Produção Nacional
Revisado por pares
Cholesteryl ester transfer protein expression attenuates atherosclerosis in ovariectomized mice
2003; Elsevier BV; Volume: 44; Issue: 1 Linguagem: Inglês
10.1194/jlr.m100440-jlr200
ISSN1539-7262
AutoresP.M. Cazita, Jairo Augusto Berti, Carolina Aoki, Magnus Gidlund, L.M. Harada, V.S. Nunes, E.C.R. Quintão, Helena C.F. Oliveira,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoReduced estrogen levels result in loss of protection from coronary heart disease in postmenopausal women. Enhanced and diminished atherosclerosis have been associated with plasma levels of cholesteryl ester transfer protein (CETP); however, little is known about the role of CETP-ovarian hormone interactions in atherogenesis. We assessed the severity of diet-induced atherosclerosis in ovariectomized (OV) CETP transgenic mice crossbred with LDL receptor knockout mice. Compared with OV CETP expressing (+), OV CETP non-expressing (−) mice had higher plasma levels of total, VLDL-, LDL-, and HDL-cholesterol, as well as higher antibodies titers against oxidized LDL. The mean aortic lesion area was 2-fold larger in OV CETP− than in OV CETP+ mice (147 ± 90 vs. 73 ± 42 × 103 μm2, respectively). Estrogen therapy in OV mice blunted the CETP dependent differences in plasma lipoproteins, oxLDL antibodies, and atherosclerosis severity. Macrophages from OV CETP+ mice took up less labeled cholesteryl ether (CEt) from acetyl-LDL than macrophages from OV CETP− mice. Estrogen replacement induced a further reduction in CEt uptake and an elevation in HDL mediated cholesterol efflux from pre-loaded OV CETP+ as compared with OV CETP− macrophages.These findings support the proposed anti-atherogenic role of CETP in specific metabolic settings. Reduced estrogen levels result in loss of protection from coronary heart disease in postmenopausal women. Enhanced and diminished atherosclerosis have been associated with plasma levels of cholesteryl ester transfer protein (CETP); however, little is known about the role of CETP-ovarian hormone interactions in atherogenesis. We assessed the severity of diet-induced atherosclerosis in ovariectomized (OV) CETP transgenic mice crossbred with LDL receptor knockout mice. Compared with OV CETP expressing (+), OV CETP non-expressing (−) mice had higher plasma levels of total, VLDL-, LDL-, and HDL-cholesterol, as well as higher antibodies titers against oxidized LDL. The mean aortic lesion area was 2-fold larger in OV CETP− than in OV CETP+ mice (147 ± 90 vs. 73 ± 42 × 103 μm2, respectively). Estrogen therapy in OV mice blunted the CETP dependent differences in plasma lipoproteins, oxLDL antibodies, and atherosclerosis severity. Macrophages from OV CETP+ mice took up less labeled cholesteryl ether (CEt) from acetyl-LDL than macrophages from OV CETP− mice. Estrogen replacement induced a further reduction in CEt uptake and an elevation in HDL mediated cholesterol efflux from pre-loaded OV CETP+ as compared with OV CETP− macrophages. These findings support the proposed anti-atherogenic role of CETP in specific metabolic settings. Remodelling of plasma lipoproteins through the transfer of neutral lipids such as cholesteryl ester (CE) and triacylglycerols (TAG) is the best characterized function of the cholesteryl ester transfer protein (CETP) (1Bruce C. Chouinard Jr., R.A. Tall A.R. Plasma lipid transfer proteins, high-density lipoproteins, and reverse cholesterol transport.Annu. Rev. Nutr. 1998; 18: 297-330Google Scholar). Epidemiological and experimental evidences have shown that CETP may play an important role in the development of atherosclerosis (2Inazu A. Koizumi J. Mabuchi H. Cholesteryl ester transfer protein and atherosclerosis.Curr. Opin. Lipidol. 