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Anacetrapib reduces (V)LDL cholesterol by inhibition of CETP activity and reduction of plasma PCSK9

2015; Elsevier BV; Volume: 56; Issue: 11 Linguagem: Inglês

10.1194/jlr.m057794

ISSN

1539-7262

Autores

Sam J.L. van der Tuin, Susan Kühnast, Jimmy F.P. Berbée, Lars Verschuren, Elsbet J. Pieterman, Louis M. Havekes, José W.A. van der Hoorn, Patrick C.N. Rensen, J. Wouter Jukema, P. Hans, Ko Willems van Dijk, Yanan Wang,

Tópico(s)

Computational Drug Discovery Methods

Resumo

Recently, we showed in APOE*3-Leiden cholesteryl ester transfer protein (E3L.CETP) mice that anacetrapib attenuated atherosclerosis development by reducing (V)LDL cholesterol [(V)LDL-C] rather than by raising HDL cholesterol. Here, we investigated the mechanism by which anacetrapib reduces (V)LDL-C and whether this effect was dependent on the inhibition of CETP. E3L.CETP mice were fed a Western-type diet alone or supplemented with anacetrapib (30 mg/kg body weight per day). Microarray analyses of livers revealed downregulation of the cholesterol biosynthesis pathway (P < 0.001) and predicted downregulation of pathways controlled by sterol regulatory element-binding proteins 1 and 2 (z-scores −2.56 and −2.90, respectively; both P < 0.001). These data suggest increased supply of cholesterol to the liver. We found that hepatic proprotein convertase subtilisin/kexin type 9 (Pcsk9) expression was decreased (−28%, P < 0.01), accompanied by decreased plasma PCSK9 levels (−47%, P < 0.001) and increased hepatic LDL receptor (LDLr) content (+64%, P < 0.01). Consistent with this, anacetrapib increased the clearance and hepatic uptake (+25%, P < 0.001) of [14C]cholesteryl oleate-labeled VLDL-mimicking particles. In E3L mice that do not express CETP, anacetrapib still decreased (V)LDL-C and plasma PCSK9 levels, indicating that these effects were independent of CETP inhibition. We conclude that anacetrapib reduces (V)LDL-C by two mechanisms: 1) inhibition of CETP activity, resulting in remodeled VLDL particles that are more susceptible to hepatic uptake; and 2) a CETP-independent reduction of plasma PCSK9 levels that has the potential to increase LDLr-mediated hepatic remnant clearance. Recently, we showed in APOE*3-Leiden cholesteryl ester transfer protein (E3L.CETP) mice that anacetrapib attenuated atherosclerosis development by reducing (V)LDL cholesterol [(V)LDL-C] rather than by raising HDL cholesterol. Here, we investigated the mechanism by which anacetrapib reduces (V)LDL-C and whether this effect was dependent on the inhibition of CETP. E3L.CETP mice were fed a Western-type diet alone or supplemented with anacetrapib (30 mg/kg body weight per day). Microarray analyses of livers revealed downregulation of the cholesterol biosynthesis pathway (P < 0.001) and predicted downregulation of pathways controlled by sterol regulatory element-binding proteins 1 and 2 (z-scores −2.56 and −2.90, respectively; both P < 0.001). These data suggest increased supply of cholesterol to the liver. We found that hepatic proprotein convertase subtilisin/kexin type 9 (Pcsk9) expression was decreased (−28%, P < 0.01), accompanied by decreased plasma PCSK9 levels (−47%, P < 0.001) and increased hepatic LDL receptor (LDLr) content (+64%, P < 0.01). Consistent with this, anacetrapib increased the clearance and hepatic uptake (+25%, P < 0.001) of [14C]cholesteryl oleate-labeled VLDL-mimicking particles. In E3L mice that do not express CETP, anacetrapib still decreased (V)LDL-C and plasma PCSK9 levels, indicating that these effects were independent of CETP inhibition. We conclude that anacetrapib reduces (V)LDL-C by two mechanisms: 1) inhibition of CETP activity, resulting in remodeled VLDL particles that are more susceptible to hepatic uptake; and 2) a CETP-independent reduction of plasma PCSK9 levels that has the potential to increase LDLr-mediated hepatic remnant clearance. Reduction in PCSK9 levels induced by anacetrapib: an off-target effect?Journal of Lipid ResearchVol. 