ABCA1 Overexpression in the Liver of LDLr-KO Mice Leads to Accumulation of Pro-atherogenic Lipoproteins and Enhanced Atherosclerosis
2006; Elsevier BV; Volume: 281; Issue: 44 Linguagem: Inglês
10.1074/jbc.m604526200
ISSN1083-351X
AutoresCharles Joyce, Elke Wagner, Federica Basso, Marcelo Amar, Lita A. Freeman, Robert D. Shamburek, Catherine L. Knapper, Jafri Syed, Justina E. Wu, Boris Vaisman, Jamila Fruchart‐Najib, Eric M. Billings, Beverly Paigen, Alan T. Remaley, Silvia Santamarina-Fojo, H Bryan Brewer,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoThe identification of ABCA1 as a key transporter responsible for cellular lipid efflux has led to considerable interest in defining its role in cholesterol metabolism and atherosclerosis. In this study, the effect of overexpressing ABCA1 in the liver of LDLr-KO mice was investigated. Compared with LDLr-KO mice, ABCA1-Tg × LDLr-KO (ABCA1-Tg) mice had significantly increased plasma cholesterol levels, mostly because of a 2.8-fold increase in cholesterol associated with a large pool of apoB-lipoproteins. ApoB synthesis was unchanged but the catabolism of 125I-apoB-VLDL and -LDL were significantly delayed, accounting for the 1.35-fold increase in plasma apoB levels in ABCA1-Tg mice. We also found rapid in vivo transfer of free cholesterol from HDL to apoB-lipoproteins in ABCA1-Tg mice, associated with a significant 2.7-fold increase in the LCAT-derived cholesteryl linoleate content found primarily in apoB-lipoproteins. ABCA1-Tg mice had 1.4-fold increased hepatic cholesterol concentrations, leading to a compensatory 71% decrease in de novo hepatic cholesterol synthesis, as well as enhanced biliary cholesterol, and bile acid secretion. CAV-1, CYP2b10, and ABCG1 were significantly induced in ABCA1-overexpressing livers; however, no differences were observed in the hepatic expression of CYP7α1, CYP27α1, or ABCG5/G8 between ABCA1-Tg and control mice. As expected from the pro-atherogenic plasma lipid profile, aortic atherosclerosis was increased 10-fold in ABCA1-Tg mice. In summary, hepatic overexpression of ABCA1 in LDLr-KO mice leads to: 1) expansion of the pro-atherogenic apoB-lipoprotein cholesterol pool size via enhanced transfer of HDL-cholesterol to apoB-lipoproteins and delayed catabolism of cholesterol-enriched apoB-lipoproteins; 2) increased cholesterol concentration in the liver, resulting in up-regulated hepatobiliary sterol secretion; and 3) significantly enhanced aortic atherosclerotic lesions. The identification of ABCA1 as a key transporter responsible for cellular lipid efflux has led to considerable interest in defining its role in cholesterol metabolism and atherosclerosis. In this study, the effect of overexpressing ABCA1 in the liver of LDLr-KO mice was investigated. Compared with LDLr-KO mice, ABCA1-Tg × LDLr-KO (ABCA1-Tg) mice had significantly increased plasma cholesterol levels, mostly because of a 2.8-fold increase in cholesterol associated with a large pool of apoB-lipoproteins. ApoB synthesis was unchanged but the catabolism of 125I-apoB-VLDL and -LDL were significantly delayed, accounting for the 1.35-fold increase in plasma apoB levels in ABCA1-Tg mice. We also found rapid in vivo transfer of free cholesterol from HDL to apoB-lipoproteins in ABCA1-Tg mice, associated with a significant 2.7-fold increase in the LCAT-derived cholesteryl linoleate content found primarily in apoB-lipoproteins. ABCA1-Tg mice had 1.4-fold increased hepatic cholesterol concentrations, leading