Cellular SR-BI and ABCA1-mediated cholesterol efflux are gender-specific in healthy subjects
2007; Elsevier BV; Volume: 49; Issue: 3 Linguagem: Inglês
10.1194/jlr.m700510-jlr200
ISSN1539-7262
AutoresGiovanna Catalano, Emilie Duchêne, Zélie Julia, Wilfried Le Goff, Éric Bruckert, M. John Chapman, Maryse Guérin,
Tópico(s)Diabetes, Cardiovascular Risks, and Lipoproteins
ResumoWe evaluated the impact of gender differences in both the quantitative and qualitative features of HDL subspecies on cellular free cholesterol efflux through the scavenger receptor class B type I (SR-BI), ABCA1, and ABCG1 pathways. For that purpose, healthy subjects (30 men and 26 women) matched for age, body mass index, triglyceride, apolipoprotein A-I, and high density lipoprotein-cholesterol (HDL-C) levels were recruited. We observed a significant increase (+14%; P < 0.03) in the capacity of whole sera from women to mediate cellular free cholesterol efflux via the SR-BI-dependent pathway compared with sera from men. Such enhanced efflux capacity resulted from a significant increase in plasma levels of large cholesteryl ester-rich HDL2 particles (+20%; P < 0.04) as well as from an enhanced capacity (+14%; P < 0.03) of these particles to mediate cellular free cholesterol efflux via SR-BI. By contrast, plasma from men displayed an enhanced free cholesterol efflux capacity (+31%; P < 0.001) via the ABCA1 transporter pathway compared with that from women, which resulted from a 2.4-fold increase in the plasma level of preβ particles (P < 0.008). Moreover, in women, SR-BI-mediated cellular free cholesterol efflux was significantly correlated with plasma HDL-C (r = 0.72, P < 0.0001), whereas this relationship was not observed in men. In conclusion, HDL-C level may not represent the absolute indicator of the efficiency of the initial step of the reverse cholesterol transport. We evaluated the impact of gender differences in both the quantitative and qualitative features of HDL subspecies on cellular free cholesterol efflux through the scavenger receptor class B type I (SR-BI), ABCA1, and ABCG1 pathways. For that purpose, healthy subjects (30 men and 26 women) matched for age, body mass index, triglyceride, apolipoprotein A-I, and high density lipoprotein-cholesterol (HDL-C) levels were recruited. We observed a significant increase (+14%; P < 0.03) in the capacity of whole sera from women to mediate cellular free cholesterol efflux via the SR-BI-dependent pathway compared with sera from men. Such enhanced efflux capacity resulted from a significant increase in plasma levels of large cholesteryl ester-rich HDL2 particles (+20%; P < 0.04) as well as from an enhanced capacity (+14%; P < 0.03) of these particles to mediate cellular free cholesterol efflux via SR-BI. By contrast, plasma from men displayed an enhanced free cholesterol efflux capacity (+31%; P < 0.001) via the ABCA1 transporter pathway compared with that from women, which resulted from a 2.4-fold increase in the plasma level of preβ particles (P < 0.008). Moreover, in women, SR-BI-mediated cellular free cholesterol efflux was significantly correlated with plasma HDL-C (r = 0.72, P < 0.0001), whereas this relationship was not observed in men. In conclusion, HDL-C level may not represent the absolute indicator of the efficiency of the initial step of the reverse cholesterol transport. Plasma HDL particles are highly heterogeneous in their structure, metabolism, and biological functions (1.Asztalos B.F. Schaefer E.J. HDL in atherosclerosis: actor or bystander?.Atheroscler. Suppl. 