56Issue 11PreviewInhibitors of proprotein convertase subtilisin/kexin type 9 (PCSK9) and cholesteryl ester transfer protein (CETP) are both under current investigation as agents with the potential to reduce atherosclerotic cardiovascular disease (ASCVD) risk. Inhibitors of PCSK9 reduce the concentration of LDL cholesterol by more than 60%, while inhibitors of CETP reduce LDL cholesterol by up to 45% and increase HDL cholesterol by up to 180%. Full-Text PDF Open Access High plasma levels of (V)LDL cholesterol [(V)LDL-C] and TGs, as well as low levels of HDL cholesterol (HDL-C), are important risk factors for cardiovascular diseases. The standard treatment for the reduction of cardiovascular disease risk is statin therapy aiming to reduce plasma (V)LDL-C. However, a substantial residual risk remains despite statin treatment. This has prompted the search for secondary treatment targets (1Baigent C. Blackwell L. Emberson J. Holland L.E. Reith C. Bhala N. Peto R. Barnes E.H. Keech A. Simes J. et al.Cholesterol Treatment Trialists' (CTT) Collaboration.Efficacy and safety of more intensive lowering of LDL cholesterol: a meta-analysis of data from 170,000 participants in 26 randomised trials. Lancet. 2010; 376: 1670-1681Google Scholar, 2Davidson M.H. Maki K.C. Pearson T.A. Pasternak R.C. Deedwania P.C. McKenney J.M. Fonarow G.C. Maron D.J. Ansell B.J. Clark L.T. et al.Results of the National Cholesterol Education (NCEP) Program Evaluation ProjecT Utilizing Novel E-Technology (NEPTUNE) II survey and implications for treatment under the recent NCEP Writing Group recommendations.Am. J. Cardiol. 2005; 96: 556-563Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). Prospective epidemiological studies indicate HDL-C as a potential target (3Di Angelantonio E. Sarwar N. Perry P. Kaptoge S. Ray K.K. Thompson A. Wood A.M. Lewington S. Sattar N. Packard C.J. et al.Major lipids, apolipoproteins, and risk of vascular disease.J. Am. Med. Assoc. 2009; 302: 1993-2000Crossref PubMed Scopus (1965) Google Scholar). The ratio of plasma (V)LDL-C to HDL-C is to a great extent affected by cholesteryl ester transfer protein (CETP). CETP facilitates the transfer of cholesteryl esters from HDL to (V)LDL in exchange for TG (4Tall A.R. Plasma cholesteryl ester transfer protein.J. Lipid Res. 1993; 34: 1255-1274Abstract Full Text PDF PubMed Google Scholar). In several mouse models, including C57Bl/6, Ldlr−/−, and APOE*3-Leiden (E3L) transgenic mice, CETP expression aggravates the development of atherosclerosis (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: 73-75Crossref PubMed Scopus (412) Google 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: 1105-1110Crossref PubMed Scopus (203) Google Scholar, 7Westerterp M. van der Hoogt C.C. de Haan W. Offerman E.H. Dallinga-Thie G.M. Jukema J.W. Havekes L.M. Rensen P.C. Cholesteryl ester transfer protein decreases high-density lipoprotein and severely aggravates atherosclerosis in APOE*3-Leiden mice.Arterioscler. Thromb. Vasc. Biol. 2006; 26: 2552-2559Crossref PubMed Scopus (179) Google Scholar). Although human studies have shown conflicting results with regard to the association between CETP deficiency and decreased cardiovascular disease risk (8de Grooth G.J. Zerba K.E. Huang S.P. Tsuchihashi Z. Kirchgessner T. Belder R. Vishnupad P. Hu B. Klerkx A.H. Zwinderman A.H. et al.The cholesteryl ester transfer protein (CETP) TaqIB polymorphism in the cholesterol and recurrent events study: no interaction with the response to pravastatin therapy and no effects on cardiovascular outcome: a prospective analysis of the CETP TaqIB polymorphism on cardiovascular outcome and interaction with cholesterol-lowering therapy.J. Am. Coll. Cardiol. 