Advertisement

AGE

, Volume 35, Issue 4, pp 1157–1172 | Cite as

MiR-146a as marker of senescence-associated pro-inflammatory status in cells involved in vascular remodelling

  • Fabiola OlivieriEmail author
  • Raffaella Lazzarini
  • Rina Recchioni
  • Fiorella Marcheselli
  • Maria Rita Rippo
  • Silvia Di Nuzzo
  • Maria Cristina Albertini
  • Laura Graciotti
  • Lucia Babini
  • Serena Mariotti
  • Giorgio Spada
  • Angela Marie Abbatecola
  • Roberto Antonicelli
  • Claudio Franceschi
  • Antonio Domenico Procopio
Article

Abstract

In order to identify new markers of vascular cell senescence with potential in vivo implications, primary cultured endothelial cells, including human umbilical vein endothelial cells (HUVECs), human aortic endothelial cells (HAECs), human coronary artery endothelial cells (HCAECs) and ex vivo circulating angiogenic cells (CACs), were analysed for microRNA (miR) expression. Among the 367 profiled miRs in HUVECs, miR-146a, miR-9, miR-204 and miR-367 showed the highest up-regulation in senescent cells. Their predicted target genes belong to nine common pathways, including Toll-like receptor signalling (TLR) that plays a pivotal role in inflammatory response, a key feature of senescence (inflammaging). MiR-146a was the most up-regulated miR in the validation analysis (>10-fold). Mimic and antagomir transfection confirmed TLR’s IL-1 receptor-associated kinase (IRAK1) protein modulation in both young and senescent cells. Significant correlations were observed among miR-146a expression and β-galactosidase expression, telomere length and telomerase activity. MiR-146a hyper-expression was also validated in senescent HAECs (>4-fold) and HCAECs (>30-fold). We recently showed that CACs from patients with chronic heart failure (CHF) presented a distinguishing feature of senescence. Therefore, we also included miR-146a expression determination in CACs from 37 CHF patients and 35 healthy control subjects (CTR) for this study. Interestingly, a 1,000-fold increased expression of miR-146a was observed in CACs of CHF patients compared to CTR, along with decreased expression of IRAK1 protein. Moreover, significant correlations among miR-146a expression, telomere length and telomerase activity were observed. Overall, our findings indicate that miR-146a is a marker of a senescence-associated pro-inflammatory status in vascular remodelling cells.

Keywords

Vascular senescence MiR-146a Circulating angiogenic cells Congestive heart failure Toll-like receptor pathway 

Supplementary material

11357_2012_9440_MOESM1_ESM.doc (42 kb)
ESM 1 (DOC 46 kb)
11357_2012_9440_MOESM2_ESM.doc (42 kb)
Table 1 IL-1 β, 1-α, -2, -6, -8, -10, -12, TNF-a, INF-γ and mieloperoxidase (MPO) concentration were measured at II, III, V, IX, XI, XII and XIII passages. Values were reported as pg/ml (DOC 41 kb)
11357_2012_9440_MOESM3_ESM.doc (264 kb)
Table 2 MiRNAs profiling results in senescent (XIII) vs. young (II) HUVECs. Each value corresponds to the fold difference expression of single microRNA, calculated as ∆∆Ct. ∆∆CT for each miR was defined as expression changes of senescent vs. young HUVEC, calculated with the following equation: [(CT senescent microRNA- median Ct values obtained in the profiling of senescent cells)-(CT young microRNA- median Ct values obtained in the profiling of young cells)] (DOC 264 kb)
11357_2012_9440_Fig8_ESM.jpg (193 kb)
Fig. 1

Markers of cellular senescence in HUVEC cells until replicative proliferation arrest: a growth curve; cell population doubling (CPD) from I to XIII passages. b SΑ−β-gal staining; percentage of positive SA-β-gal cells. c Telomere length; telomere restriction fragment (TRF) length. d Telomerase activity (TERT) (JPEG 193 kb)

11357_2012_9440_MOESM4_ESM.tif (193 kb)
High resolution image (TIFF 193 kb)
11357_2012_9440_Fig9_ESM.jpg (227 kb)
Fig. 2

IL-1 β, 1-α, -2, -6, -8, -10, -12, TNF-α, INF-γ and mieloperoxidase (MPO) release in young (II) and senescent (XIII) HUVECs (values were reported as pg/ml). T-test * p < 0.05 for all comparisons (JPEG 227 kb)

