Inhibition of CETP activity by torcetrapib reduces susceptibility to diet-induced atherosclerosis in New Zealand White rabbits
2007; Elsevier BV; Volume: 48; Issue: 6 Linguagem: Inglês
10.1194/jlr.m600332-jlr200
ISSN1539-7262
AutoresLee A. Morehouse, Eliot Sugarman, Patricia-Ann K. Bourassa, Thomas Sand, Francesca Zimetti, Feng Gao, George H. Rothblat, Anthony J. Milici,
Tópico(s)Diabetes, Cardiovascular Risks, and Lipoproteins
ResumoCholesteryl ester transfer protein (CETP) inhibitors increase high density lipoprotein-cholesterol (HDL-C) in animals and humans, but whether CETP inhibition will be antiatherogenic is still uncertain. We tested the CETP inhibitor torcetrapib in rabbits fed an atherogenic diet at a dose sufficient to increase HDL-C by at least 3-fold (207 ± 32 vs. 57 ± 6 mg/dl in controls at 16 weeks). CETP activity was inhibited by 70–80% throughout the study. Non-HDL-C increased in both groups, but there was no difference apparent by the study's end. At 16 weeks, aortic atherosclerosis was 60% lower in torcetrapib-treated animals (16.4 ± 3.4% vs. 39.8 ± 5.4% in controls) and aortic cholesterol content was reduced proportionally. Sera from a separate group of rabbits administered torcetrapib effluxed 48% more cholesterol from Fu5AH cells than did sera from control animals, possibly explaining the reduced aortic cholesterol content. Regression analyses indicated that lesion area in the torcetrapib-treated group was strongly correlated with the ratio of total plasma cholesterol to HDL-C but not with changes in other lipid or lipoprotein levels. CETP inhibition with torcetrapib retards atherosclerosis in rabbits, and the reduced lesion area is associated with increased levels of HDL-C. Cholesteryl ester transfer protein (CETP) inhibitors increase high density lipoprotein-cholesterol (HDL-C) in animals and humans, but whether CETP inhibition will be antiatherogenic is still uncertain. We tested the CETP inhibitor torcetrapib in rabbits fed an atherogenic diet at a dose sufficient to increase HDL-C by at least 3-fold (207 ± 32 vs. 57 ± 6 mg/dl in controls at 16 weeks). CETP activity was inhibited by 70–80% throughout the study. Non-HDL-C increased in both groups, but there was no difference apparent by the study's end. At 16 weeks, aortic atherosclerosis was 60% lower in torcetrapib-treated animals (16.4 ± 3.4% vs. 39.8 ± 5.4% in controls) and aortic cholesterol content was reduced proportionally. Sera from a separate group of rabbits administered torcetrapib effluxed 48% more cholesterol from Fu5AH cells than did sera from control animals, possibly explaining the reduced aortic cholesterol content. Regression analyses indicated that lesion area in the torcetrapib-treated group was strongly correlated with the ratio of total plasma cholesterol to HDL-C but not with changes in other lipid or lipoprotein levels. CETP inhibition with torcetrapib retards atherosclerosis in rabbits, and the reduced lesion area is associated with increased levels of HDL-C. apolipoprotein B area under the curve cholesteryl ester cholesteryl ester transfer protein free cholesterol fast protein liquid chromatography high density lipoprotein-cholesterol total plasma cholesterol High levels of high density lipoprotein-cholesterol (HDL-C) have been associated with a decreased incidence of coronary heart disease in epidemiological studies (1.Boden W.E. High-density lipoprotein cholesterol as an independent risk factor in cardiovascular disease: assessing the data from Framingham to the Veterans Affairs High-Density Lipoprotein Intervention Trial.Am. J. Cardiol. 2000; 86: 19L-22LAbstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar, 2.Gordon D.J. Probstfield J.L. Garrision R.J. Neaton J.D. Castelli W.P. Knoke Jr., J.D. Jacobs Jr., D.R. Bangdiwala S. Tyroler H.A. 