SIRT3 mediates the effects of PCSK9 inhibitors on inflammation, autophagy, and oxidative stress in endothelial cells

Nunzia D'Onofrio, Francesco Prattichizzo, Raffaele Marfella, Celestino Sardu, Elisa Martino, Lucia Scisciola, Lorenza Marfella, Rosalba La Grotta, Chiara Frigé, Giuseppe Paolisso, Antonio Ceriello, Maria Luisa Balestrieri, Nunzia D'Onofrio, Francesco Prattichizzo, Raffaele Marfella, Celestino Sardu, Elisa Martino, Lucia Scisciola, Lorenza Marfella, Rosalba La Grotta, Chiara Frigé, Giuseppe Paolisso, Antonio Ceriello, Maria Luisa Balestrieri

Abstract

Background: Proprotein convertase subtilisin-kexin type 9 (PCSK9) inhibitors (i) are a class of lipid-lowering drugs suggested to hold a plethora of beneficial effects independent of their LDL cholesterol-lowering properties. However, the mechanism underlying such observations is debated. Methods: Human aortic endothelial cells (TeloHAEC) were pre-treated with 100 µg/mL of the PCSK9i evolocumab and then exposed to 20 ng/mL of IL-6, a major driver of cardiovascular diseases (CVD), in both naïve state and after siRNA-mediated suppression of the NAD-dependent deacetylase sirtuin-3 (SIRT3). Inflammation, autophagy, and oxidative stress were assessed through Western Blots, ELISAs, and/or immunofluorescence coupled by flow cytometry. To explore the human relevance of the findings, we also evaluated the expression of IL-6, SIRT3, IL-1β, the ratio LC3B II/I, and PCSK9 within the plaques of patients undergoing carotid endarterectomy (n=277), testing possible correlations between these proteins. Results: PCSK9i improved a range of phenotypes including the activation of inflammatory pathways, oxidative stress, and autophagy. Indeed, treatment with PCSK9i was able to counteract the IL-6 induced increase in inflammasome activation, the accrual of autophagic cells, and mitochondrial ROS accumulation. Of note, silencing of SIRT3 reverted the beneficial effects observed with PCSK9i treatment on all these phenomena. In atheroma specimens, the expression of PCSK9 was inversely related to that of SIRT3 while positively correlating with IL-6, IL-1β, and the ratio LC3B II/I. Conclusions: Overall, these data suggest that PCSK9i bear intrinsic anti-inflammatory, anti-autophagic, and antioxidant properties in endothelial cells, and that these pleiotropic effects might be mediated, at least in part, by SIRT3. These results provide an additional mechanism supporting the emerging knowledge relative to the benefit of PCSK9i on CVD beyond LDL-lowering and uncover SIRT3 as a putative mediator of such pleiotropy.

Keywords: IL-6; LDL-cholesterol; PCSK9; ROS; SIRT3; autophagy; cardiovascular diseases; endothelial cells; inflammasome; inflammation; oxidative stress.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

© The author(s).

