Artigo Acesso aberto Revisado por pares

Alirocumab inhibits atherosclerosis, improves the plaque morphology, and enhances the effects of a statin

2014; Elsevier BV; Volume: 55; Issue: 10 Linguagem: Inglês

10.1194/jlr.m051326

ISSN

1539-7262

Autores

Susan Kühnast, José W.A. van der Hoorn, Elsbet J. Pieterman, Anita M. van den Hoek, William J. Sasiela, Viktoria Gusarova, Anusch Peyman, Hans‐Ludwig Schäfer, Uwe Schwahn, J. Wouter Jukema, P. Hans,

Tópico(s)

Protein Kinase Regulation and GTPase Signaling

Resumo

Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition is a potential novel strategy for treatment of CVD. Alirocumab is a fully human PCSK9 monoclonal antibody in phase 3 clinical development. We evaluated the antiatherogenic potential of alirocumab in APOE*3Leiden.CETP mice. Mice received a Western-type diet and were treated with alirocumab (3 or 10 mg/kg, weekly subcutaneous dosing) alone and in combination with atorvastatin (3.6 mg/kg/d) for 18 weeks. Alirocumab alone dose-dependently decreased total cholesterol (−37%; −46%, P < 0.001) and TGs (−36%; −39%, P < 0.001) and further decreased cholesterol in combination with atorvastatin (−48%; −58%, P < 0.001). Alirocumab increased hepatic LDL receptor protein levels but did not affect hepatic cholesterol and TG content. Fecal output of bile acids and neutral sterols was not changed. Alirocumab dose-dependently decreased atherosclerotic lesion size (−71%; −88%, P < 0.001) and severity and enhanced these effects when added to atorvastatin (−89%; −98%, P < 0.001). Alirocumab reduced monocyte recruitment and improved the lesion composition by increasing the smooth muscle cell and collagen content and decreasing the macrophage and necrotic core content. Alirocumab dose-dependently decreases plasma lipids and, as a result, atherosclerosis development, and it enhances the beneficial effects of atorvastatin in APOE*3Leiden.CETP mice. In addition, alirocumab improves plaque morphology. Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition is a potential novel strategy for treatment of CVD. Alirocumab is a fully human PCSK9 monoclonal antibody in phase 3 clinical development. We evaluated the antiatherogenic potential of alirocumab in APOE*3Leiden.CETP mice. Mice received a Western-type diet and were treated with alirocumab (3 or 10 mg/kg, weekly subcutaneous dosing) alone and in combination with atorvastatin (3.6 mg/kg/d) for 18 weeks. Alirocumab alone dose-dependently decreased total cholesterol (−37%; −46%, P < 0.001) and TGs (−36%; −39%, P < 0.001) and further decreased cholesterol in combination with atorvastatin (−48%; −58%, P < 0.001). Alirocumab increased hepatic LDL receptor protein levels but did not affect hepatic cholesterol and TG content. Fecal output of bile acids and neutral sterols was not changed. Alirocumab dose-dependently decreased atherosclerotic lesion size (−71%; −88%, P < 0.001) and severity and enhanced these effects when added to atorvastatin (−89%; −98%, P < 0.001). Alirocumab reduced monocyte recruitment and improved the lesion composition by increasing the smooth muscle cell and collagen content and decreasing the macrophage and necrotic core content. Alirocumab dose-dependently decreases plasma lipids and, as a result, atherosclerosis development, and it enhances the beneficial effects of atorvastatin in APOE*3Leiden.CETP mice. In addition, alirocumab improves plaque morphology. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease involved in LDL metabolism (1Horton J.D. Cohen J.C. Hobbs H.H. Molecular biology of PCSK9: its role in LDL metabolism.Trends Biochem. Sci. 2007; 32: 71-77Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar). PCSK9, previously known as neutral apoptosis regulated convertase, is mainly expressed in the liver, kidney, and intestines (2Seidah N.G. Prat A. The biology and therapeutic targeting of the proprotein convertases.Nat. Rev. Drug Discov. 