Therapeutic silencing miR-146b-5p improves cardiac remodeling in a porcine model of myocardial infarction by modulating the wound reparative phenotype

Abstract

Fibrotic remodeling is an adverse consequence of immune response-driven phenotypic modulation of cardiac cells following myocardial infarction (MI). MicroRNA-146b (miR-146b) is an active regulator of immunomodulation, but its function in the cardiac inflammatory cascade and its clinical implication in fibrotic remodeling following MI remain largely unknown. Herein, miR-146b-5p was found to be upregulated in the infarcted myocardium of mice and the serum of myocardial ischemia patients. Gain- and loss-of-function experiments demonstrated that miR-146b-5p was a hypoxia-induced regulator that governed the pro-fibrotic phenotype transition of cardiac cells. Overexpression of miR-146b-5p activated fibroblast proliferation, migration, and fibroblast-to-myofibroblast transition, impaired endothelial cell function and stress survival, and disturbed macrophage paracrine signaling. Interestingly, the opposite effects were observed when miR-146b-5p expression was inhibited. Luciferase assays and rescue studies demonstrated that the miR-146b-5p target genes mediating the above phenotypic modulations included interleukin 1 receptor associated kinase 1 (IRAK1) and carcinoembryonic antigen related cell adhesion molecule 1 (CEACAM1). Local delivery of a miR-146b-5p antagomir significantly reduced fibrosis and cell death, and upregulated capillary and reparative macrophages in the infarcted myocardium to restore cardiac remodeling and function in both mouse and porcine MI models. Local inhibition of miR-146b-5p may represent a novel therapeutic approach to treat cardiac fibrotic remodeling and dysfunction following MI.

Key words： cardiac fibrosis; microRNA; porcine model; myocardial infarction

References

1 Aurora AB, Porrello ER, Tan W, Mahmoud AI, Hill JA, Bassel-Duby R, Sadek HA, Olson EN (2014) Macrophages are required for neonatal heart regeneration. J Clin Invest 124:1382–1392

2 Baltimore D, Boldin MP, O’Connell RM, Rao DS, Taganov KD (2008) MicroRNAs: new regulators of immune cell development and function. Nat Immunol 9:839–845

3 Cheng HS, Sivachandran N, Lau A, Boudreau E, Zhao JL, Baltimore D, Delgado-Olguin P,Cybulsky MI,Fish JE (2013) MicroRNA-146 represses endothelial activation by inhibiting pro-inflammatory pathways. EMBO Mol Med 5:1017–1034

4 Cheng HS, Besla R, Li A, Chen Z, Shikatani EA, Nazari-Jahantigh M, Hammoutène A, Nguyen M, Geoffrion M, Cai L (2017) Paradoxical suppression of atherosclerosis in the absence of microRNA-146a. Circ Res 121:354–367

5 Deng F, He S, Cui S, Shi Y, Tan Y, Li Z, Huang C, Liu D, Zhi F, Peng L (2019) A molecular targeted immunotherapeutic strategy for ulcerative colitis via dual-targeting nanoparticles delivering miR-146b to intestinal macrophages. J Crohns Colitis 13:482–494

6 Desjarlais M, Dussault S, Rivard F, Harel S, Sanchez V, Hussain SNA, Rivard A (2019) Forced expression of microRNA-146b reduces TRAF6-dependent inflammation and improves ischemiainduced neovascularization in hypercholesterolemic conditions. Atherosclerosis 289:73–84

7 Di YF, Li DC, Shen YQ, Wang CL, Zhang DY, Shang AQ, Hu T(2017) MiR-146b protects cardiomyocytes injury in myocardial ischemia/reperfusion by targeting Smad4. Am J Transl Res 9:656–663

8 Dick SA, Macklin JA, Nejat S, Momen A, Clemente-Casares X, Althagafi MG, Chen J, Kantores C, Hosseinzadeh S,Aronoff L (2019) Self-renewing resident cardiac macrophages limit adverse remodeling following myocardial infarction. Nat Immunol 20:29–39