2003; 4: 21-29Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). HDL particles display a spectrum of antiatherogenic properties, including cellular cholesterol efflux, a key feature of the initial phase of the reverse cholesterol transport pathway, protection of LDL particles from oxidative stress, and anti-inflammatory, antiapoptotic, vasodilatory, antithrombotic, and anti-infectious activities (2.von Eckardstein A. Nofer J.R. Assmann G. High density lipoproteins and arteriosclerosis. Role of cholesterol efflux and reverse cholesterol transport.Arterioscler. Thromb. Vasc. Biol. 2001; 21: 13-27Crossref PubMed Scopus (638) Google Scholar). Several mechanisms of cellular cholesterol efflux have been proposed (3.Rothblat G.H. de la Llera-Moya M. Atger V. Kellner-Weibel G. Williams D.L. Phillips M.C. Cell cholesterol efflux: integration of old and new observations provides new insights.J. Lipid Res. 1999; 40: 781-796Abstract Full Text Full Text PDF PubMed Google Scholar). One is the nonspecific release of cholesterol from the cell surface membrane to extracellular acceptors mediated by passive aqueous diffusion. This efflux pathway occurs in all cell types, but it has been considered inefficient compared with receptor- or transporter-mediated efflux, although recent studies (4.Duong 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: 541-547Crossref PubMed Scopus (92) Google Scholar, 5.Adorni M.P. Zimetti F. Billheimer J.T. Wang N. Rader D.J. Phillips M.C. Rothblat G.H. The roles of different pathways in the release of cholesterol from macrophages.J. Lipid Res. 2007; 48: 2453-2462Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar) suggest that aqueous transfer mechanism may be quantitatively as important as cholesterol efflux mediated by specific transporters or receptors. ABCA1, a key membrane cholesterol transporter and a member of the large ATP binding cassette family, has been reported to play a major role in cholesterol efflux from macrophages and foam cells (6.Jessup W. Gelissen I.C. Gaus K. Kritharides L. Roles of ATP binding cassette transporters A1 and G1, scavenger receptor BI and membrane lipid domains in cholesterol export from macrophages.Curr. Opin. Lipidol. 2006; 17: 247-257Crossref PubMed Scopus (221) Google Scholar). Indeed, ABCA1 promotes phospholipid and free cholesterol efflux to lipid-poor or lipid-free apolipoprotein A-I (apoA-I), thereby allowing the formation of spherical HDL particles after the esterification of free cholesterol by LCAT. Scavenger receptor class B type I (SR-BI), a member of the CD36 family, mediates cellular cholesterol efflux to large, cholesteryl ester (CE)-rich HDL particles (7.Thuahnai S.T. Lund-Katz S. Dhanasekaran P. de la Llera-Moya M. Connelly M.A. Williams D.L. Rothblat G.H. Phillips M.C. Scavenger receptor class B type I-mediated cholesteryl ester-selective uptake and efflux of unesterified cholesterol. Influence of high density lipoprotein size and structure.J. Biol. Chem. 2004; 279: 12448-12455Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Indeed SR-BI interacts with a broad range of cholesterol acceptors but preferentially mediates the efflux of free cholesterol to large phospholipid- and CE-rich HDL particles (8.de la Llera-Moya M. Rothblat G.H. Connelly M.A. Kellner-Weibel G. Sakr S.W. Phillips M.C. Williams D.L. Scavenger receptor BI (SR-BI) mediates free cholesterol flux independently of HDL tethering to the cell surface.J. Lipid Res. 1999; 40: 575-580Abstract Full Text Full Text PDF PubMed Google Scholar). Similarly, ABCG1 facilitates cellular cholesterol export to phospholipid-rich acceptors such as HDL (9.Wang 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: 9774-9779Crossref PubMed Scopus (875) Google Scholar, 10.Gelissen I.C. Harris M. Rye K.A. Quinn C. Brown A.J. Kockx M. Cartland S. Packianathan M. Kritharides L. Jessup W. ABCA1 and ABCG1 synergize to mediate cholesterol export to