2000; 11: 389-396Google Scholar); however, the precise effects of CETP on atherogenesis are controversial. In humans, increased incidence of coronary heart disease has been associated with both CETP deficiency (3Zhong S. Sharp D.S. Grove J.S. Bruce C. Yano K. Curb J.D. Tall A.R. Increased coronary heart disease in Japanese-American men with mutation in the cholesteryl ester transfer protein gene despite increased HDL levels.J. Clin. Invest. 1996; 97: 2917-2923Google Scholar) and augmentation (4Bhatnagar D. Durrington P.N. Channon K.M. Prais H. Mackness M.I. Increased transfer of cholesteryl esters from high density lipoproteins to low density and very low density lipoproteins in patients with angiographic evidence of coronary artery disease.Atherosclerosis. 1993; 98: 25-32Google Scholar). In several animal models of atherosclerosis, the effects of CETP on vascular health are clearly dependent upon the metabolic context (5Marotti K.R. Castle C.K. Boyle T.P. Lin A.H. Murray R.W. Melchior G.W. Severe atherosclerosis in transgenic mice expressing simian cholesteryl ester transfer protein.Nature. 1993; 364: 73-75Google Scholar, 6Plump A.S. Masucci-Magoulas L. Bruce C. Bisgaier C.L. Breslow J.L. Tall A.R. Increased atherosclerosis in ApoE and LDL receptor gene knock-out mice as a result of human cholesteryl ester transfer protein transgene expression.Arterioscler. Thromb. Vasc. Biol. 1999; 19: 1105-1110Google Scholar, 7Hayek T. Masucci-Magoulas L. Jiang X. Walsh A. Rubin E. Breslow J.L. Tall A.R. Decreased early atherosclerotic lesions in hypertriglyceridemic mice expressing cholesteryl ester transfer protein transgene.J. Clin. Invest. 1995; 96: 2071-2074Google Scholar, 8Foger B. Chase M. Amar M.J. Vaisman B.L. Shamburek R.D. Paigen B. Fruchart-Najib J. Paiz J.A. Koch C.A. Hoyt R.F. Brewer Jr., H.B. Santamarina-Fojo S. Cholesteryl ester transfer protein corrects dysfunctional high density lipoproteins and reduces aortic atherosclerosis in lecithin cholesterol acyltransferase transgenic mice.J. Biol. Chem. 1999; 274: 36912-36920Google Scholar, 9Sugano M. Makino N. Sawada S. Otsuka S. Watanabe M. Okamoto H. Kamada M. Mizushima A. Effect of antisense oligonucleotides against cholesteryl ester transfer protein on the development of atherosclerosis in cholesterol-fed rabbits.J. Biol. Chem. 1998; 273: 5033-5036Google Scholar, 10Okamoto H. Yonemori F. Wakitani K. Minowa T. Maeda K. Shinkai H. A cholesteryl ester transfer protein inhibitor attenuates atherosclerosis in rabbits.Nature. 2000; 406: 203-207Google Scholar, 11Rittershaus C.W. Miller D.P. Thomas L.J. Picard M.D. Honan C.M. Emmett C.D. Pettey C.L. Adari H. Hammond R.A. Beattie D.T. Callow A.D. Marsh H.C. Ryan U.S. Vaccine-induced antibodies inhibit CETP activity in vivo and reduce aortic lesions in a rabbit model of atherosclerosis.Arterioscler. Thromb. Vasc. Biol. 2000; 20: 2106-2112Google Scholar). Various researchers are attempting to target CETP as a form of therapy (9Sugano M. Makino N. Sawada S. Otsuka S. Watanabe M. Okamoto H. Kamada M. Mizushima A. Effect of antisense oligonucleotides against cholesteryl ester transfer protein on the development of atherosclerosis in cholesterol-fed rabbits.J. Biol. Chem. 1998; 273: 5033-5036Google Scholar, 10Okamoto H. Yonemori F. Wakitani K. Minowa T. Maeda K. Shinkai H. A cholesteryl ester transfer protein inhibitor attenuates atherosclerosis in rabbits.Nature. 2000; 406: 203-207Google Scholar, 11Rittershaus C.W. Miller D.P. Thomas L.J. Picard M.D. Honan C.M. Emmett C.D. Pettey C.L. Adari H. Hammond R.A. Beattie D.T. Callow A.D. Marsh H.C. Ryan U.S. Vaccine-induced antibodies inhibit CETP activity in vivo and reduce aortic lesions in a rabbit model of atherosclerosis.Arterioscler. Thromb. Vasc. Biol. 2000; 20: 2106-2112Google Scholar, 12Hirano K. Yamashita S. Matsuzawa Y. Pros and cons of inhibiting cholesteryl ester transfer protein.Curr. Opin. Lipidol. 