Genetic and In Vitro Inhibition of PCSK9 and Calcific Aortic Valve Stenosis

Nicolas Perrot, Vincenza Valerio, Donato Moschetta, S Matthijs Boekholdt, Christian Dina, Hao Yu Chen, Erik Abner, Andreas Martinsson, Hasanga D Manikpurage, Sidwell Rigade, Romain Capoulade, Elvira Mass, Marie-Annick Clavel, Thierry Le Tourneau, David Messika-Zeitoun, Nicholas J Wareham, James C Engert, Gianluca Polvani, Philippe Pibarot, Tõnu Esko, J Gustav Smith, Patrick Mathieu, George Thanassoulis, Jean-Jacques Schott, Yohan Bossé, Marina Camera, Sébastien Thériault, Paolo Poggio, Benoit J Arsenault, Nicolas Perrot, Vincenza Valerio, Donato Moschetta, S Matthijs Boekholdt, Christian Dina, Hao Yu Chen, Erik Abner, Andreas Martinsson, Hasanga D Manikpurage, Sidwell Rigade, Romain Capoulade, Elvira Mass, Marie-Annick Clavel, Thierry Le Tourneau, David Messika-Zeitoun, Nicholas J Wareham, James C Engert, Gianluca Polvani, Philippe Pibarot, Tõnu Esko, J Gustav Smith, Patrick Mathieu, George Thanassoulis, Jean-Jacques Schott, Yohan Bossé, Marina Camera, Sébastien Thériault, Paolo Poggio, Benoit J Arsenault

Abstract

The authors investigated whether PCSK9 inhibition could represent a therapeutic strategy in calcific aortic valve stenosis (CAVS). A meta-analysis of 10 studies was performed to determine the impact of the PCSK9 R46L variant on CAVS, and the authors found that CAVS was less prevalent in carriers of this variant (odds ratio: 0.80 [95% confidence interval: 0.70 to 0.91]; p = 0.0011) compared with noncarriers. PCSK9 expression was higher in the aortic valves of patients CAVS compared with control patients. In human valve interstitials cells submitted to a pro-osteogenic medium, PCSK9 levels increased and a PCSK9 neutralizing antibody significantly reduced calcium accumulation.

Keywords: Ad DMEM, advanced Dulbecco’s modified Eagle’s medium; CAD, coronary artery disease; CAVS, calcific aortic valve stenosis; HDL-C, high-density lipoprotein cholesterol; IQR, interquartile range; LDL cholesterol; LDL-C, low-density lipoprotein cholesterol; Lp(a), lipoprotein(a); PBS, phosphate-buffered saline; PBST, 1× phosphate-buffered saline with 0.1% Triton; PCSK9, proprotein convertase subtilisin/kexin type 9; SNP, single nucleotide polymorphism; TC, total cholesterol; VIC, valve interstitial cell; VLDL-C, very-low-density lipoprotein cholesterol; aortic valve interstitial cell; apoB, apolipoprotein B; apolipoprotein B; calcific aortic valve stenosis; lipoprotein(a); proprotein convertase subtilisin/kexin type 9; wGRS, weighted genetic risk score.

© 2020 The Authors.

