 280: 2352-2360Google Scholar, 15Zhao B. Li Y. Buono C. Waldo S.W. Jones N.L. Mori M. Kruth H.S. Constitutive receptor-independent low density lipoprotein uptake and cholesterol accumulation by macrophages differentiated from human monocytes with macrophage-colony-stimulating factor (M-CSF). J. Biol. Chem. 2006; 281: 15757-15762Google Scholar). Under in vivo physiological conditions, there is no cholesterol deprivation. Therefore, we labeled LDLr−/− macrophages with [3H]cholesteryl oleate-labeled LDL or [3H]cholesterol-labeled LDL without pretreatment with LPDS. Under these conditions, most LDL cholesterol internalization (∼75%) was
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