9 Epelman S, Liu PP, Mann DL (2015) Role of innate and adaptive immune mechanisms in cardiac injury and repair. Nat Rev Immunol 15:117–129

10 Ferraro B, Leoni G, Hinkel R, Ormanns S, Paulin N, Ortega-Gomez A, Viola JR, de Jong R,Bongiovanni D, Bozoglu T (2019) Pro-angiogenic macrophage phenotype to promote myocardial repair. J Am Coll Cardiol 73:2990–3002

11 Frangogiannis NG (2012) Regulation of the inflammatory response in cardiac repair. Circ Res 110:159–173

12 Frangogiannis NG (2014) The inflammatory response in myocardial injury, repair, and remodelling. Nat Rev Cardiol 11:255–265

13 Fu X, Khalil H, Kanisicak O, Boyer JG, Vagnozzi RJ, Maliken BD, Sargent MA, Prasad V, Valiente-Alandi I, Blaxall BC (2018) Specialized fibroblast differentiated states underlie scar formation in the infarcted mouse heart. J Clin Invest 128:2127–2143

14 Gabisonia K, Prosdocimo G,Aquaro GD, Carlucci L, Zentilin L, Secco I,Ali H, Braga L, Gorgodze N, Bernini F (2019) MicroRNA therapy stimulates uncontrolled cardiac repair after myocardial infarction in pigs. Nature 569:418–422

15 Gao M, Wang X, Zhang X, Ha T, Ma H, Liu L, Kalbfleisch JH, Gao X, Kao RL, Williams DL (2015) Attenuation of cardiac dysfunction in polymicrobial sepsis by microRNA-146a is mediated via targeting of IRAK1 and TRAF6 expression. J Immunol 195:672–682

16 He X, Tang R, Sun Y, Wang YG, Zhen KY, Zhang DM, Pan WQ (2016) MicroR-146 blocks the activation of M1 macrophage by targeting signal transducer and activator of transcription 1 in hepatic schistosomiasis. eBiomedicine 13:339–347

17 Hinkel R, Penzkofer D, Zühlke S, Fischer A, Husada W, Xu Q, Baloch E, van Rooij E, Zeiher AM, Kupatt C (2013) Inhibition of MicroRNA-92a protects against ischemia/reperfusion injury in a large-animal model. Circulation 128:1066–1075

18 Horst AK (2006) Carcinoembryonic antigen-related cell adhesion molecule 1 modulates vascular remodeling in vitro and in vivo. J Clin Invest 116:1596–1605

19 Huang S, Frangogiannis NG (2018) Anti-inflammatory therapies in myocardial infarction: failures, hopes and challenges. Br J Pharmacol 175:1377–1400

20 Huang Y, Zhu C, Kondo Y,Anderson AC, Gandhi A, Russell A, Dougan SK, Petersen B,Melum E, Pertel T (2015) CEACAM1 regulates TIM-3-mediated tolerance and exhaustion. Nature 517:386–390

21 Huang S, Li X, Zheng H, Si X, Li B, Wei G, Li C, Chen Y, Chen Y, Liao W (2019) Loss of super-enhancer-regulated circRNA Nfix induces cardiac regeneration after myocardial infarction in adult mice. Circulation 139:2857–2876

22 Hullinger TG, Montgomery RL, Seto AG, Dickinson BA, Semus HM, Lynch JM, Dalby CM, Robinson K, Stack C, Latimer PA (2012) Inhibition of miR-15 protects against cardiac ischemic injury. Circ Res 110:71–81

23 Jia D, Jiang H, Weng X, Wu J,Bai P,Yang W, Wang Z, Hu K, Sun A, Ge J (2019) Interleukin-35 promotes macrophage survival and improves wound healing after myocardial infarction in mice. Circ Res 124:1323–1336

24 Kilic N,Oliveira-Ferrer L , Wurmbach J,Loges S, Chalajour F, Vahid SN, Weil J, Fernando M, Ergun S (2005) Pro-angiogenic signaling by the endothelial presence of CEACAM1. J Biol Chem 280:2361–2369