apoA-I.Arterioscler. Thromb. Vasc. Biol. 2006; 26: 534-540Crossref PubMed Scopus (337) Google Scholar).Atherogenic dyslipidemias, which are associated with an increased risk of premature atherosclerosis, are characterized by increased plasma levels of proatherogenic apoB-containing lipoproteins relative to subnormal concentrations of antiatherogenic apoA-I-containing HDL particles. Indeed, low circulating levels of high density lipoprotein-cholesterol (HDL-C) in men (<40 mg/dl) represent a strong and independent risk factor for premature atherosclerosis and coronary heart disease (11.Chapman M.J. Assmann G. Fruchart J.C. Shepherd J. Sirtori C. Raising high-density lipoprotein cholesterol with reduction of cardiovascular risk: the role of nicotinic acid. A position paper developed by the European Consensus Panel on HDL-C.Curr. Med. Res. Opin. 2004; 20: 1253-1268Crossref PubMed Scopus (238) Google Scholar). Thus, it has been suggested that increasing plasma HDL-C levels might afford clinical benefits. Indeed, in epidemiological studies, an increment of 1 mg/dl in plasma HDL-C is associated with a 2–4% reduction in the risk of cardiovascular events (12.Gordon D.J. Probstfield J.L. Garrison R.J. Neaton J.D. Castelli W.P. Knoke J.D. Jacobs Jr., D.R. Bangdiwala S. Tyroler H.A. High-density lipoprotein cholesterol and cardiovascular disease. Four prospective American studies.Circulation. 1989; 79: 8-15Crossref PubMed Scopus (2630) Google Scholar). High levels of HDL-C, apoA-I, and apoA-I-containing lipoprotein subspecies have been reported to be negatively associated with the risk of coronary heart disease. It is well established that women have consistently higher HDL-C levels, especially the HDL2 subfraction (13.Schaefer E.J. Zech L.A. Jenkins L.L. Bronzert T.J. Rubalcaba E.A. Lindgren F.T. Aamodt R.L. Brewer Jr, H.B. Human apolipoprotein A-I and A-II metabolism.J. Lipid Res. 1982; 23: 850-862Abstract Full Text PDF PubMed Google Scholar), as well as higher concentrations of HDL-apoA-I compared with men (14.Brinton E.A. Eisenberg S. Breslow J.L. Human HDL cholesterol levels are determined by apoA-I fractional catabolic rate, which correlates inversely with estimates of HDL particle size. Effects of gender, hepatic and lipoprotein lipases, triglyceride and insulin levels, and body fat distribution.Arterioscler. Thromb. 1994; 14: 707-720Crossref PubMed Scopus (246) Google Scholar) and that premenopausal women display a lower risk of developing cardiovascular diseases than similarly aged men. In addition, estrogen administration is associated with increases in HDL-C and apoA-I levels in both young premenopausal and postmenopausal women (15.Schaefer E.J. Foster D.M. Zech L.A. Lindgren F.T. Brewer Jr., H.B. Levy R.I. The effects of estrogen administration on plasma lipoprotein metabolism in premenopausal females.J. Clin. Endocrinol. Metab. 1983; 57: 262-267Crossref PubMed Scopus (280) Google Scholar, 16.Applebaum-Bowden D. McLean P. Steinmetz A. Fontana D. Matthys C. Warnick G.R. Cheung M. Albers J.J. Hazzard W.R. Lipoprotein, apolipoprotein, and lipolytic enzyme changes following estrogen administration in postmenopausal women.J. Lipid Res. 1989; 30: 1895-1906Abstract Full Text PDF PubMed Google Scholar). These data suggest that sex hormones influence the metabolism of both the lipid and protein components of HDL particles and protection from cardiovascular disease. In this context, many studies have been undertaken to evaluate the role of estradiol in this protection. It appears that the atheroprotective effect of estradiol is mainly related to a direct effect on the cells of the arterial wall (17.Arnal J.F. Gourdy P. Elhage R. Garmy-Susini B. Delmas E. Brouchet L. Castano C. Barreira Y. Couloumiers J.C. Prats H. et al.Estrogens and atherosclerosis.Eur. J. Endocrinol. 