2012; 11: 367-383Crossref PubMed Scopus (564) Google Scholar, 3Abifadel M. Varret M. Rabes J.P. Allard D. Ouguerram K. Devillers M. Cruaud C. Benjannet S. Wickham L. Erlich D. et al.Mutations in PCSK9 cause autosomal dominant hypercholesterolemia.Nat. Genet. 2003; 34: 154-156Crossref PubMed Scopus (2180) Google Scholar). Besides familial hypercholesterolemia (FH) caused by ldlr mutations and familial defective apoB100 caused by apob mutations (1Horton J.D. Cohen J.C. Hobbs H.H. Molecular biology of PCSK9: its role in LDL metabolism.Trends Biochem. Sci. 2007; 32: 71-77Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar), autosomal dominant hypercholesterolemia can be caused by gain-of-function mutations in the pcsk9 gene, now commonly referred to as FH3 (3Abifadel M. Varret M. Rabes J.P. Allard D. Ouguerram K. Devillers M. Cruaud C. Benjannet S. Wickham L. Erlich D. et al.Mutations in PCSK9 cause autosomal dominant hypercholesterolemia.Nat. Genet. 2003; 34: 154-156Crossref PubMed Scopus (2180) Google Scholar, 4Lambert G. Sjouke B. Choque B. Kastelein J.J. Hovingh G.K. The PCSK9 decade.J. Lipid Res. 2012; 53: 2515-2524Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). Conversely, loss-of-function mutations of the pcsk9 gene were associated with a reduction in LDL cholesterol (LDL-C) and protection against coronary heart disease (5Benn M. Nordestgaard B.G. Grande P. Schnohr P. Tybjaerg-Hansen A. PCSK9 R46L, low-density lipoprotein cholesterol levels, and risk of ischemic heart disease: 3 independent studies and meta-analyses.J. Am. Coll. Cardiol. 2010; 55: 2833-2842Crossref PubMed Scopus (255) Google Scholar, 6Cohen J.C. Boerwinkle E. Mosley Jr, T.H. Hobbs H.H. Sequence variations in PCSK9, low LDL, and protection against coronary heart disease.N. Engl. J. Med. 2006; 354: 1264-1272Crossref PubMed Scopus (2420) Google Scholar). In addition, familial hypobetalipoproteinemia related to loss-of-function mutations of pcsk9 resulted in very low plasma levels of LDL-C, attributed to an increased clearance rate of LDL (7Cariou B. Ouguerram K. Zair Y. Guerois R. Langhi C. Kourimate S. Benoit I. Le May C. Gayet C. Belabbas K. et al.PCSK9 dominant negative mutant results in increased LDL catabolic rate and familial hypobetalipoproteinemia.Arterioscler. Thromb. Vasc. Biol. 2009; 29: 2191-2197Crossref PubMed Scopus (114) Google Scholar). Several studies have confirmed that PCSK9 is responsible for targeting the LDL receptor (LDLR) for lysosomal degradation in the liver by preventing recycling of the receptor to the cell membrane after internalization of the LDL-bound LDLR (4Lambert G. Sjouke B. Choque B. Kastelein J.J. Hovingh G.K. The PCSK9 decade.J. Lipid Res. 2012; 53: 2515-2524Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). PCSK9 interacts with the LDLR on the cell membrane, after which the LDLR-PCSK9 complex is internalized and travels through the endosome to the lysosome for degradation (8Lagace T.A. Curtis D.E. Garuti R. McNutt M.C. Park S.W. Prather H.B. Anderson N.N. Ho Y.K. Hammer R.E. Horton J.D. Secreted PCSK9 decreases the number of LDL receptors in hepatocytes and in livers of parabiotic mice.J. Clin. Invest. 2006; 116: 2995-3005Crossref PubMed Scopus (524) Google Scholar). In a study in mice, adenovirus-mediated expression of PCSK9 increased plasma LDL-C levels, which was associated with decreased hepatic LDLR protein, although LDLR mRNA levels were unaffected (9Maxwell K.N. Breslow J.L. Adenoviral-mediated expression of Pcsk9 in mice results in a low-density lipoprotein receptor knockout phenotype.Proc. Natl. Acad. Sci. USA. 2004; 101: 7100-7105Crossref PubMed Scopus (509) Google Scholar). On the contrary, mice lacking PCSK9 have decreased plasma LDL-C as a result of increased hepatic LDLR levels (10Rashid S. Curtis D.E. Garuti R. Anderson N.N. Bashmakov