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CETP is a plasma glycoprotein that transfers cholesteryl esters (CEs), triglycerides, and phospholipids among circulating lipoproteins (10.Barter P.J. Brewer Jr., H.B. Chapman M.J. Hennekens C.H. Rader D.J. Tall A.R. Cholesteryl ester transfer protein. A novel target for raising HDL and inhibiting atherosclerosis.Arterioscler. Thromb. Vasc. Biol. 2003; 23: 160-167Crossref PubMed Scopus (718) Google Scholar, 11.de Grooth G.J. Klerkx A.H.E.M. Stroes E.S.G. Stalenhoef A.F.H. Kastelein J.J.P. Kuivenhoven J.A. A review of CETP and its relation to atherosclerosis.J. Lipid Res. 2004; 45: 1967-1974Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). CETP transfers neutral lipids down concentration gradients; as such, the physiologically relevant direction of the transfer of CE is from the CE-enriched HDL fraction to non-HDL lipoproteins, with retrograde transfer of triglycerides. In the context of reverse cholesterol transport, the transfer of CE via CETP can divert HDL-CE from the direct, hepatic, specific uptake pathway to an indirect pathway for hepatic CE delivery involving the receptor-mediated uptake of apolipoprotein B (apoB)-containing lipoproteins. Under conditions in which hepatic apoB uptake is downregulated, CETP action results in a CE enrichment of non-HDL lipoproteins, which could contribute to atherogenesis. Perhaps because of these complexities, the introduction of the CETP transgene into mice of various genetic backgrounds has not yielded a consistent effect on atherosclerosis (12.Cazita P.M. Berti J.A. Aoki C. Gidlund M. Harada L.M. Nunes V.S. Quintão E.C.R. Oliveira H.C.F. Cholesteryl ester transfer protein expression attenuates atherosclerosis in ovariectomized mice.J Lipid Res. 2003; 44: 33-40Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 13.Hayek T. Masucci-Magoulas L. Jiang X. Walsh A. Rubin E. Breslow J.L. Tall A.R. 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Med. 1998; 27: 659-667Crossref PubMed Scopus (162) Google Scholar). Therefore, although higher levels of HDL-C are associated with reduced atherosclerotic disease epidemiologically and HDL levels are increased in individuals with low CETP activity, some of the data associated with CETP deficiency suggest that it might not be associated with reduced atherosclerosis. To examine this experimentally, several groups of investigators have used the cholesterol-fed rabbit, a species that naturally expresses CETP, and have modulated CE transfer activity via antisense oligonucleotides (29.Sugano 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: 5033-5036Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar), vaccine (30.Rittershaus C.W. Miller D.P. Thomas L.J. Picard M.D. Honan C.M. 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In general, decreasing CETP expression or activity has been reported to be antiatherogenic, but taken as a whole, the results have not been convincing, especially with regard to whether HDL increase played a significant role in the reported antiatherogenic effect. In the majority of these studies, there were greater absolute decreases in non-HDL-C fractions than increases in HDL-C. In the only report claiming no effect of CETP inhibition on lesion formation, non-HDL-C levels were significantly higher than in the other studies and not significantly decreased in the CETP inhibitor group (31.Huang Z. Inazu A. Nohara A. Higashikata T. Mabuchi H. Cholesteryl ester transfer protein inhibitor (JTT-705) and the development of atherosclerosis in rabbits with severe hypercholesterolaemia.Clin. Sci. 2002; 103: 587-594Crossref PubMed Scopus (109) Google Scholar), prompting the question of whether the reduction of atherosclerosis with CETP inhibition was attributable to the increase of HDL-C or the reduction of non-HDL-C levels (33.Parini P. Rudel L.L. Is there a need for cholesteryl ester transfer protein inhibition?.Arterioscler. Thromb. Vasc. Biol. 2003; 23: 374-375Crossref PubMed Scopus (24) Google Scholar). To address this question, we tested torcetrapib, a robust CETP inhibitor, in the cholesterol-fed rabbit, achieving a multiple-fold increase in HDL-C without a significant decrease in non-HDL-C levels. Male New Zealand White rabbits (1.5–1.75 kg; Covance, Denver, PA) were housed at Pfizer, an American Association of Laboratory Animal Care-accredited facility, and an in-house committee reviewed all experimental procedures for adherence to ethical treatment standards. A scheme of the design of the