2004; 150: 113-117Crossref PubMed Scopus (50) Google Scholar). However, the mechanisms involved in the protection from cardiovascular diseases observed in women are not fully understood.The potential relevance of gender differences to the capacity of HDL to mediate cholesterol efflux is indeterminate. Therefore, the objective of this study was to evaluate whether potential differences in the quantitative and qualitative features of plasma HDL particles between healthy nondyslipidemic men and women might affect cellular free cholesterol efflux through the SR-BI, ABCA1, and ABCG1 pathways. Our data clearly demonstrate that plasma from women displayed a higher capacity to mediate cellular free cholesterol efflux via the SR-BI pathway compared with that from men. Such enhanced efflux capacity resulted from significant increases in plasma levels of large CE-rich HDL2 particles as well as from an enhanced capacity of these particles to mediate cellular free cholesterol efflux via the SR-BI pathway. By contrast, plasma from healthy nondyslipidemic men displayed an enhanced free cholesterol efflux capacity via the ABCA1 transporter pathway compared with that from women, which resulted from an increased level of poorly lipidated apoA-I particles (preβ-HDL).METHODSSubjectsPlasma was obtained from healthy volunteers (30 men and 26 women) selected specifically for their healthy nondyslipidemic status. Selection criteria for subjects were as follows: total cholesterol < 250 mg/dl, triglyceride < 150 mg/dl, LDL-C < 150 mg/dl, apoB < 140 mg/dl, and body mass index < 25 kg/m2 (Table 1). They were nonobese and free of cardiovascular disease, hypertension, diabetes, hyperlipidemia, medication use, including lipid-lowering drugs, and thyroid, renal, or liver dysfunction. Eight of the women were under contraceptive treatment; none of the postmenopausal women (n = 11) was currently receiving estrogen replacement therapy. After an overnight fast, blood samples were collected into sterile EDTA-containing tubes for the isolation of plasma or into anticoagulant-free tubes for the isolation of serum. Plasma was immediately separated from blood cells by low-speed centrifugation at 2,500 rpm for 20 min at 4°C and frozen at −80°C until used.TABLE 1.Clinical characteristics and plasma parameters in healthy nondyslipidemic subjectsCharacteristicsMen (n = 30)Women (n = 26)Age (years)46 ± 1345 ± 18Body mass index (kg/m2)24 ± 222 ± 2Cholesterol (mg/dl) Total217 ± 26219 ± 22 LDL141 ± 28140 ± 17 HDL58 ± 1360 ± 13Triglycerides (mg/dl)90 ± 2696 ± 19Apolipoproteins (mg/dl) apoA-I144 ± 26161 ± 27aP < 0.05 versus men. apoA-II44 ± 744 ± 7 apoB109 ± 13116 ± 26Cholesteryl ester transfer protein activity (%)26 ± 932 ± 8aP < 0.05 versus men.ApoA-I, apolipoprotein A-I. Values are means ± SD.a P < 0.05 versus men. Open table in a new tab This study was performed in accordance with the ethical principles set forth in the Declaration of Helsinki. Written informed consent was obtained from all subjects.Isolation of lipoprotein subfractionsLipoproteins were isolated from plasma by density gradient ultracentrifugation in a Beckman SW41 Ti rotor at 40,000 rpm for 48 h in a Beckman XL70 at 15°C and by a slight modification of the method of Chapman et al. (18.Chapman M.J. Goldstein S. Lagrange D. Laplaud P.M. A density gradient ultracentrifugal procedure for the isolation of the major lipoprotein classes from human serum.J. Lipid Res. 1981; 22: 339-358Abstract Full Text PDF PubMed Google Scholar) as described previously (19.Guerin M. Lassel T.S. Goff W.Le Farnier M. Chapman M.J. Action of atorvastatin in combined hyperlipidemia: preferential reduction of cholesteryl ester transfer