11357_2012_9440_MOESM5_ESM.tif (227 kb)
High resolution image (TIFF 227 kb)

References

  1. Albertini MC, Olivieri F, Lazzarini R, Pilolli F, Galli F, Spada G, Accorsi A, Rippo MR, Procopio AD (2011) Predicting microRNA modulation in human prostate cancer using a simple String IDentifier (SID1.0). J Biomed Inform 44:615–620. doi: org/10.1016/j.jbi.2011.02.006 PubMedCrossRefGoogle Scholar
  2. Bates DJ, Li N, Liang R, Sarojini H, An J, Masternak MM, Bartke A, Wang E (2010) MicroRNA regulation in Ames dwarf mouse liver may contribute to delayed aging. Aging Cell 9:1–18. doi: 10.1111/j.1474-9726.2009.00529.x PubMedCrossRefGoogle Scholar
  3. Bazzoni F, Rossato M, Fabbri M, Gaudiosi D, Mirolo M, Mori L, Tamassia N, Mantovani A, Cassatella MA, Locati M (2009) Induction and regulatory function of miR-9 in human monocytes and neutrophils exposed to proinflammatory signals. Proc Natl Acad Sci U S A 106:5282–5287. doi: 10.1073/pnas.0810909106 PubMedCrossRefGoogle Scholar
  4. Bhaumik D, Scott GK, Schokrpur S, Patil CK, Orjalo AV, Rodier F, Lithgow GJ, Campisi J (2009) MicroRNAs miR-146a/b negatively modulate the senescence-associated inflammatory mediators IL-6 and IL-8. Aging 1:402–411PubMedGoogle Scholar
  5. Bonifacio LN, Jarstfer MB (2010) MiRNA profile associated with replicative senescence, extended cell culture, and ectopic telomerase expression in human foreskin fibroblasts. PLoS One 5(9):e12519. doi: 10.1371/journal.pone.001251 PubMedCrossRefGoogle Scholar
  6. Campisi J, d'Adda di Fagagna F (2007) Cellular senescence: when bad things happen to good cells. Nat Rev Mol Cell Biol 8:729–740. doi: 10.1038/nrm2233 PubMedCrossRefGoogle Scholar
  7. Cawthon RM (2002) Telomere measurement by quantitative PCR. Nucleic Acids Res 15: 30(10):e47Google Scholar
  8. Christoffersen NR, Shalgi R, Frankel LB, Leucci E, Lees M, Klausen M, Pilpel Y, Nielsen FC, Oren M, Lund AH (2010) p53-independent upregulation of miR-34a during oncogene-induced senescence represses MYC. Cell Death Differ 17:236–245. doi: 10.1038/cdd.2009.109 PubMedCrossRefGoogle Scholar
  9. D'Alessandra Y, Devanna P, Limana F, Straino S, Di Carlo A, Brambilla PG, Rubino M, Carena MC, Spazzafumo L, De Simone M, Micheli B, Biglioli P, Achilli F, Martelli F, Maggiolini S, Marenzi G, Pompilio G, Capogrossi MC (2010) Circulating microRNAs are new and sensitive biomarkers of myocardial infarction. Eur Heart J 31:2765–2773. doi: 10.1093/eurheartj/ehq167 PubMedCrossRefGoogle Scholar
  10. Donato AJ, Black AD, Jablonski KL, Gano LB, Seals DR (2008) Aging is associated with greater nuclear NF kappa B, reduced I kappa B alpha, and increased expression of proinflammatory cytokines in vascular endothelial cells of healthy humans. Aging Cell 7:805–812. doi: 10.1111/j.1474-9726.2008.00438.x PubMedCrossRefGoogle Scholar
  11. Edgar R, Domrachev M, Lash AE (2002) Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res 30:207–210PubMedCrossRefGoogle Scholar
  12. Fadini GP, Baesso I, Albiero M, Sartore S, Agostini C, Avogaro A (2008) Technical notes on endothelial progenitor cells: ways to escape from the knowledge plateau. Atherosclerosis 197:496–503. doi: 10.1016/j.atherosclerosis.2007.12.039 PubMedCrossRefGoogle Scholar
  13. Freund A, Orjalo AV, Desprez PY, Campisi J (2010) Inflammatory networks during cellular senescence: causes and consequences. Trends Mol Med 16:238–246. doi: 10.1016/j.molmed.2010.03.003 PubMedCrossRefGoogle Scholar