25 Krutzfeldt J, Rajewsky N, Braich R, Rajeev KG, Tuschl T, Manoharan M, Stoffel M(2005) Silencing of microRNAs in vivo with ‘antagomirs’. Nature 438:685–689

26 Li JW, He SY, Feng ZZ, Zhao L, Jia WK, Liu P,Zhu Y, Jian Z, Xiao YB (2015) MicroRNA-146b inhibition augments hypoxia-induced cardiomyocyte apoptosis. Mol Med Rep 12:6903–6910

27 Li H, Liao Y, Gao L, Zhuang T, Huang Z, Zhu H, Ge J (2018) Coronary serum exosomes derived from patients with myocardial ischemia regulate angiogenesis through the miR-939-mediated nitric oxide signaling pathway. Theranostics 8:2079–2093

28 Liu J, Zhuang T, Pi J, Chen X, Zhang Q,Li Y, Wang H, Shen Y,Tomlinson B, Chan P (2019) Endothelial forkhead box transcription factor P1 regulates pathological cardiac remodeling through transforming growth factor-β1-endothelin-1 signal pathway . Circulation 140:665–680

29 Ma Y, Mouton AJ, Lindsey ML (2018) Cardiac macrophage biology in the steady-state heart, the aging heart, and following myocardial infarction . Transl Res 191:15–28

30 Mendell JT, Olson EN (2012) MicroRNAs in stress signaling and human disease. Cell 148:1172–1187

31 Mescher AL (2017) Macrophages and fibroblasts during inflammation and tissue repair in models of organ regeneration. Regeneration 4:39–53

32 Meyer IS, Jungmann A,Dieterich C, Zhang M, Lasitschka F, Werkmeister S, Haas J, Müller OJ, Boutros M, Nahrendorf M (2017) The cardiac microenvironment uses non-canonicalWNT signaling to activate monocytes after myocardial infarction. EMBO Mol Med 9:1279–1293

33 Nagpal V, Rai R, Place AT, Murphy SB, Verma SK, Ghosh AK, Vaughan DE (2016) MiR-125b is critical for fibroblast-to-myofibroblast transition and cardiac fibrosis. Circulation 133:291–301

34 Nahrendorf M, Swirski FK (2013) Monocyte and macrophage heterogeneity in the heart. Circ Res 112:1624–1633

35 Nakada Y, Canseco DC, Thet S,Abdisalaam S, Asaithamby A, Santos CX, Shah AM, Zhang H, Faber JE, Kinter MT (2017) Hypoxia induces heart regeneration in adult mice . Nature 541:222–227

36 Paterson MR, Kriegel AJ (2017) MiR-146a/b: a family with shared seeds and different roots. Physiol Genomics 49:243–252

37 Peng L, Zhang H, Hao Y, Xu F,Yang J,Zhang R,Lu G, Zheng Z, Cui M, Qi C (2016) Reprogramming macrophage orientation by microRNA 146b targeting transcription factor IRF5. eBiomedicine 14:83–96

38 Peres DL, Schuman ML, Aisicovich M, Toblli JE, Pirola CJ, Landa MS, Garcia SI(2018) Angiotensin II requires an intact cardiac thyrotropin-releasing hormone (TRH) system to induce cardiac hypertrophy in mouse. J Mol Cell Cardiol 124:1–11

39 Prabhu SD, Frangogiannis NG (2016a) The biological basis for cardiac repair after myocardial infarction. Circ Res 119:91–112

40 Prabhu SD, Frangogiannis NG (2016b) The biological basis for cardiac repair after myocardial infarction: from inflammation to fibrosis. Circ Res 119:91–112

41 Purcell BP, Lobb D, Charati MB, Dorsey SM, Wade RJ, Zellars KN, Doviak H, Pettaway S,Logdon CB, Shuman JA (2014) Injectable and bioresponsive hydrogels for on-demand matrix metalloproteinase inhibition. Nat Mater 13:653–661