from HDL to VLDL1 particles.Arterioscler. Thromb. Vasc. Biol. 2000; 20: 189-197Crossref PubMed Scopus (211) Google Scholar). After centrifugation, gradients were collected from the top of the tubes with an Eppendorf precision pipette in aliquots of 0.4 ml. Fractions corresponding to HDL subspecies, HDL2b (d = 1.063–1.091 g/ml), HDL2a (d = 1.091–1.110 g/ml), HDL3a (d = 1.110–1.133 g/ml), HDL3b (d = 1.133–1.156 g/ml), and HDL3c (d = 1.156–1.179 g/ml), were analyzed for their lipid and protein contents. In addition, plasma lipoproteins were also fractionated by gel filtration on two Superose 6 (Amersham Biosciences) columns connected in series using the Biologic DuoFlow Chromatography System (Bio-Rad).Lipid and protein analysisThe lipid contents of plasma and isolated lipoprotein fractions, total protein, and apoA-I, apoA-II, and apoB were quantified with an Autoanalyzer (Konelab 20). Reagent kits from Roche Diagnostics and ThermoElectron were used for the determination of total cholesterol and triglyceride levels, respectively. The levels of unesterified cholesterol and phospholipids were determined with reagent kits (Wako Diagnostics). CE mass was calculated as total cholesterol minus free cholesterol × 1.67 and thus represents the sum of the esterified cholesterol and fatty acids moieties (18.Chapman M.J. Goldstein S. Lagrange D. Laplaud P.M. A density gradient ultracentrifugal procedure for the isolation of the major lipoprotein classes from human serum.J. Lipid Res. 1981; 22: 339-358Abstract Full Text PDF PubMed Google Scholar). Bicinchoninic acid assay reagent (Pierce) was used for protein quantification. Fasting plasma LDL-C was calculated using the Friedewald formula. HDL-C levels were determined after dextran sulfate-magnesium precipitation of apolipoprotein B-containing lipoproteins. Plasma apoA-I, apoB, and apoA-II concentrations were determined using immunoturbidimetric assays (ThermoElectron reagents and calibrators; Wako Diagnostics reagents and calibrators). Lipoprotein mass was calculated as the sum of the mass of the individual lipid and protein components for each lipoprotein fraction. Molecular weights of HDL subfractions were calculated by transforming concentration data (mg/dl) into absolute molar units using molecular weights of CE, free cholesterol, phospholipid, and triglyceride of 650, 387, 750, and 850, respectively (20.Chancharme L. Therond P. Nigon F. Lepage S. Couturier M. Chapman M.J. Cholesteryl ester hydroperoxide lability is a key feature of the oxidative susceptibility of small, dense LDL.Arterioscler. Thromb. Vasc. Biol. 1999; 19: 810-820Crossref PubMed Scopus (60) Google Scholar). The protein moiety was considered to consist of two apolipoproteins, apoA-I and apoA-II, and the molecular weight of the protein moiety in each HDL subfraction was calculated using the total protein content (mg/dl) converted to molarity on the basis of the relative mass contents of apoA-I and apoA-II (21.Nobecourt E. Jacqueminet S. Hansel B. Chantepie S. Grimaldi A. Chapman M.J. Kontush A. Defective antioxidative activity of small dense HDL3 particles in type 2 diabetes: relationship to elevated oxidative stress and hyperglycaemia.Diabetologia. 2005; 48: 529-538Crossref PubMed Scopus (174) Google Scholar).Quantification of preβ-HDLThe quantification of preβ-HDL in serum was performed as described previously (22.Mweva S. Paul J.L. Cambillau M. Goudouneche D. Beaune P. Simon A. Fournier N. Comparison of different cellular models measuring in vitro the whole human serum cholesterol efflux capacity.Eur. J. Clin. Invest. 