  14. Garfinkel S, Brown S, Wessendorf JH, Maciag T (1994) Post-transcriptional regulation of interleukin 1 alpha in various strains of young and senescent human umbilical vein endothelial cells. Proc Natl Acad Sci U S A 91:1559–1563PubMedCrossRefGoogle Scholar
  15. Hackl M, Brunner S, Fortschegger K, Schreiner C, Micutkova L, Mück C, Laschober GT, Lepperdinger G, Sampson N, Berger P, Herndler-Brandstetter D, Wieser M, Kühnel H, Strasser A, Rinnerthaler M, Breitenbach M, Mildner M, Eckhart L, Tschachler E, Trost A, Bauer JW, Papak C, Trajanoski Z, Scheideler M, Grillari-Voglauer R, Grubeck-Loebenstein B, Jansen-Dürr P, Grillari J (2010) miR-17, miR-19b, miR-20a, and miR-106a are down-regulated in human aging. Aging Cell 9:291–296. doi: 10.1111/j.1474-9726.2010.00549.x PubMedCrossRefGoogle Scholar
  16. Haendeler J, Hoffmann J, Diehl JF, Vasa M, Spyridopoulos I, Zeiher AM, Dimmeler S (2004) Antioxidants inhibit nuclear export of telomerase reverse transcriptase and delay replicative senescence of endothelial cells. Circ Res 94:768–775. doi: 10.1161/01.RES.0000121104.05977.F3 PubMedCrossRefGoogle Scholar
  17. Ito T, Yagi S, Yamakuchi M (2010) MicroRNA-34a regulation of endothelial senescence. Biochem Biophys Res Commun 398:735–740. doi: 10.1016/j.bbrc.2010.07.012 | PubMedCrossRefGoogle Scholar
  18. Kovacic JC, Moreno P, Hachinski V, Nabel EG, Fuster V (2011) Cellular senescence, vascular disease, and aging: part 1 of a 2-part review. Circulation 123:1900–1910. doi: 10.1161/CIRCULATIONAHA.110.007021 PubMedCrossRefGoogle Scholar
  19. Lafferty-Whyte K, Cairney CJ, Jamieson NB, Oien KA, Keith WN (2009) Pathway analysis of senescence-associated miRNA targets reveals common processes to different senescence induction mechanisms. Biochim Biophys Acta 1792:341–352. doi: 10.1016/j.bbadis.2009.02.003 PubMedCrossRefGoogle Scholar
  20. Lewis BP, Burge CB, Bartel DP (2005) Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120:15–20. doi: 10.1016/j.cell.2004.12.035 PubMedCrossRefGoogle Scholar
  21. Li G, Luna C, Qiu J, Epstein DL, Gonzalez P (2009) Alterations in microRNA expression in stress-induced cellular senescence. Mech Ageing Dev 130:731–741. doi: 10.1016/j.mad.2009.09.002 PubMedCrossRefGoogle Scholar
  22. Li G, Luna C, Qiu J, Epstein DL, Gonzalez P (2010) Modulation of inflammatory markers by miR-146a during replicative senescence in trabecular meshwork cells. Invest Ophthalmol Vis Sci 6:2976–2985. doi: 10.1167/iovs.09-4874 CrossRefGoogle Scholar
  23. Li G, Luna C, Qiu J, Epstein DL, Gonzalez P (2011) Role of miR-204 in the regulation of apoptosis, endoplasmic reticulum stress response, and inflammation in human trabecular meshwork cells. Invest Ophthalmol Vis Sci 52:2999–3007. doi: 10.1167/iovs.10-6708 PubMedCrossRefGoogle Scholar
  24. Liang R, Bates DJ, Wang E (2009) Epigenetic control of microRNA expression and aging. Curr Genomics 10:184–193. doi: 10.2174/138920209788185225 PubMedCrossRefGoogle Scholar
  25. Lin J, Epel E, Cheon J, Kroenke C, Sinclair E, Bigos M, Wolkowitz O, Mellon S, Blackburn E (2010) Analyses and comparisons of telomerase activity and telomere length in human T and B cells: insights for epidemiology of telomere maintenance. J Immunol Methods 352:71–80. doi: 10.1016/j.jim.2009.09.012 PubMedCrossRefGoogle Scholar
