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PCSK9 inhibition fails to alter hepatic LDLR, circulating cholesterol, and atherosclerosis in the absence of ApoE

2014; Elsevier BV; Volume: 55; Issue: 11 Linguagem: Inglês

10.1194/jlr.m053207

ISSN

1539-7262

Autores

Brandon Ason, José W.A. van der Hoorn, Joyce Chan, Edward Lee, Elsbet J. Pieterman, Kathy Nguyen, Mei Di, Susan Shetterly, Jie Tang, Wen‐Chen Yeh, Margrit Schwarz, J. Wouter Jukema, Robert C. Scott, Scott M. Wasserman, P. Hans, Simon Jackson,

Tópico(s)

Diabetes, Cardiovascular Risks, and Lipoproteins

Resumo

LDL cholesterol (LDL-C) contributes to coronary heart disease. Proprotein convertase subtilisin/kexin type 9 (PCSK9) increases LDL-C by inhibiting LDL-C clearance. The therapeutic potential for PCSK9 inhibitors is highlighted by the fact that PCSK9 loss-of-function carriers exhibit 15–30% lower circulating LDL-C and a disproportionately lower risk (47–88%) of experiencing a cardiovascular event. Here, we utilized pcsk9−/− mice and an anti-PCSK9 antibody to study the role of the LDL receptor (LDLR) and ApoE in PCSK9-mediated regulation of plasma cholesterol and atherosclerotic lesion development. We found that circulating cholesterol and atherosclerotic lesions were minimally modified in pcsk9−/− mice on either an LDLR- or ApoE-deficient background. Acute administration of an anti-PCSK9 antibody did not reduce circulating cholesterol in an ApoE-deficient background, but did reduce circulating cholesterol (−45%) and TGs (−36%) in APOE*3Leiden.cholesteryl ester transfer protein (CETP) mice, which contain mouse ApoE, human mutant APOE3*Leiden, and a functional LDLR. Chronic anti-PCSK9 antibody treatment in APOE*3Leiden.CETP mice resulted in a significant reduction in atherosclerotic lesion area (−91%) and reduced lesion complexity. Taken together, these results indicate that both LDLR and ApoE are required for PCSK9 inhibitor-mediated reductions in atherosclerosis, as both are needed to increase hepatic LDLR expression. LDL cholesterol (LDL-C) contributes to coronary heart disease. Proprotein convertase subtilisin/kexin type 9 (PCSK9) increases LDL-C by inhibiting LDL-C clearance. The therapeutic potential for PCSK9 inhibitors is highlighted by the fact that PCSK9 loss-of-function carriers exhibit 15–30% lower circulating LDL-C and a disproportionately lower risk (47–88%) of experiencing a cardiovascular event. Here, we utilized pcsk9−/− mice and an anti-PCSK9 antibody to study the role of the LDL receptor (LDLR) and ApoE in PCSK9-mediated regulation of plasma cholesterol and atherosclerotic lesion development. We found that circulating cholesterol and atherosclerotic lesions were minimally modified in pcsk9−/− mice on either an LDLR- or ApoE-deficient background. Acute administration of an anti-PCSK9 antibody did not reduce circulating cholesterol in an ApoE-deficient background, but did reduce circulating cholesterol (−45%) and TGs (−36%) in APOE*3Leiden.cholesteryl ester transfer protein (CETP) mice, which contain mouse ApoE, human mutant APOE3*Leiden, and a functional LDLR. Chronic anti-PCSK9 antibody treatment in APOE*3Leiden.CETP mice resulted in a significant reduction in atherosclerotic lesion area (−91%) and reduced lesion complexity. Taken together, these results indicate that both LDLR and ApoE are required for PCSK9 inhibitor-mediated reductions in atherosclerosis, as both are needed to increase hepatic LDLR expression. High levels of circulating LDL cholesterol (LDL-C) play a key role in the initiation and development of atherosclerosis. This contributes to the development of CVD and places patients at increased risk of experiencing an adverse cardiovascular event (1Steinberg D. The pathogenesis of atherosclerosis. An interpretive history of the cholesterol controversy: part I.J. Lipid Res. 2004; 45: 1583-1593Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 2Steinberg D. The pathogenesis of atherosclerosis. 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Cardiol. 