2006; 36: 552-559Crossref PubMed Scopus (23) Google Scholar). Serum from healthy nondyslipidemic men and women (5 μl) was electrophoresed (2 h at 200 V) at 7°C on a 0.75% agarose gel in 50 mM barbital buffer (pH 8.6) on Gelbond using the Multiphor system (Amersham Pharmacia Biotech, Inc., Orsay, France) and transferred to a nitrocellulose membrane. After blocking in 5% skim milk powder buffer, the bound serum proteins were immunoreacted with a monoclonal mouse antiserum raised against human apoA-I (AbCys, Paris, France). Immunodetection of apoA-I was subsequently localized with horseradish peroxidase-conjugated goat anti-mouse IgG (Bio-Rad, Ivry-sur-Seine, France) and visualized by staining with a 4-chloro-1-naphthol solution kit (Bio-Rad). The relative abundance of the human apoA-I among the α- or preβ-HDL species was determined by scanning reflectance densitometry (Quantity One software; Bio-Rad). The amount of preβ-HDL was expressed as the percentage of total apoA-I (relative concentration) and as absolute concentration (mg/l apoA-I) by multiplying its percentage by the serum apoA-I levels. A normolipidemic serum and a hypertriglyceridemic serum were systematically included in each experiment as controls.Determination of endogenous CE transfer from HDL to apoB-containing lipoproteinsDetermination of endogenous CE transfer from HDL to apoB-containing lipoproteins was assayed by a modification of the method of Guerin et al. (23.Guerin M. Dolphin P.J. Chapman M.J. A new in vitro method for the simultaneous evaluation of cholesteryl ester exchange and mass transfer between HDL and apoB-containing lipoprotein subspecies: identification of preferential cholesteryl ester acceptors in human plasma.Arterioscler. Thromb. 1994; 14: 199-206Crossref PubMed Google Scholar), which estimates net physiological CE transfer between lipoprotein donor and acceptor particles in the plasma of individual patients. To obtain radiolabeled HDL, a single plasma from a normolipidemic healthy man was depleted of apoB-containing lipoproteins by ultracentrifugation. The d > 1.063 g/ml plasma fraction was then labeled with [3H]cholesterol overnight (4 μCi/ml). Radiolabeled [3H]HDL was then isolated from the d > 1.063 g/ml plasma fraction as described previously (23.Guerin M. Dolphin P.J. Chapman M.J. A new in vitro method for the simultaneous evaluation of cholesteryl ester exchange and mass transfer between HDL and apoB-containing lipoprotein subspecies: identification of preferential cholesteryl ester acceptors in human plasma.Arterioscler. Thromb. 1994; 14: 199-206Crossref PubMed Google Scholar). CE transfer was determined after incubation of whole plasma (200 μl) from individual subjects at 37°C or 0°C for 3 h in the presence of radiolabeled HDL (2 μg) and iodoacetate (final concentration, 1.5 mmol/l) for the inhibition of LCAT. After incubation, apoB-containing lipoproteins were precipitated using the dextran sulfate-magnesium procedure. The radioactive content of the supernatant was quantified by liquid scintillation spectrometry with a Trilux 1450 (Perkin-Elmer). Cholesteryl ester transfer protein (CETP) activity (expressed as percentage) was calculated as the amount of the label recovered in the supernatant after incubation and divided by the label present in the supernatant before incubation. The CETP-dependent CE transfer was calculated from the difference between the radioactivity transferred at 37°C and 0°C.Cell culture and lipid efflux assaysFu5AH cells were grown at 37°C in Eagle's MEM and 5% newborn calf serum, HepG2 and RAW267.4 cells in DMEM plus 10% fetal bovine serum, and CHO-K1 cells (wild-type and human ABCG1-transfected cells), kindly provided by Dr. W. Jessup, in Ham's F-12 medium (Gibco) supplemented with 10% fetal bovine serum. Lipid efflux assays were performed as described previously for the different cellular models (10.Gelissen I.C. Harris M. Rye K.A. Quinn C. Brown A.J. Kockx M. Cartland S. Packianathan M. Kritharides L. Jessup W. ABCA1 and ABCG1 synergize to mediate cholesterol export to apoA-I.Arterioscler. Thromb. Vasc. Biol. 2006; 26: 534-540Crossref PubMed Scopus (337) Google Scholar, 24.de la Llera Moya M. Atger V. Paul J.L. Fournier N. Moatti N. Giral P. Friday K.E. Rothblat G. A cell culture system for screening human serum for ability to promote cellular cholesterol efflux. Relations between serum components and efflux, esterification, and transfer.Arterioscler. Thromb. 