atherosclerosis study is shown in Fig. 1 . Rabbits (n = 47) were fed an atherogenic diet (0.2% cholesterol, 10% coconut oil, and 1.2% ethyl lactate) (Harlan-Teklad, Madison, WI) for 5 days and assigned to one of the treatment groups based on their total plasma cholesterol (TPC) responses (n = 23 or 24 per group). Control animals continued consuming the cholesterol-containing diet for an additional 16 weeks; the treated group was fed the same diet but with increasing amounts of torcetrapib [0.15, 0.3, and 0.6% (w/w), 1 week at each dose] to identify the dose necessary to achieve at least 3-fold increases of HDL. Despite the fact that the targeted level of HDL-C increase was achieved at the 0.15% dose level, the dose-escalation phase of the experiment was completed as initially planned. Because of the known variability of rabbits to an atherogenic diet, we were unsure that the starting dose of torcetrapib would maintain HDL-C levels at greater than three times control levels throughout the study. After dose escalation, rabbits were returned to the 0.15% dose level for the remaining 13 weeks of the study. This level of dietary feeding equated to ∼90 mg/kg/day torcetrapib at the start of the study, gradually declining as the rabbits gained weight to 60–65 mg/kg/day by the end of the study. There was no difference in body weight or food consumption in torcetrapib-treated rabbits relative to controls (data not shown). In preliminary studies to gauge the extent of atherosclerosis in rabbits fed this diet, the non-HDL-C levels in ∼10–15% of the rabbits began declining after 8–10 weeks, and those animals with the lowest non-HDL-C (<150 mg/dl) had no aortic lesions at 16 weeks. It was not possible to identify these abnormal responders before the start of the study, because their initial response to an atherogenic diet was not predictive of subsequent hyporesponsiveness. Because the goal of this study was to determine whether CETP inhibition was antiatherogenic, including animals with no aortic atherosclerosis in either treatment group would only serve to increase the variability in each group. Thus, before the start of the study, we chose to prospectively identify these rabbits by measuring non-HDL-C at 14 weeks and to exclude any animals whose non-HDL-C did not exceed this 150 mg/dl threshold. Torcetrapib treatment did not affect the number of animals exhibiting this atypical response (four treated vs. three controls). One control animal expired at 6 weeks as a result of a congenital heart valve defect, so the total number of rabbits completing the study was 39 (19 controls and 20 torcetrapib-treated). Rabbits were anesthetized with intravenous pentobarbital (50 mg/kg), and aortas were perfused in situ with PBS followed by 10% formalin, excised, and stored in 1% gum arabic and 30% sucrose at 4°C overnight before returning them to 10% formalin. After removal of adventitia and connective tissue, aortic lesion area was quantitated en face by individuals blinded to treatment. Aortas were opened longitudinally and mounted on black foam boards, and sequential aortic images were captured using a Spot Camera (Diagnostic Instruments, Sterling Heights, MI). Digital images were merged using AnalySIS software (Soft Imaging Systems, Lakewood, CO). A preliminary test evaluating the aortic lesion areas in the same aortas had revealed no differences in lesion quantitation regardless of whether the aortas were analyzed unstained or stained with Sudan IV, so aortas were analyzed unstained, because this also enabled subsequent aortic lipid analyses. Unstained lesions were opaque, raised, and easily distinguishable from surrounding unlesioned areas. Animals were bled from the marginal ear vein weekly during the first 2 weeks of dose escalation and approximately monthly thereafter. CE transfer activity was estimated using a fluorescent transfer assay. It was necessary to use this assay instead of the typical radiolabel transfer assays because the non-HDL fractions in the extremely hyperlipidemic samples did not precipitate after low-spin centrifugation; thus, it was difficult to determine the level of CE transfer activity by conventional