Figures

Figure 1
Figure 1
PCSK9i counteract IL-6-induced inflammation. Evaluation of (A) NLRP3 protein expression and (B) content, (C) PCSK9 protein expression, caspase-1 (D) expression and (E) content, IL-1β (F) expression and (G) content, TNF-α (H) expression and (I) content, (J) IFN-γ levels and (K) NF-κB expression levels in EC treated for 24h with 20 ng/mL IL-6 (IL-6), with 100 µg/mL PCSK9i (PCSK9i) or pre-treated for 8h with 100 µg/mL PCSK9i before being exposed to 20 ng/mL IL-6 for 24h (PCSK9i+IL-6). Control cells (Ctr) were maintained in complete culture medium with the corresponding highest volume of HBSS-10 mM Hepes. Immunoblotting analyses are represented as floating bars with line representing the mean of n = 4 independent experiments ± SD, where the densitometric intensity was calculated with ImageJ software and expressed as arbitrary units (AU). Lane 1 = Ctr; lane 2 = IL-6; lane 3 = PCSK9i; lane 4 = PCSK9i+IL-6; M = weight markers (G266, Applied Biological Materials Inc.). §p < 0.001 vs. Ctr; **p < 0.01 vs. IL-6.
Figure 2
Figure 2
PCSK9i decrease IL-6-related autophagy. Representative images by fluorescence microscopy and flow cytometry analysis of (A-C) green detection reagent, quantified as median fluorescence intensities, and representative cropped blots with relative immunoblotting analysis of (D) beclin-1, (E) ATG5, (F) LC3B II/I, and (G) p62 protein levels in EC treated for 24h with 20 ng/mL IL-6 (IL-6), with 100 µg/mL PCSK9i (PCSK9i) or pre-treated for 8h with 100 µg/mL PCSK9i before being exposed to 20 ng/mL IL-6 for 24h (PCSK9i+IL-6). Control cells (Ctr) were maintained in complete culture medium with the corresponding highest volume of HBSS-10 mM Hepes. Scale bars = 100 µm. Lane 1 = Ctr; lane 2 = IL-6; lane 3 = PCSK9i; lane 4 = PCSK9i+IL-6; M = weight markers (G266, Applied Biological Materials Inc.). Immunoblotting analyses are represented as floating bars with line representing the mean of n = 4 independent experiments ± SD, where the densitometric intensity was calculated with ImageJ software and expressed as arbitrary units (AU). §p < 0.001 vs. Ctr; **p < 0.01 vs. IL-6.
Figure 3
Figure 3
PCSK9i inhibit IL-6-induced mitochondrial damage. Representative images by fluorescence microscopy and FACS analysis of (A-C) mitochondrial ROS and (D-F) mitochondrial membrane potential detection, expressed as fluorescence intensity ± SD of n = 3 experiments, and (G) representative cropped blots with relative immunoblotting analysis of SIRT3 protein levels in EC treated for 24h with 20 ng/mL IL-6 (IL-6), with 100 µg/mL PCSK9i (PCSK9i) or pre-treated for 8h with 100 µg/mL PCSK9i before being exposed to 20 ng/mL IL-6 for 24h (PCSK9i+IL-6). Control cells (Ctr) were maintained in complete culture medium with the corresponding highest volume of HBSS-10 mM Hepes. Scale bars = 100 µm. Lane 1 = Ctr; lane 2 = IL-6; lane 3 = PCSK9i; lane 4 = PCSK9i+IL-6; M = weight markers (G266, Applied Biological Materials Inc.). Immunoblotting analysis is represented as floating bars with line representing the mean of n = 4 independent experiments ± SD, where the densitometric intensity was calculated with ImageJ software and expressed as arbitrary units (AU). §p < 0.001 vs. Ctr; **p < 0.01 vs. IL-6.
Figure 4
Figure 4