2004; 43: 854-857Crossref PubMed Scopus (67) Google Scholar, 9Ridker P.M. Pare G. Parker A.N. Zee R.Y. Miletich J.P. Chasman D.I. Polymorphism in the CETP gene region, HDL cholesterol, and risk of future myocardial infarction: genomewide analysis among 18 245 initially healthy women from the Women's Genome Health Study.Circ. Cardiovasc. Genet. 2009; 2: 26-33Crossref PubMed Scopus (163) Google Scholar), CETP inhibition is actively pursued as a potential strategy to reduce this risk (10Karalis I. Rensen P.C. Jukema J.W. Journey through cholesteryl ester transfer protein inhibition: from bench to bedside.Circ. Cardiovasc. Qual. Outcomes. 2013; 6: 360-366Crossref PubMed Scopus (11) Google Scholar). This has led to the development of pharmacological CETP inhibitors, such as torcetrapib, dalcetrapib, anacetrapib, and evacetrapib. In clinical trials, torcetrapib, anacetrapib, and evacetrapib have been shown to increase HDL-C [up to +72% (11Barter P.J. Caulfield M. Eriksson M. Grundy S.M. Kastelein J.J. Komajda M. Lopez-Sendon J. Mosca L. Tardif J.C. Waters D.D. et al.Effects of torcetrapib in patients at high risk for coronary events.N. Engl. J. Med. 2007; 357: 2109-2122Crossref PubMed Scopus (2605) Google Scholar), +139% (12Bloomfield D. Carlson G.L. Sapre A. Tribble D. McKenney J.M. Littlejohn III, T.W. Sisk C.M. Mitchel Y. Pasternak R.C. Efficacy and safety of the cholesteryl ester transfer protein inhibitor anacetrapib as monotherapy and coadministered with atorvastatin in dyslipidemic patients.Am. Heart J. 2009; 157: 352-360.e2Crossref PubMed Scopus (230) Google Scholar), and +129% (13Nicholls S.J. Brewer H.B. Kastelein J.J. Krueger K.A. Wang M.D. Shao M. Hu B. McErlean E. Nissen S.E. Effects of the CETP inhibitor evacetrapib administered as monotherapy or in combination with statins on HDL and LDL cholesterol: a randomized controlled trial.J. Am. Med. Assoc. 2011; 306: 2099-2109Crossref PubMed Scopus (368) Google Scholar), respectively] and to reduce LDL-C [down to −25% (11Barter P.J. Caulfield M. Eriksson M. Grundy S.M. Kastelein J.J. Komajda M. Lopez-Sendon J. Mosca L. Tardif J.C. Waters D.D. et al.Effects of torcetrapib in patients at high risk for coronary events.N. Engl. J. Med. 2007; 357: 2109-2122Crossref PubMed Scopus (2605) Google Scholar), −40% (12Bloomfield D. Carlson G.L. Sapre A. Tribble D. McKenney J.M. Littlejohn III, T.W. Sisk C.M. Mitchel Y. Pasternak R.C. Efficacy and safety of the cholesteryl ester transfer protein inhibitor anacetrapib as monotherapy and coadministered with atorvastatin in dyslipidemic patients.Am. Heart J. 2009; 157: 352-360.e2Crossref PubMed Scopus (230) Google Scholar), and −36% (13Nicholls S.J. Brewer H.B. Kastelein J.J. Krueger K.A. Wang M.D. Shao M. Hu B. McErlean E. Nissen S.E. Effects of the CETP inhibitor evacetrapib administered as monotherapy or in combination with statins on HDL and LDL cholesterol: a randomized controlled trial.J. Am. Med. Assoc. 