to a compensatory 71% decrease in de novo hepatic cholesterol synthesis, as well as enhanced biliary cholesterol, and bile acid secretion. CAV-1, CYP2b10, and ABCG1 were significantly induced in ABCA1-overexpressing livers; however, no differences were observed in the hepatic expression of CYP7α1, CYP27α1, or ABCG5/G8 between ABCA1-Tg and control mice. As expected from the pro-atherogenic plasma lipid profile, aortic atherosclerosis was increased 10-fold in ABCA1-Tg mice. In summary, hepatic overexpression of ABCA1 in LDLr-KO mice leads to: 1) expansion of the pro-atherogenic apoB-lipoprotein cholesterol pool size via enhanced transfer of HDL-cholesterol to apoB-lipoproteins and delayed catabolism of cholesterol-enriched apoB-lipoproteins; 2) increased cholesterol concentration in the liver, resulting in up-regulated hepatobiliary sterol secretion; and 3) significantly enhanced aortic atherosclerotic lesions. The inverse relationship between plasma high density lipoprotein-cholesterol (HDL-C) 3The abbreviations used are: HDL-C, high density lipoprotein-cholesterol; ABCA1, ATP-binding cassette transporter A1; ABCG1, ATP-binding cassette transporter G1; ACAT, acyl-CoA acyltransferase; Apo, apolipoprotein; ApoB-lps, apoB-containing lipoproteins; BA, bile acids; Cav-1, caveolin-1; CE, cholesterol ester; Cyp, cytochrome P450; FC, free cholesterol; GEO, gene expression omnibus; HMGCoA-Reductase or Hmgcr, 3-hydroxy-3-methylglutaryl-coenzyme A reductase; KO, knock-out; LCAT, lecithin:cholesterol acyltransferase; LDL-C, low density lipoprotein-cholesterol; LDL, low density lipoprotein; LDLr, LDL-receptor; LRP, LDL receptor-related protein; PL, phospholipids; SR-BI, mouse scavenger receptor B-I; RCT, reverse cholesterol transport; TC, total cholesterol; TG, triglycerides; Tg, transgenic; VLDL-C, very low density lipoprotein-cholesterol; DMEM, Dulbecco's modified Eagle's medium. 3The abbreviations used are: HDL-C, high density lipoprotein-cholesterol; ABCA1, ATP-binding cassette transporter A1; ABCG1, ATP-binding cassette transporter G1; ACAT, acyl-CoA acyltransferase; Apo, apolipoprotein; ApoB-lps, apoB-containing lipoproteins; BA, bile acids; Cav-1, caveolin-1; CE, cholesterol ester; Cyp, cytochrome P450; FC, free cholesterol; GEO, gene expression omnibus; HMGCoA-Reductase or Hmgcr, 3-hydroxy-3-methylglutaryl-coenzyme A reductase; KO, knock-out; LCAT, lecithin:cholesterol acyltransferase; LDL-C, low density lipoprotein-cholesterol; LDL, low density lipoprotein; LDLr, LDL-receptor; LRP, LDL receptor-related protein; PL, phospholipids; SR-BI, mouse scavenger receptor B-I; RCT, reverse cholesterol transport; TC, total cholesterol; TG, triglycerides; Tg, transgenic; VLDL-C, very low density lipoprotein-cholesterol; DMEM, Dulbecco's modified Eagle's medium. concentrations and the incidence of coronary artery disease is well established in humans (1Gordon D.J. 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Royer L.J. deWet J. Broccardo C. Chimini G. Francone O.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4245-4250Crossref PubMed Scopus (481) Google Scholar, 24Aiello R.J. Brees D. Bourassa P.A.K. Royer L. Lindsey S. Coskran T. Haghpassand M. Francone O.L. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 630-637Crossref PubMed Scopus (341) Google Scholar), but repopulation of ABCA1-KO mice with wild-type macrophages (24Aiello R.J. Brees D. Bourassa P.A.K. Royer L. Lindsey S. Coskran T. Haghpassand M. Francone O.L. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 630-637Crossref PubMed Scopus (341) Google Scholar) or macrophages from ABCA1-overexpressing mice (25van Eck M. Singaraja R.R. Ye D. Hildebrand R.B. James E.R. Hayden M.R. van Berkel T.J. Arterioscler. Thromb. Vasc. Biol. 2006; 26: 929-934Crossref PubMed Scopus (149) Google Scholar) led to significantly decreased atherosclerosis. In contrast, selective inactivation of macrophage ABCA1 in mice results in markedly increased atherosclerosis (24Aiello R.J. Brees D. Bourassa P.A.K. Royer L. Lindsey S. Coskran T. Haghpassand M. Francone O.L. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 630-637Crossref PubMed Scopus (341) Google Scholar, 26van Eck M. Sophie I. Bos T. Kaminski W.E. Orso E. Rothe G. Twisk J. Bottcher A. Van Amersfoort E.S. Christiansen-Weber T.A. Fung-Leung W.P. Van Berkel T.J.C. Schmitz G. Proc. Natl. Acad. Sci. 2002; 99: 6298-6303Crossref PubMed Scopus (318) Google Scholar). Transgenic mice overexpressing a human ABCA1 cDNA under the control of the apoE promoter in both liver and macrophages developed less atherosclerosis in C57Bl/6 mice when fed a high fat-high cholesterol diet containing cholate (19Joyce C. Amar M.J.A. Lambert G. Vaisman B.L. Paigen B. Najib-Fruchart J. Hoyt Jr., R.F. Neufeld E.D. Remaley A.T. Fredrickson D.S. Brewer H.B.J. Santamarina-Fojo S. Proc. Nat. Acad. Sci. U. S. 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Najib-Fruchart J. Hoyt Jr., R.F. Neufeld E.D. Remaley A.T. Fredrickson D.S. Brewer H.B.J. Santamarina-Fojo S. Proc. Nat. Acad. Sci. U. S. A. 2002; 99: 407-412Crossref PubMed Scopus (238) Google Scholar).Although the underlying mechanisms for these contrary results remain unclear, the different promoters (28Joyce C. Freeman L. Brewer Jr., H.B. Santamarina-Fojo S. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 965-971Crossref PubMed Scopus (100) Google Scholar) regulating the tissue expression levels of the human ABCA1 transgenes may provide a partial explanation for the opposing results in atherosclerosis. However, an increase in the pro-atherogenic plasma apoB-containing lipoproteins (apoB-Lps) was observed in these as well as other studies, including C57Bl/6 as well as apoE-KO mice transgenic for ABCA1 (18Vaisman B.L. Lambert G. Amar M. Joyce C. Ito T. Shamburek R.D. Cain W.J. Fruchart-Najib J. Neufeld E.B. Remaley A.T. Brewer H.B.J. Santamarina-Fojo S. J. Clin. Investig. 2001; 108: 303-309Crossref PubMed Scopus (220) Google Scholar, 19Joyce C. Amar M.J.A. Lambert G. Vaisman B.L. Paigen B. Najib-Fruchart J. Hoyt Jr., R.F. Neufeld E.D. Remaley A.T. Fredrickson D.S. Brewer H.B.J. Santamarina-Fojo S. Proc. Nat. Acad. Sci. U. S. A. 2002; 99: 407-412Crossref PubMed Scopus (238) Google Scholar, 20Singaraja R.R. Bocher V. James E.R. Clee S.M. Zhang L.H. Leavitt B.R. Tan B. Brooks-Wilson A. Kwok A. Bissada N. Yang Y.Z. Liu G. Tafuri S.R. Fievet C. Wellington C.L. Staels B. Hayden M.R. J. Biol. Chem. 2001; 276: 33969-33979Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar), and C57Bl/6 mice with low level (29Basso F.P. Freeman L. Knapper C.L. Remaley A. Stonik J. Neufeld E.B. Tansey T. Amar M.J.A. Fruchart-Najib J. Duverger N. Santamarina-Fojo S. Brewer Jr., H.B. J. Lipid Res. 2003; 44: 296-302Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar) or high level (30Wellington C.L. Brunham L.R. Zhou S. Singaraja R.R. 