Figures

Graphical abstract
Graphical abstract
Figure 1
Figure 1
Association of the PCSK9 R46L Variant With CAVS Association of the PCSK9 R46L variant with calcific aortic valve stenosis in a meta-analysis of 10 cohorts totaling 12,059 cases and 541,081 control subjects. CAVS = calcific aortic valve stenosis; CI = confidence interval; EPIC = European Prospective Investigation into Cancer and Nutrition; GERA = Genetic Epidemiology Research on Aging study; MDCS = Malmo Diet and Cancer Study; OR = odds ratio.
Figure 2
Figure 2
Impact of Individual SNPs and a wGRS on CAVS Impact of individual single nucleotide polymorphisms (SNPs) (A) and of a weighted genetic risk score of single nucleotide polymorphisms at the PCSK9 locus associated with low-density cholesterol (LDL-C) levels only (B) on calcific aortic valve stenosis in the UK Biobank. wGRS = weighted genetic risk score; other abbreviations as in Figure 1.
Figure 3
Figure 3
PCSK9 Is Highly Abundant in Calcified Leaflets (A) Representative explanted aortic valve leaflet with normal structure (control valve) and (B) representative calcified aortic valve leaflet (stenotic valve), both stained with Von Kossa to visualize calcium deposits (upper panels) and with anti-PCSK9 antibody (lower panels). PCSK9 immunohistochemical (IHC) staining is presented as 3,3′-diaminobenzidine (DAB) and hematoxylin (H) counter staining (DAB + H) and as deconvoluted image visualizing only the DAB staining. Panoramic images were taken with a 10× magnification. Black boxes indicate the higher magnification areas. (C and D) High magnification areas of aortic valve leaflets (20× left and 40× right). Arrows indicate representative PCSK9 expression in close proximity to cell nuclei. (E) Box and whisker plots represent DAB quantification by ImageJ with IHC Tool box plugin on normal (control; n = 6) and calcified (stenotic; n = 6) leaflets. (F) Box and whisker plots represent PCSK9 levels measured by enzyme-linked immunosorbent assay on control (n = 6) and stenotic (n = 6) whole tissue extracts.
Figure 4
Figure 4
PCSK9 Expression and Secretion Is Induced by Osteogenic Milieu in Valve Interstitial Cells (A) Box and whisker plots represent PCSK9 transcript levels in valve interstitial cells (VIC) cultured for 7 days in normal or osteogenic media (n = 6). RNA levels were normalized to GAPDH expression. (B) Box and whisker plots represent secreted PCSK9 levels from VICs cultured for 7 days in normal or osteogenic media (n = 4). PCSK9 levels were normalized to total protein content. (C) Mean-centered correlation between secreted PCSK9 and calcium levels in VICs. The linear correlation between the 2 variables was performed with the Pearson correlation coefficient. (D) Bar graph shows the calcification potential, after 7 days, in normal and osteogenic media (n = 3) of VICs treated with a neutralizing antibody anti-PCSK9 (NAb anti-PCSK9) or immunoglobulin G1 as control (IgG1).