2000; 11: 589-596Google Scholar, 13Thompson G.R. Barter P.J. Therapeutic approaches to reducing the LDL- and HDL-associated risks of coronary heart disease.Curr. Opin. Lipidol. 2000; 11: 567-570Google Scholar), but these approaches will be useless unless the circumstances where CETP acts as pro- or anti-atherogenic are properly clarified. Deficiency in endogenous estrogen accounts for the loss of protection against coronary heart disease after menopause or following bilateral ovariectomy (14Wenger N.K. Postmenopausal hormone use for cardioprotection: what we know and what we must learn.Curr. Opin. Cardiol. 1999; 14: 292-297Google Scholar). Estrogen deficiency per se does not alter plasma CETP activity as shown in castrated CETP transgenic mice (15Vadlamudi S. MacLean P. Green T. Shukla N. Bradfield J. Vore S. Barakat H. Role of female sex steroids in regulating cholesteryl ester transfer protein in transgenic mice.Metabolism. 1998; 47: 1048-1051Google Scholar). Also, estrogen therapy has no impact on the plasma CETP activity in humans (16Tilly-Kiesi M. Kahri J. Pyorala T. Puolakka J. Luotola H. Lappi M. Lahdenpera S. Taskinen M.R. Responses of HDL subclasses, Lp(A-I) and Lp(A-I:A-II) levels and lipolytic enzyme activities to continuous oral estrogen-progestin and transdermal estrogen with cyclic progestin regimens in postmenopausal women.Atherosclerosis. 1997; 129: 249-259Google Scholar) as well as in apolipoprotein B/CETP double transgenic mouse model (17Zuckerman S.H. Evans G.F. Schelm J.A. Eacho P.I. Sandusky G. Estrogen-mediated increases in LDL cholesterol and foam cell-containing lesions in human ApoB100xCETP transgenic mice.Arterioscler. Thromb. Vasc. Biol. 1999; 19: 1476-1483Google Scholar). The present study aimed at investigating whether CETP expression would alter the development of atherosclerosis in a moderately hypercholesterolemic mouse model lacking ovarian hormones. For this purpose, mice expressing the human CETP gene were crossbred with LDL receptor (LDLR) knockout mice. On a high fat diet, the LDLR knockout mice develop extensive aorta atherosclerosis in a pattern similar to humans (18Ishibashi S. Goldstein J.L. Brown M.S. Herz J. Burns D.K. Massive xanthomatosis and atherosclerosis in cholesterol-fed low density lipoprotein receptor-negative mice.J. Clin. Invest. 1994; 93: 1885-1893Google Scholar, 19Tangirala R.K. Rubin E.M. Palinski W. Quantitation of atherosclerosis in murine models: correlation between lesions in the aortic origin and in the entire aorta, and differences in the extent of lesions between sexes in LDL receptor-deficient and apolipoprotein E-deficient mice.J. Lipid Res. 1995; 36: 2320-2328Google Scholar). We have shown here that the expression of the CETP gene significantly reduced the development of atherosclerotic lesions in ovariectomized hypercholesterolemic mice. Furthermore, this antiatherogenic effect of CETP was blunted by the estrogen replacement therapy. The animal protocols were approved by the University of São Paulo Medical School Ethics Committee. Hemizygous human CETP transgenic mice (line 5203, C57BL6/J background) (20Jiang X.C. Agellon L.B. Walsh A. Breslow J.L. Tall A. Dietary cholesterol increases transcription of the human cholesteryl ester transfer protein gene in transgenic mice. Dependence on natural flanking sequences.J. Clin. Invest. 