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Gelfer A. Ross C. James E. Liu G. Huber M.T. Yang Y.Z. Parks R.J. Groen A. Fruchart-Najib J. Hayden M.R. J. Lipid Res. 2003; 44: 1470-1480Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) or down-regulation (31Ragozin S. Niemeier A. Laatsch A. Loeffler B. Merkel M. Beisiegel U. Heeren J. Arterioscler. Thromb. Vasc. Biol. 2005; 25: 1433-1438Crossref PubMed Scopus (54) Google Scholar), was the demonstration that hepatic ABCA1 is primarily responsible for regulating the level of plasma HDL. Recent findings in mice with selective inactivation of ABCA1 in either liver (32Timmins J.M. Lee J.Y. Boudyguina E. Kluckman K.D. Brunham L.R. Mulya A. Gebre A.K. Coutinho J.M. Colvin P.L. Smith T.L. Hayden M.R. Maeda N. Parks J.S. J. Clin. Investig. 2005; 115: 1333-1342Crossref PubMed Scopus (418) Google Scholar) or intestine (33Brunham L.R. Kruit J.K. Iqbal J. Fievet C. Timmins J.M. Pape T.D. Coburn B.A. Bissada N. Staels B. Groen A.K. Hussain M.M. Parks J.S. Kuipers F. Hayden M.R. J. Clin. Investig. 2006; 116: 1052-1062Crossref PubMed Scopus (413) Google Scholar) have confirmed the role of hepatic ABCA1 as the primary source of plasma HDL-cholesterol as well as a critical factor for the HDL plasma residence time, accounting for ∼70–80% of plasma HDL-cholesterol in mice on a chow diet, with the remaining fraction of HDL coming largely from the intestine (33Brunham L.R. Kruit J.K. Iqbal J. Fievet C. Timmins J.M. Pape T.D. Coburn B.A. Bissada N. Staels B. Groen A.K. Hussain M.M. Parks J.S. Kuipers F. Hayden M.R. J. Clin. Investig. 2006; 116: 1052-1062Crossref PubMed Scopus (413) Google Scholar).Taken together, these studies revealed that macrophage ABCA1 expression does not contribute to plasma HDL levels but does control the development of atherosclerosis in apoE-KO and LDLr-KO mice, while the role of hepatic ABCA1 expression in the development of atherosclerosis and the regulation of non-HDL lipoprotein formation has not yet been fully established.In the present study, LDLr-KO mice overexpressing human ABCA1 in the liver were used to investigate the effect of hepatic ABCA1 on hepatic cholesterol balance, apoB-Lp metabolism and the development of atherosclerosis. We report that hepatic overexpression of ABCA1 in LDLr-KO mice consuming a chow diet leads to significantly increased hepatic cholesterol levels, resulting in the induction of compensatory sterol secretion and reduced cholesterol synthesis. The plasma lipoprotein profile in ABCA1-transgenic LDLr-KO mice was found to be highly pro-atherogenic with accumulation of cholesterol-enriched apoB-Lps because of delayed apoB-Lp catabolism and rapid transfer of free cholesterol from HDL to apoB-Lps. In accordance with the plasma lipoprotein profile changes, aortic atherosclerosis was significantly increased in ABCA1-Tg mice compared with LDLr-KO mice. These results establish that hepatic up-regulation of ABCA1 in LDLr-KO mice, a mouse model with genetic abnormality in apoB-clearance, is pro-atherogenic and further support the concept that genetic background as well as tissue-specific regulation of ABCA1 are critical factors that modulate the atheroprotective properties of ABCA1.EXPERIMENTAL PROCEDURESAnimals—Human ABCA1-Tg mice were produced with a construct containing the human ABCA1 cDNA driven by the murine apoE promoter, as previously described (18Vaisman B.L. Lambert G. Amar M. Joyce C. Ito T. Shamburek R.D. Cain W.J. Fruchart-Najib J. Neufeld E.B. Remaley A.T. Brewer H.B.J. Santamarina-Fojo S. J. Clin. Investig. 