2011; 306: 2099-2109Crossref PubMed Scopus (368) Google Scholar), respectively], whereas dalcetrapib only increased HDL-C [up to +40% (14Schwartz G.G. Olsson A.G. Abt M. Ballantyne C.M. Barter P.J. Brumm J. Chaitman B.R. Holme I.M. Kallend D. Leiter L.A. et al.Effects of dalcetrapib in patients with a recent acute coronary syndrome.N. Engl. J. Med. 2012; 367: 2089-2099Crossref PubMed Scopus (1554) Google Scholar)]. Although torcetrapib showed favorable effects on the lipoprotein profile, it failed in phase III clinical development due to increased risk of major cardiovascular events and mortality. These adverse effects were ascribed to an off-target effect (11Barter P.J. Caulfield M. Eriksson M. Grundy S.M. Kastelein J.J. Komajda M. Lopez-Sendon J. Mosca L. Tardif J.C. Waters D.D. et al.Effects of torcetrapib in patients at high risk for coronary events.N. Engl. J. Med. 2007; 357: 2109-2122Crossref PubMed Scopus (2605) Google Scholar) and proinflammatory lesions (15de Haan W. de Vries-van der Weij J. van der Hoorn J.W. Gautier T. van der Hoogt C.C. Westerterp M. Romijn J.A. Jukema J.W. Havekes L.M. Princen H.M. et al.Torcetrapib does not reduce atherosclerosis beyond atorvastatin and induces more proinflammatory lesions than atorvastatin.Circulation. 2008; 117: 2515-2522Crossref PubMed Scopus (80) Google Scholar). A large phase III clinical trial with dalcetrapib was prematurely terminated due to a lack of clinical benefit (14Schwartz G.G. Olsson A.G. Abt M. Ballantyne C.M. Barter P.J. Brumm J. Chaitman B.R. Holme I.M. Kallend D. Leiter L.A. et al.Effects of dalcetrapib in patients with a recent acute coronary syndrome.N. Engl. J. Med. 2012; 367: 2089-2099Crossref PubMed Scopus (1554) Google Scholar). Nonetheless, the effects of anacetrapib and evacetrapib on cardiovascular outcomes are currently being evaluated in phase III clinical trials (16U.S. National Institutes of Health. ClinicalTrials.gov. Accessed September 29, 2014, at http://www.clinicaltrials.gov.Google Scholar). Neither compound shows increased blood pressure as observed with torcetrapib (12Bloomfield D. Carlson G.L. Sapre A. Tribble D. McKenney J.M. Littlejohn III, T.W. Sisk C.M. Mitchel Y. Pasternak R.C. Efficacy and safety of the cholesteryl ester transfer protein inhibitor anacetrapib as monotherapy and coadministered with atorvastatin in dyslipidemic patients.Am. Heart J. 2009; 157: 352-360.e2Crossref PubMed Scopus (230) Google Scholar, 13Nicholls S.J. Brewer H.B. Kastelein J.J. Krueger K.A. Wang M.D. Shao M. Hu B. McErlean E. Nissen S.E. Effects of the CETP inhibitor evacetrapib administered as monotherapy or in combination with statins on HDL and LDL cholesterol: a randomized controlled trial.J. Am. Med. Assoc. 2011; 306: 2099-2109Crossref PubMed Scopus (368) Google Scholar), and both compounds are more potent in increasing HDL-C and reducing LDL-C as compared with torcetrapib and dalcetrapib. While regular C57BL/6J mice have a very rapid clearance of apoB-containing lipoproteins, E3L mice have an attenuated clearance and increased TG level, thereby mimicking the slower clearance observed in humans, particularly in patients with familial dyslipoproteinemia (17de Knijff P. van den Maagdenberg A.M. Stalenhoef A.F. Leuven J.A. Demacker P.N. Kuyt L.P. Frants R.R. Havekes L.M. Familial dysbetalipoproteinemia associated with apolipoprotein E3-Leiden in an extended multigeneration pedigree.J. Clin. Invest. 1991; 88: 643-655Crossref PubMed Scopus (88) Google Scholar). Similarly as in familial dyslipoproteinemia patients, in E3L and E3L.CETP mice as a model for mixed dyslipoproteinemia, a major part of plasma cholesterol is contained in the VLDL (remnant) particles, leading to the formation of β-VLDL particles, which further increases after cholesterol feeding. The E3L.CETP mouse model, unlike Apoe−/− and Ldlr−/− mice, responds in a human-like way to the lipid-lowering effects of statins (18de Haan W. van der Hoogt C.C. Westerterp M. Hoekstra M. Dallinga-Thie G.M. Princen H.M. Romijn J.A. Jukema J.W. Havekes L.M. Rensen P.C. Atorvastatin increases HDL cholesterol by reducing CETP expression in cholesterol-fed APOE*3-Leiden.CETP mice.Atherosclerosis. 