1992; 90: 1290-1295Google Scholar) expressing a human CETP minigene under the control of natural flanking sequences were derived from Dr. Alan R. Tall's colony (Columbia University, New York, NY) and crossbred with LDLR knockout mice purchased from Jackson Laboratory (Bar Harbor, ME). The pups' tail tips were utilized for screening for the presence of the CETP gene promoter by polymerase chain reaction (PCR) DNA amplification of the −538 to −222 CETP promoter region (GeneBank U71187). Tail blood was also drawn for determining plasma CETP activity (21Berti J.A. Amaral M.E. Boschero A.C. Nunes V.S. Harada L.M. Castilho L.N. Oliveira H.C. Thyroid hormone increases plasma cholesteryl ester transfer protein activity and plasma high-density lipoprotein removal rate in transgenic mice.Metabolism. 2001; 50: 530-536Google Scholar). Female littermates, 8–12 weeks of age, heterozygous for the LDLR null allele expressing CETP (+) or not (−) were bilaterally ovariectomized (OV) or sham-operated (Sham). All mice were anesthetized for surgery using ketamine (50 mg/kg, ip, Ketalar, Parke-Davis, São Paulo, Brazil) and xylazine (16 mg/kg, ip, Rompum, Bayer S.A., São Paulo, Brazil). The success of the ovariectomy was checked by analyzing vaginal smear during 5 consecutive days after the surgery. OV mice presented only the diestrus pattern while in Sham mice the four stages of the estrous cycle (proestrus, estrus, metestrus, and diestrus) were clearly verified (22Montes G.S. Luque E.H. Effects of ovarian steroids on vaginal smears in the rat.Acta Anat. (Basel). 1988; 133: 192-199Google Scholar). Five days after surgery, all animals were placed on an atherogenic high fat and high cholesterol (HFHC) diet containing 15% fat, 1.25% cholesterol, and 0.5% cholic acid (Cat. # 611208, Dyets, Inc. Bethlehem, PA) for 19 weeks. It has been previously demonstrated that LDLR deficient mice exhibited similar distribution pattern and histological features of the atherosclerotic lesions when fed cholate-free or cholate-containing high fat and high cholesterol diets (23Lichtman A.H. Clinton S.K. Iiyama K. Connelly P.W. Libby P. Cybulsky M.I. Hyperlipidemia and atherosclerotic lesion development in LDL receptor- deficient mice fed defined semipurified diets with and without cholate.Arterioscler. Thromb. Vasc. Biol. 1999; 19: 1938-1944Google Scholar). Blood samples drawn from mice fasted for 6 h on the chow diet (5 days after surgery), corresponding to a baseline period and after 19 weeks on the HFHC diet, were collected into pre-cooled tubes containing 1 mM EDTA and centrifuged at 2,500 g at 4°C for 10 min. Aliquots of plasma were stored at −70°C until analysis. In order to compare OV and estrogen treated OV mice, a second experiment was performed. Estrogen replacement was done utilizing 60-day release pellets that released 6 μg/day of 17-β-estradiol (E2) or placebo (Innovative Research, Toledo, OH) subcutaneously implanted in the midle of the HFHC diet period. At the end of this experiment, the uterus weight was monitored exactly as described by Marsh et al. (24Marsh M.M. Walker V.R. Curtiss L.K. Banka C.L. Protection against atherosclerosis by estrogen is independent of plasma cholesterol levels in LDL receptor-deficient mice.J. Lipid Res. 1999; 40: 893-900Google Scholar). Uterus from estrogen deficient mice were consistently smaller ( 0.15 g) (P < 0.01). Based on the uterus weight criterion, two mice were excluded from OV CETP+ placebo and one mouse in each of the other three groups: OV CETP− placebo, OV CETP+ E2, and OV CETP− E2. Mice body weight (g ± SD) at the end of the studies was slightly but significantly higher in OV than in Sham groups (P < 0.05): 22.7 ± 1.2 (CETP+ Sham) versus 24.4 ± 1.3 (CETP+ OV) and 24.1 ± 2.0 (CETP− Sham) versus 25.3 ± 1.2 (CETP− OV). In estrogen treated mice final weights were: OV CETP+ (23.9 ± 1.1) versus OV CETP− (24.6 ± 0.7); OV CETP+ E2 (22.7 ± 1.1) versus OV CETP− E2 (22.8 ± 1.7). Mice were anesthetized and their hearts were perfused in situ with phosphate buffered saline (PBS) followed by 10% PBS buffered formaldehyde, after which they were excised and fixed in 10% formaldehyde for at least 2 days. The hearts were then embedded sequentially in 5%, 10%, and 25% gelatin. Processing and staining were carried out according to Paigen et al. (25Paigen B. Morrow A. Holmes P.A. Mitchell D. Williams R.A. Quantitative assessment of atherosclerotic lesions in mice.Atherosclerosis. 