2001; 108: 303-309Crossref PubMed Scopus (220) Google Scholar). These mice were crossed to homozygosity with C57Bl/6 LDLr-KO mice. Expression of human ABCA1 was determined by dot blot hybridization, as previously described (19Joyce C. Amar M.J.A. Lambert G. Vaisman B.L. Paigen B. Najib-Fruchart J. Hoyt Jr., R.F. Neufeld E.D. Remaley A.T. Fredrickson D.S. Brewer H.B.J. Santamarina-Fojo S. Proc. Nat. Acad. Sci. U. S. A. 2002; 99: 407-412Crossref PubMed Scopus (238) Google Scholar). LDLr genotype status was determined by PCR screening, using mouse LDLr-specific PCR primers that generate a 383-bp PCR amplicon (5′-accccaagacgtgctcccaggatga-3′ and 5′-cgcagtgctcctcatctgacttgt-3′), and neo-specific PCR primers that generate a 200-bp amplicon (5′-aggatctcgtcgtgacccatggcga-3′ and 5′-gagcggcgataccgtaaagcacgagg-3′). LDLr-KO and human ABCA1-Tg mice were maintained on a chow diet containing 0.02% cholesterol and 4% fat, or were placed on a Western Diet (TD88137; Harlan Teklad; Madison, WI), containing 0.2% cholesterol and 21.2% fat for either 4, 9, or 12 weeks prior to sacrifice. The mice were housed under protocols approved by the Animal Care and Use Committee of the National Heart, Lung, and Blood Institute.Real-time PCR—Total RNA from liver, small intestine, kidney, heart, adrenals, testis, lung, brain, and spleen from age-matched male mice was isolated using TRIzol (Invitrogen, Carlsbad, CA) and further purified using RNeasy RNA-binding columns with on-column DNase treatment (Qiagen, Valencia, CA). Total RNA from peritoneal macrophages was extracted from a pool of macrophages (n = 6 male mice per genotype) after allowing the cells to attach for 3 h on Primaria cell culture dishes (BD Biosciences, Franklin Lakes, NJ) in DMEM containing 10% fetal bovine serum, 4.5 g of glucose/liter and penicillin/streptomycin/l-glutamine (Sigma). Cells were washed, and RNA was isolated with RNeasy RNA-binding columns followed by an on-column DNase treatment (Qiagen).Purified RNA (2 μg) was reverse-transcribed (TaqMan Reverse Transcription Core Reagents, ABI, Foster City, CA), and PCR was performed on the SDS7300 (ABI). Each primer and probe set was tested for linearity in the respective tissues (0.1–100 ng of cDNA) in a total volume of 25 μl (TaqMan PCR Mastermix, ABI). Final PCR was done using 3 and 30 ng of cDNA in duplicates for each sample concentration. Predeveloped TaqMan primer and probe sets were purchased from ABI: mAbca1, Mm00442646_m1; hABCA1, Hs00194045_m1 (ex 30–31), Hs01059118_m1 (ex 3–4), Hs00442663_m1 (ex 49–50); mAbcb4, Mm00435630_m1; mAbcb11, Mm00445168_m1; mAbcg1, Mm01351001_m1; mAbcg5, Mm00446249_m1; mAbcg8, Mm00445970_m1; mCyp7α, Mm00484152_m1; mCav1, Mm00483057_m1 and mCyp2b10, Mm00456591_m1. β-Actin (4352341E, ABI) was used as endogenous control.Western Blot Analysis—Protein analyses of m, hABCA1, mABCG1, mCYP27α1, mLRP, mSR-BI, and mCAV-1 were performed by homogenizing ∼100 mg of snap-frozen liver samples in cell lysis buffer containing 25 mm Tris, 150 mm NaCl, 1 mm EDTA, 1% Triton X-100, and 1× protease inhibitor (Roche Applied Science, Indianapolis, IN) as well as 50 μm calpain inhibitor II (Sigma). Macrophage protein was isolated from pools of macrophages of six male mice from each group, after allowing the cells to attach to Primaria cell culture dishes (BD Biosciences) for 3 h in DMEM containing 10% fetal bovine serum, 4.5g glucose/liter, and penicillin/streptomycin/l-glutamine (Sigma). Cells were washed, and protein was isolated as described above.Protein concentrations were determined by BCA assay (Pierce), and 5–20 μg of protein were separated under reducing conditions on a Novex 3–8% Tris-acetate gel or 4–12% Bis-Tris gel (Invitrogen, Carlsbad, CA). After transfer to polyvinylidene difluoride membranes (Invitrogen), blots were incubated with the following antibodies: rabbit anti-mouse LRP (Resarch Diagnostics Inc.), rabbit anti-human/mouse ABCA1 and SR-BI (Novus, Littleton, CO), rabbit anti-human/mouse ABCG1 (E-20; Santa Cruz Biotechnology) and rabbit anti-human/mouse caveolin-1 (Abcam Inc., Cambridge, MA) according to the manufacturer's protocol. Rabbit anti-human/mouse CYP27α1 was provided by Dr. David Russell, University of Texas Southwestern Medical Center, Dallas, TX and was diluted 1:2000 before use (1 h at room temperature). Corresponding horseradish peroxidase-linked secondary antibodies were purchased from Amersham Biosciences and from Santa Cruz Biotechnology for all other primary antibodies. Visualization of chemiluminescence was performed with Western Lightning Reagent (PerkinElmer Life Sciences). Anti-Human/mouse β-actin (BioLegend, San Diego, CA) was used to control for equal loading and transfer. If not otherwise indicated, equal amounts of protein were analyzed. Band intensities were quantified using the Image Quant 5.2 software (Molecular Dynamics, Sunnyvale, CA).Efflux Studies—Mouse peritoneal macrophages were seeded at a density of ∼400,000 cells per well, using a 24-well Primaria cell culture plate (BD Biosciences), and maintained in DMEM medium containing 10% LPDS, 0.3 μm 8-Br-cAMP, and penicillin/streptomycin/l-glutamine (Sigma). Macrophages were loaded with medium containing 10 μg/ml [3H]CE-labeled acetylated-LDL for 24 h. Efflux of [3H]cholesterol was performed as previously described (34Remaley A.T. Schumacher U.K. Stonik J.A. Farsi B.D. Nazih H. Brewer H.B.J. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 1813-1821Crossref PubMed Scopus (191) Google Scholar), using either HDL (50 μg/ml), apoA-I (10 μg/ml), or bovine serum albumin alone (0.2%) as acceptors. Data were normalized to cell protein and expressed as percent of total counts. Preparation of hepatocytes was performed as previously described (29Basso F.P. Freeman L. Knapper C.L. Remaley A. Stonik J. Neufeld E.B. Tansey T. Amar M.J.A. Fruchart-Najib J. Duverger N. Santamarina-Fojo S. Brewer Jr., H.B. J. Lipid Res. 2003; 44: 296-302Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar).Analysis of Plasma Lipids, Lipoproteins, and Apolipoproteins—EDTA-plasma samples were prepared from LDLr-KO, ABCA1-Tg × LDLr-KO, LDLr+/– and ABCA1-Tg × LDLr+/– mice after a 4-h fasting period. Plasma lipoprotein fractions were analyzed by FPLC (100–400 μl of pooled plasma) as described (18Vaisman B.L. Lambert G. Amar M. Joyce C. Ito T. Shamburek R.D. Cain W.J. Fruchart-Najib J. Neufeld E.B. Remaley A.T. Brewer H.B.J. Santamarina-Fojo S. J. Clin. Investig. 2001; 108: 303-309Crossref PubMed Scopus (220) Google Scholar, 19Joyce C. Amar M.J.A. Lambert G. Vaisman B.L. Paigen B. Najib-Fruchart J. Hoyt Jr., R.F. Neufeld E.D. Remaley A.T. Fredrickson D.S. Brewer H.B.J. Santamarina-Fojo S. Proc. Nat. Acad. Sci. U. S. 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Chem. 1997; 272: 17972-17980Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar), using polyclonal antibodies raised against purified mouse apoB. In addition, mouse apolipoproteins A-I, A-II, B, and E were also identified by immunostaining plasma lipoprot
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