2008; 197: 57-63Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), fibrates (19van der Hoogt C.C. de Haan W. Westerterp M. Hoekstra M. Dallinga-Thie G.M. Romijn J.A. Princen H.M. Jukema J.W. Havekes L.M. Rensen P.C. Fenofibrate increases HDL-cholesterol by reducing cholesteryl ester transfer protein expression.J. Lipid Res. 2007; 48: 1763-1771Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), niacin (20Kühnast S. Louwe M.C. Heemskerk M.M. Pieterman E.J. van Klinken J.B. van den Berg S.A. Smit J.W. Havekes L.M. Rensen P.C. van der Hoorn J.W. et al.Niacin reduces atherosclerosis development in APOE*3Leiden.CETP mice mainly by reducing nonHDL-cholesterol.PLoS One. 2013; 8: e66467Crossref PubMed Scopus (32) Google Scholar, 21van der Hoorn J.W. de Haan W. Berbee J.F. Havekes L.M. Jukema J.W. Rensen P.C. Princen H.M. Niacin increases HDL by reducing hepatic expression and plasma levels of cholesteryl ester transfer protein in APOE*3Leiden.CETP mice.Arterioscler. Thromb. Vasc. Biol. 2008; 28: 2016-2022Crossref PubMed Scopus (152) Google Scholar), torcetrapib (15de Haan W. de Vries-van der Weij J. van der Hoorn J.W. Gautier T. van der Hoogt C.C. Westerterp M. Romijn J.A. Jukema J.W. Havekes L.M. Princen H.M. et al.Torcetrapib does not reduce atherosclerosis beyond atorvastatin and induces more proinflammatory lesions than atorvastatin.Circulation. 2008; 117: 2515-2522Crossref PubMed Scopus (80) Google Scholar), anacetrapib (22Kühnast S. van der Tuin S.J. van der Hoorn J.W. van Klinken J.B. Simic B. Pieterman E. Havekes L.M. Landmesser U. Lüscher T.F. Willems van Dijk K. et al.Anacetrapib reduces progression of atherosclerosis, mainly by reducing non-HDL-cholesterol, improves lesion stability and adds to the beneficial effects of atorvastatin.Eur. Heart J. 2015; 36: 39-48Crossref PubMed Scopus (57) Google Scholar), and anti-PCSK9mabs (23Ason B. van der Hoorn J.W. Chan J. Lee E. Pieterman E.J. Nguyen K.K. Di M. Shetterly S. Tang J. Yeh W.C. et al.PCSK9 inhibition fails to alter hepatic LDLR, circulating cholesterol, and atherosclerosis in the absence of ApoE.J. Lipid Res. 2014; 55: 2370-2379Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), with respect to both direction and magnitude of the change. In conclusion, E3L.CETP mice have a more human-like lipoprotein metabolism when compared with C57Bl/6, Apoe−/−, or Ldlr−/− mice. Recently, we have shown that anacetrapib treatment increased HDL-C, reduced (V)LDL-C and TG, and dose-dependently reduced atherosclerotic lesion size and severity in E3L.CETP mice. ANCOVA showed that the effect on lesion size was mainly explained by a reduction in (V)LDL-C (22Kühnast S. van der Tuin S.J. van der Hoorn J.W. van Klinken J.B. Simic B. Pieterman E. Havekes L.M. Landmesser U. Lüscher T.F. Willems van Dijk K. et al.Anacetrapib reduces progression of atherosclerosis, mainly by reducing non-HDL-cholesterol, improves lesion stability and adds to the beneficial effects of atorvastatin.Eur. Heart J. 2015; 36: 39-48Crossref PubMed Scopus (57) Google Scholar). However, the mechanism by which anacetrapib reduces plasma (V)LDL-C and TG is not fully understood. To elucidate this, we performed microarrays on the livers from this latter study, identifying pathways affected by anacetrapib. To confirm physiological consequences of these identified pathways, we performed a VLDL production experiment and studied the clearance