Suppression of SIRT3 abolishes the effects of PCSK9i effects on inflammation and mitochondrial ROS. (A) Representative cropped blots with relative immunoblotting analysis of SIRT3 protein levels in EC treated with the empty transfection reagent (Vehicle) or transfected with NT-siRNA (NT) or with SIRT3 siRNA (SIRT3-/-). Lane 1 = Ctr; lane 2 = Vehicle; lane 3 = NT; lane 4 = SIRT3-/-; M = weight markers (G266, Applied Biological Materials Inc.). (B) Representative cropped blots with relative immunoblotting analysis of PCSK9 protein levels, assessment of (C) NLRP3 and (D) caspase-1 content and (E) representative images by fluorescence microscopy and (F,G) FACS analysis of mitochondrial ROS evaluated in EC transfected with NT-siRNA (NT) or with SIRT3 siRNA (SIRT3-/-) and then exposed for 24h to 20 ng/mL IL-6 (NT or SIRT3-/-+IL-6), to 100 µg/mL PCSK9i (NT or SIRT3-/-+PCSK9i) or to 100 µg/mL PCSK9i for 8h before exposure to 20 ng/mL IL-6 for 24h (NT or SIRT3-/-+PCSK9i+IL-6). Control cells (Ctr) were maintained in complete culture medium with the corresponding highest volume of HBSS-10 mM Hepes. Scale bars = 100 µm. Lane 1 = Ctr; lane 2 = NT; lane 3 = NT+IL-6; lane 4 = NT+PCSK9i; lane 5 = NT+PCSK9i+IL-6; lane 6 = SIRT3-/-; lane 7 = SIRT3-/-+IL-6; lane 8 = SIRT3-/-+PCSK9i; lane 9 = SIRT3-/-+PCSK9i+IL-6; M = weight markers (G266, Applied Biological Materials Inc.). Immunoblotting analysis is represented as floating bars with line representing the mean of n = 4 independent experiments ± SD, where the densitometric intensity was calculated with ImageJ software and expressed as arbitrary units (AU). §p < 0.001 vs. NT; **p < 0.01 vs. NT+IL-6.
Figure 5
Figure 5
Downregulation of SIRT3 decreases the ability of PCSK9i to impede mitochondrial depolarization. (A) Representative images by fluorescence microscopy and (B,C) flow cytometry analysis of mitochondrial membrane potential detection, expressed as fluorescence intensity ± SD of n = 3 experiments, in EC transfected with NT-siRNA (NT) or with SIRT3 siRNA (SIRT3-/-) and then exposed for 24h to 20 ng/mL IL-6 (NT or SIRT3-/-+IL-6), to 100 µg/mL PCSK9i (NT or SIRT3-/-+PCSK9i) or to 100 µg/mL PCSK9i for 8h before exposure to 20 ng/mL IL-6 for 24h (NT or SIRT3-/-+PCSK9i+IL-6). Control cells (Ctr) were maintained in complete culture medium with the corresponding highest volume of HBSS-10 mM Hepes. Scale bars = 100 µm. §p<0.001 vs. NT; **p < 0.01 vs. NT+IL-6.
Figure 6
Figure 6
SIRT3 suppression denies the ability of PCSK9i to counteract autophagy. (A) Representative images by fluorescence microscopy and (B,C) flow cytometry analysis of green detection reagent, quantified as median fluorescence intensities, and representative cropped blots with relative immunoblotting analysis of (D) beclin-1 and (E) LC3B II/I protein levels in EC transfected with NT-siRNA (NT) or with SIRT3 siRNA (SIRT3-/-) and then exposed for 24h to 20 ng/mL IL-6 (NT or SIRT3-/-+IL-6), to 100 µg/mL PCSK9i (NT or SIRT3-/-+PCSK9i) or to 100 µg/mL PCSK9i for 8h before exposure to 20 ng/mL IL-6 for 24h (NT or SIRT3-/-+PCSK9i+IL-6). Control cells (Ctr) were maintained in complete culture medium with the corresponding highest volume of HBSS-10 mM Hepes. Scale bars = 100 µm. Lane 1 = Ctr; lane 2 = NT; lane 3 = NT+IL-6; lane 4 = NT+PCSK9i; lane 5 = NT+PCSK9i+IL-6; lane 6 = SIRT3-/-; lane 7 = SIRT3-/-+IL-6; lane 8 = SIRT3-/-+PCSK9i; lane 9 = SIRT3-/-+PCSK9i+IL-6; M = weight markers (G266, Applied Biological Materials Inc.). Immunoblotting analyses are represented as floating bars with line representing the median of n = 4 independent experiments ± SD, where the densitometric intensity was calculated with ImageJ software and expressed as arbitrary units (AU). §p < 0.001 vs. NT; **p < 0.01 vs. NT+IL-6.