1994; 14: 1056-1065Crossref PubMed Google Scholar, 25.Le Goff W. Peng D.Q. Settle M. Brubaker G. Morton R.E. Smith J.D. Cyclosporin A traps ABCA1 at the plasma membrane and inhibits ABCA1-mediated lipid efflux to apolipoprotein A-I.Arterioscler. Thromb. Vasc. Biol. 2004; 24: 2155-2161Crossref PubMed Scopus (74) Google Scholar) in cultured rat hepatoma Fu5AH cells (24.de la Llera Moya M. Atger V. Paul J.L. Fournier N. Moatti N. Giral P. Friday K.E. Rothblat G. A cell culture system for screening human serum for ability to promote cellular cholesterol efflux. Relations between serum components and efflux, esterification, and transfer.Arterioscler. Thromb. 1994; 14: 1056-1065Crossref PubMed Google Scholar) expressing high levels of SR-BI, in mouse macrophage RAW264.7 cells expressing ABCA1 after stimulation with 8-Br cAMP (25.Le Goff W. Peng D.Q. Settle M. Brubaker G. Morton R.E. Smith J.D. Cyclosporin A traps ABCA1 at the plasma membrane and inhibits ABCA1-mediated lipid efflux to apolipoprotein A-I.Arterioscler. Thromb. Vasc. Biol. 2004; 24: 2155-2161Crossref PubMed Scopus (74) Google Scholar), and in CHO cells overexpressing the human ABCG1 gene (10.Gelissen I.C. Harris M. Rye K.A. Quinn C. Brown A.J. Kockx M. Cartland S. Packianathan M. Kritharides L. Jessup W. ABCA1 and ABCG1 synergize to mediate cholesterol export to apoA-I.Arterioscler. Thromb. Vasc. Biol. 2006; 26: 534-540Crossref PubMed Scopus (337) Google Scholar).Fu5AH and HepG2 cellsAfter plating, cells were labeled by incubation with [3H]cholesterol (1 μCi/ml) for 48 h in Eagle's MEM for Fu5AH or in DMEM for HepG2. Subsequently, cells were incubated for 24 h in the presence of BSA (0.5%) and newborn calf serum (25%) for Fu5AH or in fetal bovine serum (25%) for HepG2 to allow equilibration of the label. After equilibration, cholesterol acceptors (2.5% diluted serum, 2.5% diluted apoB-depleted serum, or 10 μg phospholipid/ml isolated HDL subfractions) were added in serum-free medium and incubated with cells for 4 h at 37°C. For cholesterol efflux studies performed in the presence of rabbit antiserum directed to human SR-BI (Cla-1) (26.Treguier M. Moreau M. Sposito A. Chapman M.J. Huby T. LDL particle subspecies are distinct in their capacity to mediate free cholesterol efflux via the SR-BI/Cla-1 receptor.Biochim. Biophys. Acta. 2007; 1771: 129-138Crossref PubMed Scopus (22) Google Scholar), the assay was carried out as described above but the cells were preincubated with a 1:50 dilution of whole rabbit antiserum for 30 min before the efflux period.RAW264.7 cellsThe day after cell plating, cells were loaded and labeled with acetylated LDL (50 μg/ml) and 0.5 μCi/ml [3H]cholesterol for 24 h in serum-free DMEM containing glucose (4.5 g/l) and BSA (0.2%) (DGGB). After incubation, RAW264.7 cells were incubated with DGGB in the absence or presence of 8-Br cAMP (0.3 mM) for 16 h to induce ABCA1 expression. Cholesterol acceptors (2.5% diluted serum or 30 μg apoA-I/ml isolated HDL subfractions) were added to RAW264.7 cells in serum-free DMEM for 4 h at 37°C in the presence or absence of 0.3 mM 8-Br cAMP.CHO-K1 cells (wild type and human ABCG1)Two days after plating, cellular cholesterol was labeled by incubation