methods. BODIPY-CE and apoA-I-containing emulsion particles were generated by a direct sonication procedure (34.Lloyd D.B. Lira M.E. Wood L.S. Durham L.K. Freeman T.B. Preston G.M. Qiu X. Sugarman E. Bonnette P. Lanzetti A. et al.Cholesteryl ester transfer protein variants have differential stability but uniform inhibition by torcetrapib.J. Biol. Chem. 2005; 280: 14918-14922Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Briefly, 7 mg of phosphatidylcholine and 0.75 mg of triolein (Avanti Polar Lipids, Alabaster, AL) were mixed with 3 mg of BODIPY-CE (Invitrogen, Carlsbad, CA) in chloroform and vacuum-dried at 60°C. Lipids were solubilized at 65°C in phosphate-buffered saline by sonication for 2 min under a stream of nitrogen. The preparation was cooled to 45°C, and 5 mg of apoA-I (Biodesign, Saco, ME) was added. The preparation was resonicated (at 25% of full power) for 20 min at 45°C, pausing after each minute to cool the probe. The sonicate was spun for 30 min at 3,000 g, adjusted to 1.12 g/ml using NaBr, and layered below a solution of 1.10 g/ml NaBr. After spinning for 48 h at 100,000 g, any unemulsified lipid, unincorporated protein, and small dense particles were discarded. The fluorescently labeled particles were dialyzed in phosphate-buffered saline with 0.02% (w/v) sodium azide. To assay for CETP activity in isolated plasma samples, fluorescent donor particles were incubated in PBS with 20% plasma at 37°C, and the transfer of BODIPY-CE to endogenous lipoproteins was monitored at 485 nm excitation and 520 nm emission in a Gemini plate reader (Molecular Devices, Sunnyvale, CA). Inhibition of transfer was calculated by comparing the plasma CE transfer rate from each treated rabbit to the mean CE transfer rate observed in the control group. This assay had been characterized in numerous experiments, including those in which the biotinylated, fluorescent donor particles were incubated with human plasma and the appearance of BODIPY-CE in the endogenous lipoprotein fraction was monitored. After precipitation of biotinylated donor particles with streptavidin beads, BODIPY-CE was present in all plasma lipoprotein classes, and its accumulation in endogenous lipoproteins was both time- and CETP-dependent. Side-by-side comparisons of this fluorometric assay with a radiolabeled assay using human and rabbit plasma yielded excellent correlations between the two methods (r = 0.98 and 0.91 for humans and rabbits, respectively). Lipoprotein cholesterol content was determined using gel filtration on a fast protein liquid chromatography (FPLC) unit (Gilson, Middleton, WI) equipped with in-line, postcolumn cholesterol detection (35.März W. Siekmeier R. Scharnagl H. Seiffert U.B. Gross W. Fast lipoprotein chromatography: new method of analysis for plasma lipoproteins.Clin. Chem. 1993; 39: 2276-2281Crossref PubMed Scopus (51) Google Scholar). ApoA-I was determined using an immunoturbidity assay (Sigma-Aldrich, St. Louis, MO) with human apoA-I standards on a Roche-Hitachi autoanalyzer (Roche Diagnostics, Indianapolis, IN). Aortic free cholesterol (FC) and CE contents were determined by Lipomics (West Sacramento, CA) (36.Watkins S.M. Reifsnyder P.R. Pan H. German J.B. Leiter E.H. Lipid metabolome-wide effects of the PPAR-γ agonist rosiglitazone.J. Lipid Res. 2002; 43: 1809-1817Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). Triglycerides were measured using a kit (Wako Chemical, Richmond, VA) adapted to a microtiter plate format. Plasma levels of torcetrapib were measured as described previously (37.Lee S.D. Wasan K.M. Calcagni A. Avery M. McCush F. Chen C. The in vitro plasma distribution of a novel cholesteryl ester transfer protein inhibitor, torcetrapib, is influenced by differences in plasma lipid concentrations.Pharm. Res. 2006; 23: 1025-1030Crossref PubMed Scopus (5) Google Scholar). Sera were isolated from separate groups of rabbits fed the atherogenic diet with or without torcetrapib for 2 weeks. The cholesterol efflux potential of the sera was determined using an in vitro assay similar to one that has been described previously (38.Zimetti F. Weibel G.K. Duong M. Rothblat G.H. Measurement of