Y. Ho Y.K. Hammer R.E. Moon Y.A. Horton J.D. Decreased plasma cholesterol and hypersensitivity to statins in mice lacking Pcsk9.Proc. Natl. Acad. Sci. USA. 2005; 102: 5374-5379Crossref PubMed Scopus (561) Google Scholar). A recent study in wild-type, APOE−/−, and LDLR−/− mice with or without expression of PCSK9 revealed a direct relationship between PCSK9 and atherosclerosis development, mainly mediated via the LDLR, and suggests that PCSK9 inhibition will be beneficial in reducing atherosclerosis (11Denis M. Marcinkiewicz J. Zaid A. Gauthier D. Poirier S. Lazure C. Seidah N.G. Prat A. Gene inactivation of proprotein convertase subtilisin/kexin type 9 reduces atherosclerosis in mice.Circulation. 2012; 125: 894-901Crossref PubMed Scopus (172) Google Scholar). Although statins remain the most effective treatment option for CVD, there remains a substantial persistent cardiovascular risk, and, despite statin treatment, some patients cannot reach the recommended LDL-C target (12Davidson 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, 13Mihaylova B. Emberson J. Blackwell L. Keech A. Simes J. Barnes E.H. Voysey M. Gray A. Collins R. Baigent C. The effects of lowering LDL cholesterol with statin therapy in people at low risk of vascular disease: meta-analysis of individual data from 27 randomised trials.Lancet. 2012; 380: 581-590Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). Recent outcome studies and post hoc analyses indicate that therapeutic regimens that further lower LDL-C lead to further reductions in cardiovascular events (14Cannon C.P. Braunwald E. McCabe C.H. Rader D.J. Rouleau J.L. Belder R. Joyal S.V. Hill K.A. Pfeffer M.A. Skene A.M. Intensive versus moderate lipid lowering with statins after acute coronary syndromes.N. Engl. J. Med. 2004; 350: 1495-1504Crossref PubMed Scopus (4317) Google Scholar, 15LaRosa J.C. Grundy S.M. Waters D.D. Shear C. Barter P. Fruchart J.C. Gotto A.M. Greten H. Kastelein J.J. Shepherd J. et al.Intensive lipid lowering with atorvastatin in patients with stable coronary disease.N. Engl. J. Med. 2005; 352: 1425-1435Crossref PubMed Scopus (3015) Google Scholar, 16Ridker P.M. Danielson E. Fonseca F.A. Genest J. Gotto Jr, A.M. Kastelein J.J. Koenig W. Libby P. Lorenzatti A.J. MacFadyen J.G. et al.Rosuvastatin to prevent vascular events in men and women with elevated C-reactive protein.N. Engl. J. Med. 2008; 359: 2195-2207Crossref PubMed Scopus (5285) Google Scholar), and, consequently, cholesterol management guidelines have evolved to more rigorous goals (17Grundy S.M. Cleeman J.I. Merz C.N. Brewer Jr, H.B. Clark L.T. Hunninghake D.B. Pasternak R.C. Smith Jr, S.C. Stone N.J. Implications of recent clinical trials for the National Cholesterol Education Program Adult Treatment Panel III guidelines.Circulation. 2004; 110: 227-239Crossref PubMed Scopus (5069) Google Scholar, 18Reiner Z. Catapano A.L. De Backer G. Graham I. Taskinen M.R. Wiklund O. Agewall S. Alegria E. Chapman M.J. Durrington P. et al.ESC/EAS Guidelines for the management of dyslipidaemias: the Task Force for the management of dyslipidaemias of the European Society of Cardiology (ESC) and the European Atherosclerosis Society (EAS).Eur. Heart J. 2011; 32: 1769-1818Crossref PubMed Scopus (2541) Google Scholar, 19Expert Dyslipidemia Panel, Grundy SM. 2013. An International Atherosclerosis Society Position Paper: global recommendations for the management of dyslipidemia. J. Clin. Lipidol. 