of cells with serum-free Ham's F-12 medium and 1 μCi/ml [3H]cholesterol. Equilibration of the label was performed for 90 min in serum-free medium and BSA (0.1%). After equilibration of labeling, acceptors (2.5% diluted serum or 5 μg phospholipid/ml isolated HDL subfractions) were added to the cells in serum-free medium containing BSA (0.1%) for 4 h at 37°C. Net efflux was expressed as the difference between efflux to human ABCG1-transfected CHO-K1 cells and wild-type CHO-K1 cells.All efflux experiments were performed in triplicate for each sample. Fractional cholesterol efflux (expressed as a percentage) was calculated as the amount of the label recovered in the medium divided by the total label in each well (radioactivity in the medium + radioactivity in the cells) obtained after lipid extraction from cells in a mixture of 3:2 hexane-isopropanol (v/v). The background cholesterol efflux obtained in the absence of any acceptor was subtracted from the efflux values obtained with the test samples. Plasma coming from a single normolipidemic subject not included in the study was prepared in aliquots, frozen, and used as an internal standard for each experiment. All efflux values were normalized with the fractional efflux obtained with the 2.5% diluted standard plasma. The capacity of HDL subfractions, HDL2 and HDL3, to mediate free cholesterol efflux is expressed as percentage of cholesterol efflux per mole of acceptor particle.Statistical analysesDifferences in cellular cholesterol efflux to the various acceptors from healthy nondyslipidemic men or women were tested for significance by ANOVA. Results were considered statistically significant at P < 0.05.RESULTSPlasma lipid and clinical characteristics of healthy subjectsAs shown in Table 1, no significant differences in mean age, body mass index, plasma lipid profile, and apoB and apoA-II levels were observed between men and women. By contrast, women displayed significant increases in both plasma apoA-I levels (+12%; P < 0.02) and endogenous CETP activity (+23%; P < 0.05) compared with men. Based on those parameters, the population is representative of a general population of healthy nondyslipidemic subjects. As expected, analysis of the distribution of HDL-C levels showed (Fig. 1) that even if the arithmetic mean value for HDL-C level is not significantly different between genders, men and women exhibit distinct distributions around this value, with the HDL-C peak value being shifted toward higher values in women compared with men.Gender-specific effects of sera on cellular free cholesterol efflux via SR-BIWe measured the efflux capacity of whole serum from healthy nondyslipidemic women and men in different cellular models, each representative of a specific cholesterol efflux pathway (10.Gelissen I.C. Harris M. Rye K.A. Quinn C. Brown A.J. Kockx M. Cartland S. Packianathan M. Kritharides L. Jessup W. ABCA1 and ABCG1 synergize to mediate cholesterol export to apoA-I.Arterioscler. Thromb. Vasc. Biol. 2006; 26: 534-540Crossref PubMed Scopus (337) Google Scholar, 24.de la Llera Moya M. Atger V. Paul J.L. Fournier N. Moatti N. Giral P. Friday K.E. Rothblat G. A cell culture system for screening human serum for ability to promote cellular cholesterol efflux. Relations between serum components and efflux, esterification, and transfer.Arterioscler. Thromb. 1994; 14: 1056-1065Crossref PubMed Google Scholar, 25.Le Goff W. Peng D.Q. Settle M. Brubaker G. Morton R.E. Smith J.D. Cyclosporin A traps ABCA1 at the plasma membrane and inhibits ABCA1-mediated lipid efflux to apolipoprotein A-I.Arterioscler. Thromb. Vasc. Biol. 2004; 24: 2155-2161Crossref PubMed Scopus (74) Google Scholar). Using 40-f
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