of VLDL-mimicking particles. By using E3L mice with or without CETP expression (7Westerterp M. van der Hoogt C.C. de Haan W. Offerman E.H. Dallinga-Thie G.M. Jukema J.W. Havekes L.M. Rensen P.C. Cholesteryl ester transfer protein decreases high-density lipoprotein and severely aggravates atherosclerosis in APOE*3-Leiden mice.Arterioscler. Thromb. Vasc. Biol. 2006; 26: 2552-2559Crossref PubMed Scopus (179) Google Scholar), we also determined whether these effects of anacetrapib were CETP dependent. Liver pieces were obtained from a previous experiment performed by Kühnast et al. (22Kühnast S. van der Tuin S.J. van der Hoorn J.W. van Klinken J.B. Simic B. Pieterman E. Havekes L.M. Landmesser U. Lüscher T.F. Willems van Dijk K. et al.Anacetrapib reduces progression of atherosclerosis, mainly by reducing non-HDL-cholesterol, improves lesion stability and adds to the beneficial effects of atorvastatin.Eur. Heart J. 2015; 36: 39-48Crossref PubMed Scopus (57) Google Scholar), investigating the effects of anacetrapib on atherosclerosis in female E3L.CETP mice (22Kühnast S. van der Tuin S.J. van der Hoorn J.W. van Klinken J.B. Simic B. Pieterman E. Havekes L.M. Landmesser U. Lüscher T.F. Willems van Dijk K. et al.Anacetrapib reduces progression of atherosclerosis, mainly by reducing non-HDL-cholesterol, improves lesion stability and adds to the beneficial effects of atorvastatin.Eur. Heart J. 2015; 36: 39-48Crossref PubMed Scopus (57) Google Scholar). In this study, mice were treated with a semisynthetic cholesterol-rich diet, containing 15% (w/w) cacao butter, 1% corn oil, and 0.1% cholesterol (Western-type diet; AB-Diets, Woerden, The Netherlands) with or without anacetrapib (30 mg/kg body weight per day) for 21 weeks. Total RNA was extracted from these liver pieces using the Nucleospin RNAII kit (Macherey-Nagel) according to the manufacturer's protocol. The microarray, including quality control, RNA labeling, hybridization, and data extraction, was performed by ServiceXS B.V. (Leiden, The Netherlands). To perform real-time quantitative PCR (qPCR) for validation, RNA quality was verified by the lab-on-a-chip method using ExperionTM RNA StdSens analyses kit (Bio-Rad). Total RNA was reverse transcribed with iScript cDNA synthesis kit (Bio-Rad), and qPCR was performed using a CFX96TM Touch Real-Time PCR Detection System (Bio-Rad). Gene expression was normalized to beta-2 microglobulin and hypoxanthine-guanine phosphoribosyltransferase. Relative expression was calculated as compared with the control group using Bio-Rad CFX ManagerTM software 3.0 (Bio-Rad). The probe-level background subtracted expression values were used as input for lumi package (24Du P. Kibbe W.A. Lin S.M. lumi: a pipeline for processing Illumina microarray.Bioinformatics. 2008; 24: 1547-1548Crossref PubMed Scopus (1621) Google Scholar) of the R/Bioconductor (http://www.bioconductor.org; http://www.r-project.org) to perform quality control and a quantile normalization. Unexpressed probes (P > 0.01 in all experiments) were removed from further analyses. Differentially expressed probes were identified using the limma package of R/Bioconductor (25Wettenhall J.M. Smyth G.K. limmaGUI: a graphical user interface for linear modeling of microarray data.Bioinformatics. 