7: 561–565.Google Scholar). The upregulation of the LDLR after statin treatment is accompanied by an upregulation of PCSK9, which in turn promotes LDLR degradation (20Dubuc G. Chamberland A. Wassef H. Davignon J. Seidah N.G. Bernier L. Prat A. Statins upregulate PCSK9, the gene encoding the proprotein convertase neural apoptosis-regulated convertase-1 implicated in familial hypercholesterolemia.Arterioscler. Thromb. Vasc. Biol. 2004; 24: 1454-1459Crossref PubMed Scopus (517) Google Scholar, 21Mayne J. Dewpura T. Raymond A. Cousins M. Chaplin A. Lahey K.A. Lahaye S.A. Mbikay M. Ooi T.C. Chretien M. Plasma PCSK9 levels are significantly modified by statins and fibrates in humans.Lipids Health Dis. 2008; 7: 22Crossref PubMed Scopus (189) Google Scholar, 22Careskey H.E. Davis R.A. Alborn W.E. Troutt J.S. Cao G. Konrad R.J. Atorvastatin increases human serum levels of proprotein convertase subtilisin/kexin type 9.J. Lipid Res. 2008; 49: 394-398Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). In humans, the ∼35% to 50% decrease in LDL-C after atorvastatin treatment (10 to 40 mg) was accompanied by a ∼7% to 35% increase in circulating PCSK9 levels (21Mayne J. Dewpura T. Raymond A. Cousins M. Chaplin A. Lahey K.A. Lahaye S.A. Mbikay M. Ooi T.C. Chretien M. Plasma PCSK9 levels are significantly modified by statins and fibrates in humans.Lipids Health Dis. 2008; 7: 22Crossref PubMed Scopus (189) Google Scholar, 22Careskey H.E. Davis R.A. Alborn W.E. Troutt J.S. Cao G. Konrad R.J. Atorvastatin increases human serum levels of proprotein convertase subtilisin/kexin type 9.J. Lipid Res. 2008; 49: 394-398Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). Inhibition of PCSK9 is, therefore, a potential novel strategy for treatment of CVD, specifically in combination with statin treatment. Several approaches to inhibit PCSK9, including monoclonal antibodies, gene silencing, and mimetic peptides, are currently being investigated (4Lambert G. Sjouke B. Choque B. Kastelein J.J. Hovingh G.K. The PCSK9 decade.J. Lipid Res. 2012; 53: 2515-2524Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). The anti-PCSK9 monoclonal antibody alirocumab is a lead compound in this class and is currently being tested in phase 3 clinical trials. Alirocumab, also known as SAR236553/REGN727, is a fully human, monoclonal antibody that lowers plasma LDL-C in normocholesterolemic volunteers (23Stein E.A. Mellis S. Yancopoulos G.D. Stahl N. Logan D. Smith W.B. Lisbon E. Gutierrez M. Webb C. Wu R. et al.Effect of a monoclonal antibody to PCSK9 on LDL cholesterol.N. Engl. J. Med. 2012; 366: 1108-1118Crossref PubMed Scopus (578) Google Scholar) and hypercholesterolemic patients on stable statin dose (24Stein E.A. Gipe D. Bergeron J. Gaudet D. Weiss R. Dufour R. Wu R. Pordy R. Effect of a monoclonal antibody to PCSK9, REGN727/SAR236553, to reduce low-density lipoprotein cholesterol in patients with heterozygous familial hypercholesterolaemia on stable statin dose with or without ezetimibe therapy: a phase 2 randomised controlled trial.Lancet. 2012; 380: 29-36Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar, 25McKenney J.M. Koren M.J. Kereiakes D.J. Hanotin C. Ferrand A.C. Stein E.A. Safety and efficacy of a monoclonal antibody to proprotein convertase subtilisin/kexin type 9 serine protease, SAR236553/REGN727, in patients with primary hypercholesterolemia receiving ongoing stable atorvastatin therapy.J. Am. Coll. Cardiol. 2012; 59: 2344-2353Crossref PubMed Scopus (459) Google Scholar). In patients with hypercholesterolemia, alirocumab in combination with low- and high-dose atorvastatin decreased LDL-C to a greater extent than titration to high-dose atorvastatin, and considerably more patients who received the combination treatments reached LDL-C goals of <100 mg/dl or <70 mg/dl compared with patients who received atorvastatin treatment alone (26Roth E.M. McKenney J.M. Hanotin C. Asset G. Stein E.A. Atorvastatin with or without an antibody to PCSK9 in primary hypercholesterolemia.N. Engl. J. Med. 2012; 367: 1891-1900Crossref PubMed Scopus (429) Google Scholar). Phase 2 trials demonstrated reductions in LDL-C of 40% to 72% across a dose range of 50 to 150 mg administered every 2 weeks, and of 32% to 48% with doses 200 to 300 mg administered every 4 weeks (23Stein E.A. Mellis