References

    1. Sabatine MS. PCSK9 inhibitors: clinical evidence and implementation. Nat Rev Cardiol. 2019;16:155–165.
    1. Schwartz GG, Gabriel Steg P, Bhatt DL, Bittner VA, Diaz R, Goodman SG. et al. Clinical Efficacy and Safety of Alirocumab After Acute Coronary Syndrome According to Achieved Level of Low-Density Lipoprotein Cholesterol. A Propensity Score-Matched Analysis of the ODYSSEY OUTCOMES Trial. Circulation. 2021;143:1109–1122.
    1. Giunzioni I, Tavori H, Covarrubias R, Major AS, Ding L, Zhang Y. et al. Local effects of human PCSK9 on the atherosclerotic lesion. J Pathol. 2016;238:52–62.
    1. Liu A, Frostegard J. PCSK9 plays a novel immunological role in oxidized LDL-induced dendritic cell maturation and activation of T cells from human blood and atherosclerotic plaque. J Intern Med. 2018;284:193–210.
    1. Ding Z, Wang X, Liu S, Shahanawaz J, Theus S, Fan Y. et al. PCSK9 expression in the ischaemic heart and its relationship to infarct size, cardiac function, and development of autophagy. Cardiovasc Res. 2018;114:1738–1751.
    1. Huang G, Lu X, Zhou H, Li R, Huang Q, Xiong X. et al. PCSK9 inhibition protects against myocardial ischemia-reperfusion injury via suppressing autophagy. Microvasc Res. 2022;142:104371.
    1. Urban D, Poss J, Bohm M, Laufs U. Targeting the proprotein convertase subtilisin/kexin type 9 for the treatment of dyslipidemia and atherosclerosis. J Am Coll Cardiol. 2013;62:1401–1408.
    1. Li J, Liang X, Wang Y, Xu Z, Li G. Investigation of highly expressed PCSK9 in atherosclerotic plaques and ox-LDL-induced endothelial cell apoptosis. Mol Med Rep. 2017;16:1817–1825.
    1. Ding Z, Wang X, Liu S, Zhou S, Kore RA, Mu S. et al. NLRP3 inflammasome via IL-1beta regulates PCSK9 secretion. Theranostics. 2020;10:7100–7110.
    1. Feingold KR, Moser AH, Shigenaga JK, Patzek SM, Grunfeld C. Inflammation stimulates the expression of PCSK9. Biochem Biophys Res Commun. 2008;374:341–344.
    1. Tang ZH, Peng J, Ren Z, Yang J, Li TT, Li TH. et al. New role of PCSK9 in atherosclerotic inflammation promotion involving the TLR4/NF-kappaB pathway. Atherosclerosis. 2017;262:113–122.
    1. Ricci C, Ruscica M, Camera M, Rossetti L, Macchi C, Colciago A. et al. PCSK9 induces a pro-inflammatory response in macrophages. Sci Rep. 2018;8:2267.
    1. Cheng JM, Oemrawsingh RM, Garcia-Garcia HM, Boersma E, van Geuns RJ, Serruys PW. et al. PCSK9 in relation to coronary plaque inflammation: Results of the ATHEROREMO-IVUS study. Atherosclerosis. 2016;248:117–122.
    1. Li S, Guo YL, Xu RX, Zhang Y, Zhu CG, Sun J. et al. Association of plasma PCSK9 levels with white blood cell count and its subsets in patients with stable coronary artery disease. Atherosclerosis. 2014;234:441–445.
    1. Ueland T, Kleveland O, Michelsen AE, Wiseth R, Damas JK, Aukrust P. et al. Serum PCSK9 is modified by interleukin-6 receptor antagonism in patients with hypercholesterolaemia following non-ST-elevation myocardial infarction. Open Heart. 2018 Sep 18;5(2):e000765.
    1. Ridker PM, Rane M. Interleukin-6 Signaling and Anti-Interleukin-6 Therapeutics in Cardiovascular Disease. Circ Res. 2021;128:1728–1746.
    1. Lindkvist M, Zegeye MM, Grenegard M, Ljungberg LU. Pleiotropic, Unique and Shared Responses Elicited by IL-6 Family Cytokines in Human Vascular Endothelial Cells. Int J Mol Sci. 2022 Jan 27;23(3):1448.
    1. Wassmann S, Stumpf M, Strehlow K, Schmid A, Schieffer B, Bohm M. et al. Interleukin-6 induces oxidative stress and endothelial dysfunction by overexpression of the angiotensin II type 1 receptor. Circ Res. 2004;94:534–541.