cholesterol bidirectional flux between cells and lipoproteins.J. Lipid Res. 2006; 47: 605-613Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Fu5AH hepatoma cells were maintained on MEM (Mediatech Cellgro, Herndon, VA) containing 5% calf serum (Sigma-Aldrich) and gentamycin. Cells at 90% confluence were trypsinized and plated at 0.6 × 106 cells/well on 12-well plates. Cell number was determined using a Z1 Coulter Particle Counter (Hialeah, FL). The medium was supplemented with the ACAT inhibitor CP-113,818 (2 μg/ml) during labeling, equilibration, and the flux stages of the experiment. Cells were labeled for 24 h with [3H]cholesterol (Perkin-Elmer Analytical Sciences, Boston, MA) in medium supplemented with 2.5% calf serum. After an equilibration period in 0.2% BSA-containing medium, efflux was measured by incubating the cells with medium containing 2.5% rabbit sera for 8 h. To inhibit de novo cholesterol synthesis, mevinolin (5 μg/ml) was added to the efflux medium. Cholesterol efflux was measured by the release of [3H]cholesterol into the medium and expressed as a percentage of that previously incorporated into cell monolayers. Cholesterol efflux in this cell line is predominantly via a scavenger receptor class B type I-dependent mechanism (39.Yancey P.G. Kawashiri M.A. Moore R. Glick J.M. Williams D.L. Connelly M.A. Rader D.J. Rothblat G.H. In vivo modulation of HDL phospholipid has opposing effects on SR-BI- and ABCA1-mediated cholesterol efflux.J. Lipid Res. 2004; 45: 337-346Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Statistical analyses were done using LabStats, a Microsoft Excel add-in software package developed at Pfizer. The results were checked for consistency using a commercially available software package (MathSoft, Inc., Cambridge, MA). Area under the curve (AUC) values for lipid and lipoprotein parameters were calculated for each animal and used in linear regression analyses. Differences between the mean AUC values of lipid and lipoproteins in control and torcetrapib-treated groups were evaluated using Student's t-test on log-transformed data, except where noted. The first 3 weeks of this 16 week study consisted of a dose-escalation phase to select the dose of torcetrapib that resulted in a multiple-fold increase of HDL-C. This was achieved at the 0.15% dose level (∼90 mg/kg/day); the mean HDL-C in rabbits given 0.15% torcetrapib during the first week was 200 mg/dl, compared with a mean of ∼60 mg/dl for control rabbits. After completion of the dose-escalation phase, the treated rabbits were fed the 0.15% diet for the remaining 13 weeks of the experiment. This dose of torcetrapib resulted in a sustained inhibition of CE transfer throughout the experiment (Table 1); CE transfer was at least 70% inhibited throughout the duration of the study. Because blood was obtained just before the feeding the daily food ration, these transfer inhibition values in all likelihood represented a minimum, as plasma levels of torcetrapib and the resulting inhibition would be expected to have been greater throughout the remainder of the day.TABLE 1.Cholesteryl ester transfer protein activity and lipid, lipoprotein, and apolipoprotein valuesGroupWeek NumberPlasma TorcetrapibCE TransferTPCHDLNon-HDLTPC/HDL RatioTriglycerideApoA-IControl (n = 19)0ND1.16 ± 0.11122 ± 111ND1.67 ± 0.05204 ± 1957 ± 4148 ± 183.7 ± 0.353 ± 333 ± 22ND1.69 ± 0.04285 ± 3149 ± 3237 ± 316.3 ± 0.850 ± 534 ± 25.5ND3.68 ± 0.13430 ± 4948 ± 3382 ± 489.2 ± 1.065 ± 1030 ± 511ND2.60 ± 0.32524 ± 7255 ± 4469 ± 699.3 ± 1.1114 ± 1426 ± 416ND3.37 ± 0.12703 ± 10957 ± 6645 ± 10412.0 ± 1.395 ± 1627 ± 5 AUC6,240 ± 600700 ± 355,540 ± 580120 ± 101,100 ± 120420 ± 50Torcetrapib (n = 20)0ND1.32 ± 0.10116 ± 1010.85 ± 0.040.33 ± 0.01280 ± 18200 ± 1580 ± 91.4 ± 0.122 ± 246 ± 322.05 ± 0.240.34 ± 0.01401 ± 32255 ± 22145 ± 171.6 ± 0.127 ± 249 ± 25.51.81 ± 0.270.67 ± 0.05500 ± 66270 ± 35230 ± 392.0 ± 0.235 ± 371 ± 8111.42 ± 0.100.58 ± 0.05788 ± 113323 ± 51465 ± 702.8 ± 0.284 ± 1179 ± 9161.47 ± 0.110.99 ± 0.10897 ± 122207 ± 32690 ± 1045.3 ± 0.781 ± 1775 ± 12 AUC8,530 ± 1,0203,750 ± 4704,770 ± 63036 ± 2740 ± 8
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