2004; 20: 3705-3706Crossref PubMed Scopus (570) Google Scholar). The calculated P values 2 or <2 indicates significant activation or inhibition of a pathway or process. To investigate the effects of anacetrapib on VLDL production and clearance, female E3L (26van den Maagdenberg A.M. Hofker M.H. Krimpenfort P.J. de Bruijn I. van Vlijmen B. van der Boom H. Havekes L.M. Frants R.R. Transgenic mice carrying the apolipoprotein E3-Leiden gene exhibit hyperlipoproteinemia.J. Biol. Chem. 1993; 268: 10540-10545Abstract Full Text PDF PubMed Google Scholar) and E3L.CETP (7Westerterp M. van der Hoogt C.C. de Haan W. Offerman E.H. Dallinga-Thie G.M. Jukema J.W. Havekes L.M. Rensen P.C. Cholesteryl ester transfer protein decreases high-density lipoprotein and severely aggravates atherosclerosis in APOE*3-Leiden mice.Arterioscler. Thromb. Vasc. Biol. 2006; 26: 2552-2559Crossref PubMed Scopus (179) Google Scholar) transgenic mice, 8–10 weeks of age, were fed a Western-type diet for a run-in period of 3–4 weeks. They were then matched based on plasma total cholesterol (TC), HDL-C, TG, body weight, and age into two groups receiving either no treatment (control) or anacetrapib (30 mg/kg body weight per day) for 4 weeks, after which VLDL production (E3L.CETP only) or clearance (E3L and E3L.CETP) was determined. After both experiments, the mice were euthanized by CO2 asphyxiation. The mice were housed under standard conditions with a 12 h light-dark cycle and had free access to food and water during the experiment. Body weight and food intake were monitored during the study. The Institutional Ethics Committee for Animal Procedures from the Leiden University Medical Center, Leiden, The Netherlands, approved the protocol. Blood was collected after a 4 h fasting period in heparin-coated capillaries via tail vein bleeding, and plasma was isolated. TC and TG were determined using enzymatic kits (Roche) according to the manufacturer's protocol. To measure HDL-C, apoB-containing particles were precipitated from plasma with 20% polyethylene glycol 6000 (Sigma Aldrich) in 200 mM glycine buffer (pH 10), and HDL-C was measured in the supernatant (22Kühnast S. van der Tuin S.J. van der Hoorn J.W. van Klinken J.B. Simic B. Pieterman E. Havekes L.M. Landmesser U. Lüscher T.F. Willems van Dijk K. et al.Anacetrapib reduces progression of atherosclerosis, mainly by reducing non-HDL-cholesterol, improves lesion stability and adds to the beneficial effects of atorvastatin.Eur. Heart J. 2015; 36: 39-48Crossref PubMed Scopus (57) Google Scholar). The distribution of cholesterol over plasma lipoproteins was determined by fast-performance liquid chromatography as previously described (7Westerterp M. van der Hoogt C.C. de Haan W. Offerman E.H. Dallinga-Thie G.M. Jukema J.W. Havekes L.M. Rensen P.C. Cholesteryl ester transfer protein decreases high-density lipoprotein and severely aggravates atherosclerosis in APOE*3-Leiden mice.Arterioscler. Thromb. Vasc. Biol. 2006; 26: 2552-2559Crossref PubMed Scopus (179) Google Scholar). Snap-frozen mouse livers were lysed in ice-cold lysis buffer containing 50 mM HEPES (pH 7.6), 50 mM NaF, 50 mM KCl, 5 mM NaPPi, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 5 mM β-glycerophosphate, 1 mM sodium vanadate, 1% NP40, and protease inhibitors cocktail (Roche). Thereafter, protein level was determined using the BCA Protein Assay Kit (Pierce) according to the manufacturer's instructions. Laemmli buffer (Sigma-Aldrich) was added to samples containing equal amounts of protein. Samples were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and blotted to polyvinylidene difluoride. Blots were incubated with goat-anti-mouse LDL receptor (LDLr; 1:1,000, AF2255, R and D Systems), rabbit-anti-mouse low density lipoprotein receptor-related protein 1 (LRP1; 1:20,000, ab92544, Abcam), and mouse-anti-α-tubulin (1:1,000, T5168, Sigma-Aldrich) and subsequently incubated with the appropriate secondary antibody. Bands were visualized by enhanced chemiluminescence with Pierce ECL 2 substrate