    1. Hu F, Song D, Yan Y, Huang C, Shen C, Lan J. et al. IL-6 regulates autophagy and chemotherapy resistance by promoting BECN1 phosphorylation. Nat Commun. 2021;12:3651.
    1. Yamamoto H, Schoonjans K, Auwerx J. Sirtuin functions in health and disease. Mol Endocrinol. 2007;21:1745–1755.
    1. Onyango P, Celic I, McCaffery JM, Boeke JD, Feinberg AP. SIRT3, a human SIR2 homologue, is an NAD-dependent deacetylase localized to mitochondria. Proc Natl Acad Sci U S A. 2002;99:13653–13658.
    1. Sundaresan NR, Samant SA, Pillai VB, Rajamohan SB, Gupta MP. SIRT3 is a stress-responsive deacetylase in cardiomyocytes that protects cells from stress-mediated cell death by deacetylation of Ku70. Mol Cell Biol. 2008;28:6384–6401.
    1. Zhang J, Xiang H, Liu J, Chen Y, He RR, Liu B. Mitochondrial Sirtuin 3: New emerging biological function and therapeutic target. Theranostics. 2020;10:8315–8342.
    1. Zhang S, Wu X, Wang J, Shi Y, Hu Q, Cui W. et al. Adiponectin/AdiopR1 signaling prevents mitochondrial dysfunction and oxidative injury after traumatic brain injury in a SIRT3 dependent manner. Redox Biol. 2022;54:102390.
    1. Boniakowski AM, denDekker AD, Davis FM, Joshi A, Kimball AS, Schaller M. et al. SIRT3 Regulates Macrophage-Mediated Inflammation in Diabetic Wound Repair. J Invest Dermatol. 2019;139:2528–2537. e2522.
    1. Li S, Dou X, Ning H, Song Q, Wei W, Zhang X. et al. Sirtuin 3 acts as a negative regulator of autophagy dictating hepatocyte susceptibility to lipotoxicity. Hepatology. 2017;66:936–952.
    1. Sun W, Liu C, Chen Q, Liu N, Yan Y, Liu B. SIRT3: A New Regulator of Cardiovascular Diseases. Oxid Med Cell Longev. 2018;2018:7293861.
    1. Toulassi IA, Al Saedi UA, Gutlapalli SD, Poudel S, Kondapaneni V, Zeb M. et al. A Paradigm Shift in the Management of Atherosclerosis: Protective Role of Sirtuins in Atherosclerosis. Cureus. 2021;13:e12735.
    1. Sabatine MS, Giugliano RP, Keech AC, Honarpour N, Wiviott SD, Murphy SA. et al. Evolocumab and Clinical Outcomes in Patients with Cardiovascular Disease. N Engl J Med. 2017;376:1713–1722.
    1. Rodgers MA, Bowman JW, Liang Q, Jung JU. Regulation where autophagy intersects the inflammasome. Antioxid Redox Signal. 2014;20:495–506.
    1. Mach F, Baigent C, Catapano AL. et al. 2019 ESC/EAS Guidelines for the management of dyslipidaemias: lipid modification to reduce cardiovascular risk. Eur Heart J. 2020;41:4255.
    1. Ding Z, Liu S, Wang X, Theus S, Deng X, Fan Y. et al. PCSK9 regulates expression of scavenger receptors and ox-LDL uptake in macrophages. Cardiovasc Res. 2018;114:1145–1153.
    1. Sahebkar A, Di Giosia P, Stamerra CA, Grassi D, Pedone C, Ferretti G. et al. Effect of monoclonal antibodies to PCSK9 on high-sensitivity C-reactive protein levels: a meta-analysis of 16 randomized controlled treatment arms. Br J Clin Pharmacol. 2016;81:1175–1190.
    1. Levstek T, Podkrajsek N, Rehberger Likozar A, Sebestjen M, Trebusak Podkrajsek K. The Influence of Treatment with PCSK9 Inhibitors and Variants in the CRP (rs1800947), TNFA (rs1800629), and IL6 (rs1800795) Genes on the Corresponding Inflammatory Markers in Patients with Very High Lipoprotein(a) Levels. J Cardiovasc Dev Dis. 2022 Apr 22;9(5):127.
    1. Basiak M, Kosowski M, Hachula M, Okopien B. Impact of PCSK9 Inhibition on Proinflammatory Cytokines and Matrix Metalloproteinases Release in Patients with Mixed Hyperlipidemia and Vulnerable Atherosclerotic Plaque. Pharmaceuticals (Basel) 2022 Jun 27;15(7):802.