1987; 68: 231-240Google Scholar). The lipid-stained lesions were quantified as described by Rubin et al. (26Rubin E.M. Krauss R.M. Spangler E.A. Verstuyft J.G. Clift S.M. Inhibition of early atherogenesis in transgenic mice by human apolipoprotein AI.Nature. 1991; 353: 265-267Google Scholar) using Image Pro Plus software (version 3.0) for image analysis (Media Cybernetics, Silver Spring, MD). The slides were read by an investigator who was unaware of the treatments. The area of the lesions was expressed as the sum of the lesions in six 10-μm sections, 80 μm distant from each other in a total aorta length of 480 μm. Because several other studies revealed a predilection for development of lesions in the aortic root, the segment that was chosen for analysis extended from beyond the aortic sinus up to the point where the aorta first becomes rounded (26Rubin E.M. Krauss R.M. Spangler E.A. Verstuyft J.G. Clift S.M. Inhibition of early atherogenesis in transgenic mice by human apolipoprotein AI.Nature. 1991; 353: 265-267Google Scholar). CETP activity, which reflects the plasma CETP concentration (27McPherson R. Mann C.J. Tall A.R. Hogue M. Martin L. Milne R.W. Marcel Y.L. Plasma concentrations of cholesteryl ester transfer protein in hyperlipoproteinemia. Relation to cholesteryl ester transfer protein activity and other lipoprotein variables.Arterioscler. Thromb. 1991; 11: 797-804Google Scholar), was measured using exogenous substrates as previously described (21Berti J.A. Amaral M.E. Boschero A.C. Nunes V.S. Harada L.M. Castilho L.N. Oliveira H.C. Thyroid hormone increases plasma cholesteryl ester transfer protein activity and plasma high-density lipoprotein removal rate in transgenic mice.Metabolism. 2001; 50: 530-536Google Scholar). Lecithin cholesterol acyl transferase (LCAT) mediated cholesterol esterification reaction was measured using endogenous substrates (28Dobiasova M. Stribrna J. Pritchard P.H. Frohlich J.J. Cholesterol esterification rate in plasma depleted of very low and low density lipoproteins is controlled by the proportion of HDL2 and HDL3 subclasses: study in hypertensive and normal middle-aged and septuagenarian men.J. Lipid Res. 1992; 33: 1411-1418Google Scholar). Plasma phospholipid transfer protein (PLTP) activity was measured by the method of Albers et al. (29Albers J.J. Pitman W. Wolfbauer G. Cheung M.C. Kennedy H. Tu A.Y. Marcovina S.M. Paigen B. Relationship between phospholipid transfer protein activity and HDL level and size among inbred mouse strains.J. Lipid Res. 1999; 40: 295-301Google Scholar) that utilize exogenous substrates. Lipoproteins from the pooled plasma of mice were separated by fast protein liquid chromatography (FPLC) using a HR10/30 Superose 6 column (Amersham-Pharmacia Biotech., Uppsala, Sweden) as described (30Jiao S. Cole T.G. Kitchens R.T. Pfleger B. Schonfeld G. Genetic heterogeneity of lipoproteins in inbred strains of mice: analysis by gel-permeation chromatography.Metabolism. 1990; 39: 155-160Google Scholar). Total cholesterol (Chod-Pap, Merck S.A., São Paulo, Brazil) and triacylglycerols (Enz-Color, Biodiagnostica, Paraná, Brazil) were determined by enzymatic methods according to the manufacturer's instructions. Antibodies against holo-oxidized LDL (oxLDL) or antibodies anti-apoB epitope derived from oxLDL (apoB-D) were measured in mouse plasma by ELISA (31Damasceno N.R. Goto H. Rodrigues F.M. Dias C.T. Okawabata F.S. Abdalla D.S. Gidlund M. Soy protein isolate reduces the oxidizability of LDL and the generation of oxidized LDL autoantibodies in rabbits with diet-induced atherosclerosis.J. Nutr. 2000; 130: 2641-2647Google Scholar). ApoB-D is a 22 amino acid peptide from a region of apoB-100 not accessible to trypsin (32Boschcov P. Juliano L. Juliano M.A. Gidlund M. Development of a peptide-based ELISA for the detection of antibodies against oxidized low density lipoprotein (oxLDL).Atherosclerosis. 