2014; 63: 2531-2540Crossref PubMed Scopus (327) Google Scholar, 42Hooper A.J. Burnett J.R. Anti-PCSK9 therapies for the treatment of hypercholesterolemia.Expert Opin. Biol. Ther. 2013; 13: 429-435Crossref PubMed Scopus (40) Google Scholar). ApoE, like ApoB, is present in lipoproteins and functions as a ligand of the LDLR and is important for the clearance of TG-rich lipoproteins. The decrease in HDL cholesterol (HDL-C) in pcsk9−/− mice has been attributed to the binding of ApoE containing HDL to the upregulated LDLR (11Rashid 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). Even with a functional LDLR and ApoB, mutations in APOE in humans can lead to hypercholesterolemia (43Awan Z. Choi H.Y. Stitziel N. Ruel I. Bamimore M.A. Husa R. Gagnon M.H. Wang R.H. 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Genetic heterogeneity in familial dysbetalipoproteinemia. The E2(lys146—-gln) variant results in a dominant mode of inheritance.J. Lipid Res. 1990; 31: 45-53Abstract Full Text PDF PubMed Google Scholar). To date the role of ApoE in the lipid lowering and athero-protective effects of PCSK9 inhibition is unclear. PCSK9 overexpression in an apoe-deficient background has been reported to be proatherogenic, while PCSK9 deletion in apoe-deficient mice leads to a reduction in the amount of cholesterol ester found within the aorta, even though the plaque size and total plasma cholesterol levels remain unchanged (47Denis 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). The contribution of cholesterol ester content to atherosclerotic lesion development in the absence of changes in lesion area are unknown, but these data hint that a functional ApoE-LDLR pathway is essential for PCSK9-mediated changes in atherosclerosis that are driven by decreases in plasma cholesterol. To investigate this, we utilized both genetically engineered knockout mice (pcsk9−/−) and an anti-PCSK9 antibody to examine the effect of PCSK9 inhibition on plasma lipoproteins and atherosclerotic lesion development in mice lacking the LDLR or ApoE, as well as in APOE*3Leiden.cholesteryl ester transfer protein (CETP) mice (47Denis 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), which have mouse ApoE and LDLR but hampered clearance of the ApoB-containing lipoproteins due to the expression of human mutant APOE*3Leiden (48van 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). The APOE*3Leiden.CETP mice are a well-established mouse model for familial dysbetalipoproteinemia with human-like lipoprotein metabolism and atherosclerosis development, which respond in a human-like manner to both lipid lowering as well as HDL-raising drugs (like statins, fibrates, niacin, etc.) used in the treatment of CVD (49Zadelaar 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, 50van 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, 51van 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, 52de 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). The fully human PCSK9-targeting antibody, mAb1, was generated as described previously (25Chan J.C. Piper D.E. Cao Q. Liu D. King C. Wang W. Tang J. Liu Q. Higbee J. Xia Z. et al.A proprotein convertase subtilisin/kexin type 9 neutralizing antibody reduces serum cholesterol in mice and nonhuman primates.Proc. Natl. Acad. Sci. USA. 2009; 106: 9820-9825Crossref PubMed Scopus (330) Google Scholar). Briefly, mice engineered to express human IgG antibodies were immunized with human PCSK9. Determination of binding affinity, screening for cross reactivity to mouse PCSK9, and activity in a cell-based LDL uptake assay led to mAb1 selection. cDNA sequences encoding the variable domains of heavy and light chains of mAb1 were fused to constant domains of mouse IgG1 heavy chain and mouse lambda light chain. The resulting cDNA sequences encoding the chimeric mAb1 (CmAb1) heavy chains and light chains were inserted into pTT5 expression plasmid separately. CmAb1 mouse IgG1 was expressed by cotransfecting 293 6E cells with pTT5 plasmids containing light chain and heavy chain sequences. Expressed chimeric antibody was purified by capturing on a MabSelect SuRe column and polished on a SP-Sepharose column as previously described (25Chan J.C. Piper D.E. Cao Q. Liu D. King C. Wang W. Tang J. Liu Q. Higbee J. Xia Z. et al.A proprotein convertase subtilisin/kexin type 9 neutralizing antibody reduces serum cholesterol in mice and nonhuman