S. Yancopoulos G.D. Stahl N. Logan D. Smith W.B. Lisbon E. Gutierrez M. Webb C. Wu R. et al.Effect of a monoclonal antibody to PCSK9 on LDL cholesterol.N. Engl. J. Med. 2012; 366: 1108-1118Crossref PubMed Scopus (578) Google Scholar, 24Stein E.A. Gipe D. Bergeron J. Gaudet D. Weiss R. Dufour R. Wu R. Pordy R. Effect of a monoclonal antibody to PCSK9, REGN727/SAR236553, to reduce low-density lipoprotein cholesterol in patients with heterozygous familial hypercholesterolaemia on stable statin dose with or without ezetimibe therapy: a phase 2 randomised controlled trial.Lancet. 2012; 380: 29-36Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar, 25McKenney J.M. Koren M.J. Kereiakes D.J. Hanotin C. Ferrand A.C. Stein E.A. Safety and efficacy of a monoclonal antibody to proprotein convertase subtilisin/kexin type 9 serine protease, SAR236553/REGN727, in patients with primary hypercholesterolemia receiving ongoing stable atorvastatin therapy.J. Am. Coll. Cardiol. 2012; 59: 2344-2353Crossref PubMed Scopus (459) Google Scholar). The aim of this study was to investigate the effects of two dosages of alirocumab alone and in combination with atorvastatin on plasma lipids, atherosclerosis development, and lesion composition in APOE*3Leiden.CETP mice (27Westerterp 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 (180) Google Scholar). This is a well-established model for hyperlipidemia and atherosclerosis with all features of familial dysbetalipoproteinemia (FD) in humans, which is characterized by accumulation of remnant lipoproteins and an increased VLDL cholesterol to LDL-C ratio (28de 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). APOE*3Leiden mice have an impaired clearance of (V)LDL and increased TG levels and are thereby mimicking the slow clearance observed in humans, in contrast to normal wild-type mice, which have a very rapid clearance of apoB-containing lipoproteins (29van Vlijmen B.J. van den Maagdenberg A.M. Gijbels M.J. van der Boom H. HogenEsch H. Frants R.R. Hofker M.H. Havekes L.M. Diet-induced hyperlipoproteinemia and atherosclerosis in apolipoprotein E3-Leiden transgenic mice.J. Clin. Invest. 1994; 93: 1403-1410Crossref PubMed Scopus (220) Google Scholar). The lipoprotein profile in APOE*3Leiden.CETP mice reflects that of FD patients with a similar response to lipid-modifying therapies (30Zadelaar S. Kleemann R. Verschuren L. de Vries-van der Weij J. van der Hoorn J. Princen H.M. Kooistra T. Mouse models for atherosclerosis and pharmaceutical modifiers.Arterioscler. Thromb. Vasc. Biol. 2007; 27: 1706-1721Crossref PubMed Scopus (436) Google Scholar), including statins (31de 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 (32van 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 (33van 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), and cholesteryl ester transfer protein inhibitors (34de 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). This is illustrated by a comparable reduction in cholesterol in all apoB-containing lipoprotein subfractions with statin treatment (35Kawashiri M.A. Kobayashi J. Nohara A. Noguchi T. Tada H. Nakanishi C. Inazu A. Mabuchi H. Yamagishi M. Impact of bezafibrate and atorvastatin on lipoprotein subclass in patients with type III hyperlipoproteinemia: result from a crossover study.Clin. Chim. Acta. 2011; 412: 1068-1075Crossref PubMed Scopus (12) Google Scholar). We hypothesized that alirocumab alone could reduce progression of atherosclerosis and add to the atheroprotective effects of atorvastatin. Inhibition of atherosclerosis by atorvastatin in APOE*3Leiden.CETP mice has been observed previously (34de 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, 36Kühnast S. van der Hoorn J.W. van den Hoek A.M. Havekes L.M. Liau G. Jukema J.W. Princen H.M. Aliskiren inhibits atherosclerosis development and improves plaque stability in APOE*3Leiden.CETP transgenic mice with or without treatment with atorvastatin.J. Hypertens. 