42 Shen Y,Lynch JJ, Shannon RP, Wiedmann RT (1999) A novel heart failure model induced by sequential coronary artery occlusions and tachycardiac stress in awake pigs. Am J Physiol-Heart C 277:H388–H398

43 Shiraishi M,Shintani Y, Shintani Y, Ishida H, Saba R, Yamaguchi A, Adachi H, Yashiro K, Suzuki K (2016) Alternatively activated macrophages determine repair of the infarcted adult murine heart. J Clin Invest 126:2151–2166

44 Sutton MG, Sharpe N (2000) Left ventricular remodeling after myocardial infarction: pathophysiology and therapy. Circulation 101:2981–2988

45 Swirski FK, Nahrendorf M (2013) Macrophage-stem cell crosstalk after myocardial infarction*. J Am Coll Cardiol 62:1902–1904

46 Taganov KD, Boldin MP, Chang KJ, Baltimore D (2006) NF-kappaBdependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses . Proc Natl Acad Sci USA 103:12481–12486

47 Thum T, Gross C, Fiedler J, Fischer T, Kissler S, Bussen M, Galuppo P,Just S, Rottbauer W,Frantz S (2008) MicroRNA-21 contributes to myocardial disease by stimulating MAP kinase signalling in fibroblasts. Nature 456:980–984

48 Torres WM, Jacobs J, Doviak H, Barlow SC, Zile MR, Shazly T, Spinale FG (2018) Regional and temporal changes in left ventricular strain and stiffness in a porcine model of myocardial infarction. Am J Physiol Heart Circ Physiol 315:H958–H967

49 Wang J, Wang Y, Han J, Li Y, Xie C, Xie L,Shi J, Zhang J,Yang B,Chen D (2015) Integrated analysis of microRNA and mRNA expression profiles in the left atrium of patients with nonvalvular paroxysmal atrial fibrillation: Role of miR-146b-5p in atrial fibrosis. Heart Rhythm 12:1018–1026

50 Westman PC, Lipinski MJ, Luger D, Waksman R, Bonow RO, Wu E,Epstein SE (2016) Inflammation as a driver of adverse left ventricular remodeling after acute myocardial infarction. J Am Coll Cardiol 67:2050–2060

51 Yang F,Chen Q, He S, Yang M, Maguire EM, An W,Afzal TA, Luong LA, Zhang L, Xiao Q(2018a) miR-22 Is a novel mediator of vascular smooth muscle cell phenotypic modulation and neointima formation. Circulation 137:1824–1841

52 Yang L, Wang B, Zhou Q,Wang Y, Liu X,Liu Z,Zhan Z (2018b) MicroRNA-21 prevents excessive inflammation and cardiac dysfunction after myocardial infarction through targeting KBTBD7. Cell Death Dis 9:769

53 Yuan J, Liu H, Gao W, Zhang L, Ye Y, Yuan L, Ding Z, Wu J, Kang L, Zhang X (2018) MicroRNA-378 suppresses myocardial fibrosis through a paracrine mechanism at the early stage of cardiac hypertrophy following mechanical stress. Theranostics 8:2565–2582

54 Zhang L, Fu Y, Wang H, Guan Y, Zhu W,Guo M, Zheng N, Wu Z (2019) Severe fever with thrombocytopenia syndrome virusinduced macrophage differentiation is regulated by miR-146. Front Immunol 10:1095

55 Zhu H, Sun A, Zhu H, Li Z, Huang Z, Zhang S, Ma X, Zou Y,Hu K, Ge J (2014a) Aldehyde dehydrogenase-2 is a host factor required for effective bone marrow mesenchymal stem cell therapy. Arterioscler Thromb Vasc Biol 34:894–901

56 Zhu H,Sun A, Zou Y, Ge J (2014b) Inducible metabolic adaptation promotes mesenchymal stem cell therapy for ischemia. Arterioscler Thromb Vasc Biol 34:870–876