References

    1. Otto C.M. Calcific aortic stenosis--time to look more closely at the valve. N Engl J Med. 2008;359:1395–1398.
    1. Rossebo A.B., Pedersen T.R., Boman K. Intensive lipid lowering with simvastatin and ezetimibe in aortic stenosis. N Engl J Med. 2008;359:1343–1356.
    1. Chan K.L., Teo K., Dumesnil J.G., Ni A., Tam J. Effect of lipid lowering with rosuvastatin on progression of aortic stenosis: results of the aortic stenosis progression observation: measuring effects of rosuvastatin (ASTRONOMER) trial. Circulation. 2010;121:306–314.
    1. Cowell S.J., Newby D.E., Burton J. Aortic valve calcification on computed tomography predicts the severity of aortic stenosis. Clin Radiol. 2003;58:712–716.
    1. Arsenault B.J., Boekholdt S.M., Mora S. Impact of high-dose atorvastatin therapy and clinical risk factors on incident aortic valve stenosis in patients with cardiovascular disease (from TNT, IDEAL, and SPARCL) Am J Cardiol. 2014;113:1378–1382.
    1. Perrot N., Boekholdt S.M., Mathieu P., Wareham N.J., Khaw K.-T., Arsenault B.J. Life's simple 7 and calcific aortic valve stenosis incidence in apparently healthy men and women. Int J Cardiol. 2018;269:226–228.
    1. Lagace T.A., Curtis D.E., Garuti R. Secreted PCSK9 decreases the number of LDL receptors in hepatocytes and in livers of parabiotic mice. J Clin Invest. 2006;116:2995–3005.
    1. Cohen J.C., Boerwinkle E., Mosley T.H., Jr., Hobbs H.H. Sequence variations in PCSK9, low LDL, and protection against coronary heart disease. N Engl J Med. 2006;354:1264–1272.
    1. Benn 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;55:2833–2842.
    1. Sabatine M.S., Giugliano R.P., Keech A.C. Evolocumab and clinical outcomes in patients with cardiovascular disease. N Engl J Med. 2017;376:1713–1722.
    1. Schwartz G.G., Steg P.G., Szarek M. Alirocumab and cardiovascular outcomes after acute coronary syndrome. N Engl J Med. 2018;379:2097–2107.
    1. Smith J.G., Luk K., Schulz C.A. Association of low-density lipoprotein cholesterol-related genetic variants with aortic valve calcium and incident aortic stenosis. JAMA. 2014;312:1764–1771.
    1. Langsted A.G., Nordestgaard B.G., Benn M., Tybjærg-Hansen A.R., Kamstrup P.R. PCSK9 R46L loss-of-function mutation reduces lipoprotein(a), LDL cholesterol, and risk of aortic valve stenosis. J Clin Endocrinol Metab. 2016;101:3281–3287.
    1. Willer C., Schmidt E., Sengupta S. Discovery and refinement of loci associated with lipid levels. Nat Genet. 2013;45:1274–1283.
    1. Day N., Oakes S., Luben R. EPIC-Norfolk: study design and characteristics of the cohort. European Prospective Investigation of Cancer. Br J Cancer. 1999;80(Suppl 1):95–103.
    1. Arsenault B.J., Boekholdt S.M., Dube M.P. Lipoprotein(a) levels, genotype, and incident aortic valve stenosis: a prospective Mendelian randomization study and replication in a case-control cohort. Circ Cardiovasc Genet. 2014;7:304–310.
    1. Gurdasani D., Sjouke B., Tsimikas S. Lipoprotein(a) and risk of coronary, cerebrovascular, and peripheral artery disease: the EPIC-Norfolk prospective population study. Arterioscler Thromb Vasc Biol. 2012;32:3058–3065.
    1. Sudlow C., Gallacher J., Allen N. UK Biobank: an open access resource for identifying the causes of a wide range of complex diseases of middle and old age. PLoS Med. 2015;12
    1. Poggio P., Branchetti E., Grau J.B. Osteopontin-CD44v6 interaction mediates calcium deposition via phospho-Akt in valve interstitial cells from patients with noncalcified aortic valve sclerosis. Arterioscler Thromb Vasc Biol. 2014;34:2086–2094.
    1. Rao A.S., Lindholm D., Rivas M.A., Knowles J.W., Montgomery S.B., Ingelsson E. Large-scale phenome-wide association study of variants demonstrates protection against ischemic stroke. Circ Genom Precis Med. 2018;11
    1. Verbeek R., Boyer M., Boekholdt S.M. Carriers of the PCSK9 R46L variant are characterized by an antiatherogenic lipoprotein profile assessed by nuclear magnetic resonance spectroscopy—brief report. Arterioscler Thromb Vasc Biol. 2017;37:43–48.
    1. Sliz E.V., Kettunen J.O., Holmes M.Y. Metabolomic consequences of genetic inhibition of PCSK9 compared with statin treatment. Circulation. 2018;138:2499–2512.
    1. Poggio P., Songia P., Cavallotti L. PCSK9 involvement in aortic valve calcification. J Am Coll Cardiol. 2018;72:3225–3227.
    1. O'Donoghue M.L., Fazio S., Giugliano R.P. Lipoprotein(a), PCSK9 inhibition and cardiovascular risk: insights from the FOURIER trial. Circulation. 2019;139:1483–1492.
    1. Stiekema L.C.A., Stroes E.S.G., Verweij S.L. Persistent arterial wall inflammation in patients with elevated lipoprotein(a) despite strong low-density lipoprotein cholesterol reduction by proprotein convertase subtilisin/kexin type 9 antibody treatment. Eur Heart J. 2019;40:2775–2781.
    1. Salaun E., Mahjoub H., Dahou A. Hemodynamic deterioration of surgically implanted bioprosthetic aortic valves. J Am Coll Cardiol. 2018;72:241–251.
    1. Nsaibia M.J., Mahmut A., Mahjoub H. Association between plasma lipoprotein levels and bioprosthetic valve structural degeneration. Heart. 2016;102:1915.
    1. Bergmark B.A., O'Donoghue M.L., Murphy S.A. An exploratory analysis of proprotein convertase subtilisin/kexin type 9 inhibition and aortic stenosis in the FOURIER trial. JAMA Cardiol. 2020;6:709–713.

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