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
ISSN1539-7262
AutoresSusan 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
ResumoProprotein 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. 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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. 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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
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