2000; 151: 224Google Scholar). Polystyrene microtiter plates (Costar, Cambridge, MA) were coated with 1 μg/ml of human oxLDL (20 mM Cu2+, 24 h) or 0.1 μg/ml of apoB-D in carbonate/bicarbonate buffer (20 μl/well), pH 9.4, and kept overnight at 4°C. The plates were blocked with a 5% solution of fat-free milk (Molico/Nestlé, SP, Brazil), and then incubated for 2 h at room temperature followed by washing four times with PBS (100 μl). Plasma samples (20 μl) were added and the plates were incubated overnight at 4°C followed by washing with 1% Tween 20 in PBS. A peroxidase-conjugated rabbit anti-mouse IgG antibody (20 μl; 1:1,500) was added, and after 1 h at room temperature, the plates were washed. Finally, 75 μl of substrate solution (250 mg of tetramethylbenzidine diluted in 50 ml of DMSO, 10 μl of 30% H2O2, 12 ml of citrate buffer, pH 5.5) were added and, after incubation at room temperature for 15 min, the reaction was stopped by adding 25 μl of 2.0 M sulfuric acid. The optical density (OD) was then measured in a microplate reader (Titertek Multiskan MCC/340P, model 2.20, Labsystems, Finland) at 450 nm. Results were expressed in relation to total plasma IgG concentration. For detection of total IgG levels, the plates were not treated with oxLDL but instead they were incubated with the individual plasma samples diluted 20,000× in carbonate buffer. The procedure then followed exactly as described above. Peritoneal macrophages were harvested from OV and estrogen treated OV CETP+ and CETP− mice in PBS (0.8% NaCl, 0.06% Na2HPO4, 0.02% KCl, and 0.04% KH2PO4), pH 7.4. Pelleted cells obtained after centrifugation at 500 g, 4°C for 3 min were resuspended at a final concentration of 3 × 106 cells/ml in RPMI 1640 medium containing 20% (v/v) fetal calf serum (FCS), penicillin (100 U/ml), streptomycin (100 U/ml), and fungizone (2.5 μg/ml). An aliquot of 0.5 ml was transferred into 24-well tissue culture plates and incubated in a humidified incubator with 5% CO2 atmosphere at 37°C. To remove non-adherent cells, after 2 h incubation, each plate was washed twice with RPMI 1640 medium without FCS and used for subsequent experiments. Control incubations in wells without cells were performed, and their values subtracted from the experimental values. Adhered macrophages were loaded with CE according to the method described by Brown et al. (33Brown M.S. Ho Y.K. Goldstein J.L. The cholesteryl ester cycle in macrophages foam cells. Continual hydrolysis and re-esterification of cytoplasmatic cholesteryl ester.J. Biol. Chem. 1980; 255: 9344-9352Google Scholar). Briefly, macrophages were incubated in RPMI 1640 medium containing 2 mg/ml fatty acid-free BSA in the presence of [14C]cholesteryl oleate-labeled acetylated LDL ([14C]CE-acLDL, 50 μg of protein/ml) for 24 h, and washed once with DMEM (Dulbelcco's Minimum Essential Medium) containing antibiotics. [14C]CE-acLDL loaded macrophages were incubated for 6 h with DMEM containing 2 mg/ml BSA in the presence of human HDL (100 μg protein /ml) as cellular cholesterol acceptor and the medium was collected for radioactivity counting (Ultima Gold Packard, Meriden, CT) in a β scintillation counter (LS6000-TA8, Beckman Instruments, Palo Alto, CA). Cells were washed with PBS and dissolved in 0.2 N NaOH for the measurement of the radioactivity that remained in the cells and protein