2012; 30: 107-116Crossref PubMed Scopus (22) Google Scholar). Ninety female APOE*3Leiden.CETP transgenic mice on a C57/bl6 background (9 to 13 weeks of age) (27Westerterp 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 (180) Google Scholar) received a semisynthetic cholesterol-rich diet for a run-in period of 3 weeks. After matching based on body weight, plasma total cholesterol (TC), TG, and age, mice (n = 15 per group) received a Western-type diet (WTD) alone or were treated with two dosages of alirocumab (3 or 10 mg/kg) alone or in combination with atorvastatin (3.6 mg/kg/d) for 18 weeks, and an arm with atorvastatin alone was added. Alirocumab (provided by Regeneron) was administered via weekly subcutaneous injections. All animals were euthanized by CO2 inhalation. Livers and hearts were isolated to assess hepatic LDLR protein levels, lipid content, atherosclerosis development, and plaque composition. Animal experiments were approved by the Institutional Animal Care and Use Committee of The Netherlands Organization for Applied Research. Plasma TC and TG were determined every 2 to 4 weeks, and average plasma TC and TG were calculated by total exposure over number of weeks. Lipoprotein profiles for TC were measured after lipoprotein separation by fast protein LC (FPLC) (27Westerterp 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 (180) Google Scholar). Alirocumab levels were measured by a human Fc enzyme-linked immunosorbent assay. LDLR protein level was evaluated by Western blotting using goat anti-mouse LDLR and rabbit anti-goat HRP, or mouse anti-α-tubulin and horse anti-mouse HRP; blots were developed with West Femto Super Signal ECL (Thermo Scientific) and subjected to the Chemi-Doc-it imaging system. Intensities of protein bands were quantified using Image J software. Hearts were isolated, fixed in formalin, and embedded in paraffin. They were then sectioned perpendicular to the axis of the aorta, starting within the heart and working in the direction of the aortic arch. Once the aortic root was identified by the appearance of aortic valve leaflets and smooth muscle cells (SMCs) instead of collagen-rich tissue, serial cross-sections (5 µm thick with intervals of 50 µm) were taken and mounted on aminopropyl-triethoxy-silane (APES)-coated slides These sections were stained with hematoxylin-phloxine-saffron (HPS) for histological analysis. For each mouse, atherosclerosis was measured in four subsequent cross-sections. Each section consisted of three segments. The average total lesion area per cross-section was then calculated (36Kühnast S. van der Hoorn J.W. van den Hoek A.M. Havekes L.M. Liau G. Jukema J.W. Princen H.M. Aliskiren inhibits atherosclerosis development and improves plaque stability in APOE*3Leiden.CETP transgenic mice with or without treatment with atorvastatin.J. Hypertens. 2012; 30: 107-116Crossref PubMed Scopus (22) Google Scholar, 37Delsing D.J. Offerman E.H. van Duyvenvoorde W. van der Boom H. de Wit E.C. Gijbels M.J. van der Laarse A. Jukema J.W. Havekes L.M. Princen H.M. Acyl-CoA:cholesterol acyltransferase inhibitor avasimibe reduces atherosclerosis in addition to its cholesterol-lowering effect in ApoE*3-Leiden mice.Circulation. 2001; 103: 1778-1786Crossref PubMed Scopus (106) Google Scholar). For determination of lesion severity, the lesions were classified into five categories according to the American Heart Association classification (38Stary H.C. Chandler A.B. Dinsmore R.E. Fuster V. Glagov S. Insull Jr, W. Rosenfeld M.E. Schwartz C.J. Wagner W.D. Wissler R.W. A definition of advanced types of atherosclerotic lesions and a histological classification of atherosclerosis. A report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association.Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1512-1531Crossref PubMed Scopus (979) Google Scholar): 0) no lesion, I) early fatty streak, II) regular fatty streak, III) mild plaque, IV) moderate plaque, and V) severe plaque. The percentage of each lesion type was calculated, where type I–III lesions were classified as mild lesions, and type IV–V lesions were classified as severe lesions (36Kühnast S. van der Hoorn J.W. van den Hoek A.M. Havekes L.M. Liau G. Jukema J.W. Princen H.M. Aliskiren inhibits atherosclerosis development and improves plaque stability in APOE*3Leiden.CETP transgenic mice with or without treatment with atorvastatin.J. Hypertens. 2012; 30: 107-116Crossref PubMed Scopus (22) Google Scholar, 37Delsing D.J. Offerman E.H. van Duyvenvoorde W. van der Boom H. de Wit E.C. Gijbels M.J. van der Laarse A. Jukema J.W. Havekes L.M. Princen H.M. Acyl-CoA:cholesterol acyltransferase inhibitor avasimibe reduces atherosclerosis in addition to its cholesterol-lowering effect in ApoE*3-Leiden mice.Circulation. 2001; 103: 1778-1786Crossref PubMed Scopus (106) Google Scholar). Total plaque load in the thoracic aorta (from the aortic origin to the diaphragm) was determined after Oil Red O staining for lipids as described previously (39Verschuren L. Kleemann R. Offerman E.H. Szalai A.J. Emeis S.J. Princen H.M. Kooistra T. Effect of low dose atorvastatin versus diet-induced cholesterol lowering on atherosclerotic lesion progression and inflammation in apolipoprotein E*3-Leiden transgenic mice.Arterioscler. Thromb. Vasc. Biol. 2005; 25: 161-167Crossref PubMed Scopus (70) Google Scholar). In the aortic root, lesion composition was determined for the severe lesions (type IV–V) as a percentage of lesion area after immunostaining with anti-human α-actin (1:800; Monosan, Uden, The Netherlands) for SMCs and anti-mouse Mac-3 (1:25; BD Pharmingen, The Netherlands) for macrophages followed by sirius red staining for collagen. Necrotic area was measured in macrophage/collagen staining. (36Kühnast S. van der Hoorn J.W. van den Hoek A.M. Havekes L.M. Liau G. Jukema J.W. Princen H.M. Aliskiren inhibits atherosclerosis development and improves plaque stability in APOE*3Leiden.CETP transgenic mice with or without treatment with atorvastatin.J. Hypertens. 2012; 30: 107-116Crossref PubMed Scopus (22) Google Scholar, 37Delsing D.J. Offerman E.H. van Duyvenvoorde W. van der Boom H. de Wit E.C. Gijbels M.J. van der Laarse A. Jukema J.W. Havekes L.M. Princen H.M. Acyl-CoA:cholesterol acyltransferase inhibitor avasimibe reduces atherosclerosis in addition to its cholesterol-lowering effect in ApoE*3-Leiden mice.Circulation. 2001; 103: 1778-1786Crossref PubMed Scopus (106) Google Scholar, 40Kühnast 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). In each segment used for lesion quantification, the number of monocytes adhering to the endothelium and the numbers of T cells in the total aortic root area were counted after immunostaining with AIA 31240 antibody (1:1000; Accurate Chemical and Scientific, New York, NY) and CD3 (1:500; AbD Serotec, Oxford, UK), respectively. Rat anti-mouse CD54 antibody, GTX76543 (GeneTex Inc., San Antonio, TX) was used for immunostaining of intercellular adhesion molecule 1 (ICAM-1) (41Verschuren L. de Vries-van der Weij J. Zadelaar S. Kleemann R. Kooistra T. LXR agonist suppresses atherosclerotic lesion growth and promotes lesion regression in apoE*3Leiden mice: time course and mechanisms.J. Lipid Res. 2009; 50: 301-311Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Significance of differences between the groups was calculated nonparametrically using a Kruskal-Wallis test for independent samples, followed by a Mann-Whitney U test for independent samples. Linear regression analyses were used to assess correlations between variables. IBM SPSS Statistics 20 for Windows (SPSS, Chicago, IL) was used for statistical analyses. All groups were c

Referência(s)