primates.Proc. Natl. Acad. Sci. USA. 2009; 106: 9820-9825Crossref PubMed Scopus (330) Google Scholar). Binding of mAb1 and CmAb1 to mouse PCSK9 was measured in a kinetic binding assay by BIAcore. Mouse anti-His antibody (Qiagen, Valencia, CA) was immobilized on all four flow cells of a CM5 chip using amine coupling reagents (GE Healthcare, Piscataway, NJ) with an approximate density of 5,000–6,000 RU. His-tagged PCSK9 was captured on the second and fourth flow cells at an approximate density of 130 RU for mouse PCSK9. Flow cells one and three were used as background controls. Anti-PCSK9 antibody at 100 nM was diluted in PBS plus 0.1 mg/ml BSA, 0.005% P20, and injected over the captured PCSK9 surface with a 50 ul/min flow rate (5 min association and 5 min dissociation). CmAb1 showed very similar binding activity compared with mAb1 (25Chan J.C. Piper D.E. Cao Q. Liu D. King C. Wang W. Tang J. Liu Q. Higbee J. Xia Z. et al.A proprotein convertase subtilisin/kexin type 9 neutralizing antibody reduces serum cholesterol in mice and nonhuman primates.Proc. Natl. Acad. Sci. USA. 2009; 106: 9820-9825Crossref PubMed Scopus (330) Google Scholar). Control mouse IgG1 was raised against a PeptiBody peptide AGP-3. The resulting antibody was produced in stably transfected Chinese hamster ovary cells and purified using the same method as CmAb1. All animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee at Amgen for work performed at Amgen and by the Institutional Animal Care and Use Committee of the Netherlands Organization for Applied Research for work performed at TNO Metabolic Health Research. All mice were housed and maintained under standard environmental conditions with a 12 h light-dark cycle and had free access to food and water. All mice were in a C57Bl/6 background. Ldlr−/− and apoE−/− mice were obtained from Jackson Laboratories. Each strain was crossed with pcsk9−/− mice (Ozgene Pty Ltd, Bentley, Australia) to generate ldlr+/−/pcsk9+/− and apoe+/−/pcsk9+/− colonies. These pcsk9+/− colonies were crossed again to generate the male double knockouts (ldlr−/−/pcsk9−/− and apoe−/−/pcsk9−/−) and the respective littermate controls (ldlr−/−/pcsk9+/+ andapoe−/−/pcsk9+/+) used for these experiments. Mice (male) on the ldlr−/− background were fed an atherogenic diet (Research Diets D12108C) containing 40% kcal from fat and 1.25% cholesterol. Mice (male) on the apoe−/− background were fed chow diet (Harlan 2020X). Female APOE*3Leiden.CETP transgenic mice (11–13 weeks of age) (53Westerterp 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), expressing human CETP under control of its natural flanking regions, were used. APOE*3Leiden.CETP transgenic mice were fed a semi-synthetic cholesterol-rich diet containing 15% (w/w) cacao butter and 0.15% cholesterol [Western-type diet (WTD); Hope Farms, Woerden, The Netherlands] for a run-in period of 3–4 weeks to increase plasma total cholesterol (TC) levels to approximately 650 mg/dl. Mice were matched based on body weight, TC, TGs, and age. In pharmacologic inhibitory studies, antibodies were administered by sc injection (10 mg/kg) every 10 days for 14 weeks, to examine effects on atherosclerotic plaque development. Whole blood was collected by tail nick, vena cava, or cardiac puncture. At study termination, animals were euthanized either by CO2 asphyxiation or by exsanguination under anesthesia (100 mg/kg ketamine, 5 mg/kg diazepam). For liver collection, sections of the right medial or left lobe were excised, flash frozen, and stored until further use. For heart and aorta isolation, hearts were either isolated and placed directly in formalin or animals were perfused by gravity flow under anesthesia. Perfusion was performed by inserting a 25 gauge needle into the apex of the left ventricle and nicking the right atrium. Animals were perfused with saline for 10 min followed by 4% paraformaldehyde for 10 min for fixation. Hearts and aortas were removed, immersed in 4% paraformaldehyde, and stored at 4°C. Mouse serum or EDTA plasma was obtained from whole blood collected via centrifugation. Serum or plasma cholesterol and TGs were analyzed using either a Cobas Integra 400 chemistr

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