content. Efflux was defined as the amount of radioactivity in the medium expressed as a percentage of that in the medium plus cells. Blank values were obtained by the incubation of labeled cells in medium containing only 2 mg/ml BSA and no lipoprotein. Adhered macrophages were incubated in RPMI 1640 medium containing 10% (v/v) lipoprotein deficient serum (3.5 mg protein/ml of medium) in the presence of acLDL labeled with [3H]cholesteryl oleoyl ether ([3H]CEt-acLDL), 50 μg of protein/ml, for 6 h at 37°C in a humidified incubator with 5% CO2 atmosphere. At the end of the incubation, cells were washed with PBS and solubilized in 0.2 N NaOH for the measurement of the cell-associated radioactivity and protein content. Cellular CEt uptake was defined as the amount of radioactivity in the cells expressed as a percentage of that offered to the cells per mg of cellular protein. In this study, we compared the plasma lipoprotein profiles, lipid transfer protein activities, oxLDL antibodies titers, and the extent of atherosclerotic lesions in control Sham, ovariectomized (OV), and estrogen treated OV mice that expressed the human CETP transgene or not. The baseline (chow diet) plasma lipid and lipoprotein profile determined 5 days after the surgery are shown on Table 1. Total cholesterol (TC) and triacylglycerol (TAG) concentrations were similar among all experimental groups. As expected, the HDL-cholesterol (HDL-C) concentrations were higher and LDL-C lower in CETP− compared with CETP+ mice in both Sham and ovariectomized groups.TABLE 1Plasma lipid and lipoprotein concentrations from CETP+ and CETP− mice 5 days after Sham and ovariectomy on a chow dietShamOvariectomizedCETP+CETP−CETP+CETP−mg/dlTC110 ± 10109 ± 10106 ± 14103 ± 16VLDL-C17 ± 416 ± 3 17 ± 315 ± 3LDL-C45 ± 1aP < 0.001 (Sham CETP+ vs. Sham CETP−; Ov CETP+ vs. OVCETP−).36 ± 1 43 ± 1aP < 0.001 (Sham CETP+ vs. Sham CETP−; Ov CETP+ vs. OVCETP−).34 ± 1HDL-C48 ± 3bP < 0.05 (Sham CETP+ vs. Sham CETP−; Ov CETP+ vs. OVCETP−).57 ± 4 46 ± 3bP < 0.05 (Sham CETP+ vs. Sham CETP−; Ov CETP+ vs. OVCETP−).54 ± 4Non-HDL-C62 ± 2bP < 0.05 (Sham CETP+ vs. Sham CETP−; Ov CETP+ vs. OVCETP−).52 ± 3 60 ± 3bP < 0.05 (Sham CETP+ vs. Sham CETP−; Ov CETP+ vs. OVCETP−).49 ± 4TAG61 ± 1459 ± 14 66 ± 1556 ± 16Mean ± SD (n = 8–12). TC, total cholesterol; TAG, triacylglycerol. The cholesterol distribution in the plasma lipoproteins was calculated as the area under the peaks of the FPLC profiles of pooled plasma samples (n = 3). Statistical comparisons by one way ANOVA followed by Student-Newman-Keuls test.a P < 0.001 (Sham CETP+ vs. Sham CETP−; Ov CETP+ vs. OVCETP−).b P < 0.05 (Sham CETP+ vs. Sham CETP−; Ov CETP+ vs. OVCETP−). Open table in a new tab Mean ± SD (n = 8–12). TC, total cholesterol; TAG, triacylglycerol. The cholesterol distribution in the plasma lipoproteins was calculated as the area under the peaks of the FPLC profiles of pooled plasma samples (n = 3). Statistical comparisons by one way ANOVA followed by Student-Newman-Keuls test. After 19 weeks on the atherogenic diet (Table 2), the TC concentrations rose in all groups. The TC and absolute cholesterol distributions in plasma lipoproteins were not different in Sham CETP+ and Sham CETP− mice, whereas TC, LDL-, HDL-, and non-HDL-C concentrations were significantly lower in OV CETP+ than in OV CETP− mice; however, the TC/HDL-C and non-HDL-C/HDL-C ratios, 3.0 and 2.0 respectively, were similar in all four groups. Plasma triacylglycerol (TAG) concentrations were not altered.TABLE 2Plasma lipid and lipoprotein concentrations, lipid transfer protein activities, oxLDL, and apoB-D antibodies titers in Sham and ovariectomized CETP+ and CETP− mice fed a high fat and high cholesterol diet for 19 weeksShamOvariectomizedCETP+CETP−CETP+CETP−mg/dlLipidsTC 194 ± 15200 ± 37227 ± 40 aP < 0.001 (OV CETP+ vs. Sham CETP+, Sham CETP−).266 ± 54 bP < 0.01 (OV CETP− vs. OV CETP+, Sham CETP+, Sham CETP−).VLDL-C 56 ± 460 ± 0.2 60 ± 372 ± 4 bP < 0.01 (OV CETP− vs. OV CETP+, Sham CETP+, Sham CETP−).LDL-C 73 ± 0.374 ± 3 90 ± 3 aP < 0.001 (OV CETP+ vs. Sham CETP+, Sham CETP−).100 ± 3 bP < 0.01 (OV CETP− vs. OV CETP+, Sham CETP+, Sham CETP−).HDL-C 65 ± 466 ± 3 77 ± 3 aP < 0.001 (OV CETP+ vs. Sham CETP+, Sham CETP−).94 ± 2 bP < 0.01 (OV CETP− vs. OV CETP+, Sham CETP+, Sham CETP−).Non-HDL-C 129 ± 4134 ± 3150 ± 3 aP < 0.001 (OV CETP+ vs. Sham CETP+, Sham CETP−).172 ± 2 bP < 0.01 (OV CETP− vs. OV CETP+, Sham CETP+, Sham CETP−).TAG 67 ± 1464 ± 14 55 ± 2544 ± 25%Protein activitiesCETP 48 ± 16— 55 ± 9—PLTP 21 ± 5.320 ± 1.3 19 ± 4.420 ± 1.3LCAT 5 ± 2 cP < 0.01 (Sham CETP+ vs. Sham CETP−).3 ± 1 14 ± 3 dP < 0.01 (OV CETP+ vs. OV CETP−, Sham CETP+, Sham CETP−).9 ± 3 eP < 0.001 (OV CETP− vs. Sham CETP+, Sham CETP−).AntibodiesoxLDL1.9 ± 0.8 1.7 ± 1.3 1.8 ± 1.1 2.7 ± 1.3 fP = 0.06 (OV CETP− vs. OV CETP+).ApoB-D1.9 ± 1.0 1.6 ± 0.9 1.2 ± 0.6 2.5 ± 1.0 bP < 0.01 (OV CETP− vs. OV CETP+, Sham CETP+, Sham CETP−).Mean ± SD (n = 8–12). The cholesterol distribution in the plasma lipoproteins was calculated as the area under the peaks of the FPLC profiles of pooled plasma samples (n = 3). CETP as a percentage of CE transfer over 2 h, PLTP as percentage of PL transfer over 1 h, and LCAT as percentage of CE formation over 30 min. Antibodies as optical density at 450 nm corrected by total IgG. Statistical comparisons by one way ANOVA followed by Student-Newman-Keuls test.a P < 0.001 (OV CETP+ vs. Sham CETP+, Sham CETP−).b P < 0.01 (OV CETP− vs. OV CETP+, Sham CETP+, Sham CETP−).c P < 0.01 (Sham CETP+ vs. Sham CETP−).d P < 0.01 (OV CETP+ vs. OV CETP−, Sham CETP+, Sham CETP−).e P < 0.001 (OV CETP− vs. Sham CETP+, Sham CETP−).f P = 0.06 (OV CETP− vs. OV CETP+). Open table in a new tab Mean ± SD (n = 8–12). The cholesterol distribution in the plasma lipoproteins was calculated as the area under the peaks of the FPLC profiles of pooled plasma samples (n = 3). CETP as a percentage of CE transfer over 2 h, PLTP as percentage of PL transfer over 1 h, and LCAT as percentage of CE formation over 30 min. Antibodies as optical density at 450 nm corrected by total IgG. Statistical comparisons by one way ANOVA followed by Student-Newman-Keuls test. The plasma activities of CETP, PLTP, and LCAT are also shown on Table 2. Neither CETP nor PLTP activities changed after ovariectomy. Thus, PLTP activity is not influenced by ovariectomy or by the expression of the CETP gene. In agreement with a previous report (34Oliveira H.C. Ma L. Milne R. Marcovina S.M. Inazu A. Mabuchi H. Tall A.R. Cholesteryl ester transfer protein activity enhances plasma cholesteryl ester formation. Studies in CETP transgenic mice and human genetic CETP deficiency.Arterioscler. Thromb. Vasc. Biol. 1997; 17: 1045-1052Google Scholar), the LCAT-dependent cholesterol esterification rate was higher in CETP+ than in CETP− mice in both Sham and OV mice. Comparison of the two ovariectomized groups with their respective Sham groups showed that ovariectomy markedly increased the plasma cholesterol esterification rate. This result is compatible with an increased cholesterol esterification rate shown in postmenopausal women (35Lewis-Barned N.J. Sutherland W.H. Walker R.J. Walker H.L. De Jong S.A. Edwards E.A. Markham V.H. Plasma cholesterol esterification and transfer, the menopause, and hormone replacement therapy in women.J. Clin. Endocrinol. Metab. 1999; 84: 3534-3538Google Scholar). Since estrogens protect LDL particles against oxidation (36Sack M.N. Rader D.J. Cannon 3rd R.O. Oestrogen and inhibition of oxidation of low
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