following the manufacturer's protocol and quantified using Image J software as previously described (27Kühnast S. van der Hoorn J.W. Pieterman E.J. van den Hoek A.M. Sasiela W.J. Gusarova V. Peyman A. Schäfer H.L. Schwahn U. Jukema J.W. et al.Alirocumab inhibits atherosclerosis, improves the plaque morphology, and enhances the effects of a statin.J. Lipid Res. 2014; 55: 2103-2112Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Plasma proprotein convertase subtilisin/kexin type 9 (PCSK9) was measured by using ELISA (MCP900, R and D Systems) according to the manufacturer's instructions. Mice (n = 8/9 per group) were anesthetized with 6.25 mg/kg acepromazine (Alfasan), 6.25 mg/kg midazolam (Roche), and 0.31 mg/kg fentanyl (Janssen-Cilag) after a 4 h fast. A basal blood sample was taken from the tail, and the mice received an intravenous injection of 100 µl PBS containing 100 µCi Tran35S label (MP Biomedicals) via the tail vein. After 30 min, animals received an intravenous injection of Tyloxapol (Triton WR-1339, Sigma-Aldrich; 500 mg/kg body weight), as a 10% (w/w) solution in sterile saline, to prevent systemic lipolysis of newly secreted hepatic VLDL-TG (28Aalto-Setälä K. Fisher E.A. Chen X. Chajek-Shaul T. Hayek T. Zechner R. Walsh A. Ramakrishnan R. Ginsberg H.N. Breslow J.L. Mechanism of hypertriglyceridemia in human apolipoprotein (apo) CIII transgenic mice. Diminished very low density lipoprotein fractional catabolic rate associated with increased apo CIII and reduced apo E on the particles.J. Clin. Invest. 1992; 90: 1889-1900Crossref PubMed Scopus (400) Google Scholar). At indicated time points up to 90 min after Tyloxapol injection, blood was taken and plasma TG concentration was determined. After 120 min, mice were euthanized, and blood was collected for isolation of the VLDL fraction by density gradient ultracentrifugation (29Redgrave T.G. Roberts D.C. West C.E. Separation of plasma lipoproteins by density-gradient ultracentrifugation.Anal. Biochem. 1975; 65: 42-49Crossref PubMed Scopus (870) Google Scholar). Tran35S activity was measured in the VLDL fraction, and VLDL-apoB production rate was calculated as dpm/h (30Li X. Catalina F. Grundy S.M. Patel S. Method to measure apolipoprotein B-48 and B-100 secretion rates in an individual mouse: evidence for a very rapid turnover of VLDL and preferential removal of B-48- relative to B-100-containing lipoproteins.J. Lipid Res. 1996; 37: 210-220Abstract Full Text PDF PubMed Google Scholar). Glycerol tri(9,10(n)[3H])oleate ([3H]TO) and [1α,2α (n)-14C]cholesteryl oleate ([14C]CO) double-radiolabeled VLDL-mimicking particles (mean diameter 80 nm) were prepared as previously described (31Rensen P.C. van Dijk M.C. Havenaar E.C. Bijsterbosch M.K. Kruijt J.K. van Berkel T.J. Selective liver targeting of antivirals by recombinant chylomicrons–a new therapeutic approach to hepatitis B.Nat. Med. 1995; 1: 221-225Crossref PubMed Scopus (133) Google Scholar). In short, [3H]TO (100 µCi) and [14C]CO (10 µCi) tracers were added to a mixture of TO (70 mg), egg yolk phosphatidylcholine (22.7 mg), lysophosphatidylcholine (2.3 mg), CO (3.0 mg), and cholesterol (2.0 mg). Particles were prepared from this mixture at 54°C using a Soniprep 150 (MSE Scientific Instruments) at 10 µm output. After two consecutive density gradient ultracentrifugation steps, VLDL-mimicking particles (average size of 80 nm) were isolated. The particles were stored at 4°C under argon and intravenously injected into mice within 5 days. These VLDL-mimicking particles acquire a wide range on interchangeable apolipoproteins from serum

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