    1. Hoogeveen RM, Opstal TSJ, Kaiser Y, Stiekema LCA, Kroon J, Knol RJJ. et al. PCSK9 Antibody Alirocumab Attenuates Arterial Wall Inflammation Without Changes in Circulating Inflammatory Markers. JACC Cardiovasc Imaging. 2019;12:2571–2573.
    1. Wang X, Li X, Liu S, Brickell AN, Zhang J, Wu Z. et al. PCSK9 regulates pyroptosis via mtDNA damage in chronic myocardial ischemia. Basic Res Cardiol. 2020;115:66.
    1. Tyagi A, Nguyen CU, Chong T, Michel CR, Fritz KS, Reisdorph N. et al. SIRT3 deficiency-induced mitochondrial dysfunction and inflammasome formation in the brain. Sci Rep. 2018;8:17547.
    1. Lee S, Jeon YM, Jo M, Kim HJ. Overexpression of SIRT3 Suppresses Oxidative Stress-induced Neurotoxicity and Mitochondrial Dysfunction in Dopaminergic Neuronal Cells. Exp Neurobiol. 2021;30:341–355.
    1. Zhou D, Jiang Y. Sirtuin 3 attenuates neuroinflammation-induced apoptosis in BV-2 microglia. Aging (Albany NY) 2019;11:9075–9089.
    1. Guo Y, Jia X, Cui Y, Song Y, Wang S, Geng Y. et al. Sirt3-mediated mitophagy regulates AGEs-induced BMSCs senescence and senile osteoporosis. Redox Biol. 2021;41:101915.
    1. Ding Z, Liu S, Wang X, Deng X, Fan Y, Sun C. et al. Hemodynamic shear stress via ROS modulates PCSK9 expression in human vascular endothelial and smooth muscle cells and along the mouse aorta. Antioxid Redox Signal. 2015;22:760–771.
    1. Cammisotto V, Pastori D, Nocella C, Bartimoccia S, Castellani V, Marchese C. et al. PCSK9 Regulates Nox2-Mediated Platelet Activation via CD36 Receptor in Patients with Atrial Fibrillation. Antioxidants (Basel) 2020 Apr 2;9(4):296.
    1. Safaeian L, Mirian M, Bahrizadeh S. Evolocumab, a PCSK9 inhibitor, protects human endothelial cells against H2O2-induced oxidative stress. Arch Physiol Biochem. 2022 Dec;128(6):1681–1686.
    1. Cammisotto V, Baratta F, Castellani V, Bartimoccia S, Nocella C, D'Erasmo L. et al. Proprotein Convertase Subtilisin Kexin Type 9 Inhibitors Reduce Platelet Activation Modulating ox-LDL Pathways. Int J Mol Sci. 2021 Jul 3;22(13):7193.
    1. Feng X, Su H, He X, Chen JX, Zeng H. SIRT3 Deficiency Sensitizes Angiotensin-II-Induced Renal Fibrosis. Cells. 2020 Nov 20;9(11):2510.
    1. Schwartz GG, Szarek M, Bittner VA, Diaz R, Goodman SG, Jukema JW. et al. Lipoprotein(a) and Benefit of PCSK9 Inhibition in Patients With Nominally Controlled LDL Cholesterol. J Am Coll Cardiol. 2021;78:421–433.
    1. Silverman MG, Ference BA, Im K, Wiviott SD, Giugliano RP, Grundy SM. et al. Association Between Lowering LDL-C and Cardiovascular Risk Reduction Among Different Therapeutic Interventions: A Systematic Review and Meta-analysis. JAMA. 2016;316:1289–1297.
    1. D'Onofrio N, Sardu C, Trotta MC, Scisciola L, Turriziani F, Ferraraccio F. et al. Sodium-glucose co-transporter2 expression and inflammatory activity in diabetic atherosclerotic plaques: Effects of sodium-glucose co-transporter2 inhibitor treatment. Mol Metab. 2021 Dec;54:101337.
    1. Verhoeven BA, Moll FL, Koekkoek JA, van der Wal AC, de Kleijn DP, de Vries JP. et al. Statin treatment is not associated with consistent alterations in inflammatory status of carotid atherosclerotic plaques: A retrospective study in 378 patients undergoing carotid endarterectomy. Stroke. 2006;37:2054–2060.
    1. Martinet W, De Meyer GR. Autophagy in atherosclerosis: a cell survival and death phenomenon with therapeutic potential. Circ Res. 2009 Feb 13;104(3):304–17.
    1. Hassanpour M, Rahbarghazi R, Nouri M, Aghamohammadzadeh N, Safaei N, Ahmadi M. Role of autophagy in atherosclerosis: foe or friend? J Inflamm (Lond) 2019 May 2;16:8.

Source: PubMed

Sponsorer och medarbetare

Medicinska tillstånd

Läkemedelsinterventioner

3
Prenumerera