  26. Ma X, Becker Buscaglia LE, Barker JR, Li Y (2011) MicroRNAs in NF-{kappa}B signaling. J Mol Cell Biol 3:159–166. doi: 10.1093/jmcb/mjr007 PubMedCrossRefGoogle Scholar
  27. Maes OC, Sarojini H, Wang E (2009) Stepwise up-regulation of microRNA expression levels from replicating to reversible and irreversible growth arrest states in WI-38 human fibroblasts. J Cell Physiol 221:109–119. doi: 10.1002/jcp. 21834 PubMedCrossRefGoogle Scholar
  28. Magenta A, Cencioni C, Fasanaro P, Zaccagnini G, Greco S, Sarra-Ferraris G, Antonini A, Martelli F, Capogrossi MC (2011) miR-200c is upregulated by oxidative stress and induces endothelial cell apoptosis and senescence via ZEB1 inhibition. Cell Death Differ 18(10):1628–1639. doi: 10.1038/cdd.2011.42 PubMedCrossRefGoogle Scholar
  29. Martinez I, Almstead LL, Di Maio D (2011) MicroRNAs and senescence. Aging 3:77–78PubMedGoogle Scholar
  30. Menghini R, Casagrande V, Cardellini M, Martelli E, Terrinoni A, Amati F, Vasa-Nicotera M, Ippoliti A, Novelli G, Melino G, Lauro R, Federici M (2009) MicroRNA 217 modulates endothelial cell senescence via silent information regulator 1. Circulation 120:1524–1532, CIRCULATIONAHA.109.864629v1PubMedCrossRefGoogle Scholar
  31. Meyer SU, Pfaffl MW, Ulbrich SE (2010) Normalization strategies for microRNA profiling experiments: a 'normal' way to a hidden layer of complexity? Biotechnol Lett 32:1777–1788. doi: 10.1007/s10529-010-0380-z PubMedCrossRefGoogle Scholar
  32. Minamino T, Komuro I (2007) Vascular cell senescence: contribution to atherosclerosis. Circ Res 100(1):15–26. doi: 10.1161/01.RES.0000256837.40544.4a PubMedCrossRefGoogle Scholar
  33. Minamino T, Miyauchi H, Yoshida T, Ishida Y, Yoshida H, Komuro I (2002) Endothelial cell senescence in human atherosclerosis: role of telomere in endothelial dysfunction. Circulation 105(13):1541–1544. doi: 10.1161/01.CIR.0000013836.85741.17 PubMedCrossRefGoogle Scholar
  34. Nahid MA, Satoh M, Chan EK (2011) MicroRNA in TLR signaling and endotoxin tolerance. Cell Mol Immunol 8:388–403. doi: 10.1038/cmi.2011.26 PubMedCrossRefGoogle Scholar
  35. Nazari-Jahantigh M, Wei Y, Schober A (2012) The role of microRNAs in arterial remodelling. Thromb Haemost 107(4):611–618PubMedCrossRefGoogle Scholar
  36. Olivieri F, Lorenzi M, Antonicelli R, Testa R, Sirolla C, Cardelli M, Mariotti S, Marchegiani F, Marra M, Spazzafumo L, Bonfigli AR, Procopio A (2009) Leukocyte telomere shortening in elderly Type2DM patients with previous myocardial infarction. Atherosclerosis 206:588–593. doi: 10.1016/j.atherosclerosis.2009.03.034 PubMedCrossRefGoogle Scholar
  37. Olivieri F, Antonicelli R, Recchioni R, Mariotti S, Marcheselli F, Lisa R, Spazzafumo L, Galeazzi R, Caraceni D, Testa R, Latini R, Procopio AD (2012) Telomere/telomerase system impairment in circulating angiogenic cells of geriatric patients with heart failure. Int J Cardiol. doi: 10.1016/j.ijcard.2011.06.091
  38. Papadopoulos GL, Alexiou P, Maragkakis M, Reczko M, Hatzigeorgiou AG (2009) DIANA-mirPath: Integrating human and mouse microRNAs in pathways. Bioinformatics 25:1991–1993. doi: 10.1093/bioinformatics/btp299 PubMedCrossRefGoogle Scholar
  39. Passos JF, Simillion C, Hallinan J, Wipat A, von Zglinicki T (2009) Cellular senescence: unravelling complexity. Age 31:353–363. doi: 10.1007/s11357-009-9108-1 PubMedCrossRefGoogle Scholar
  40. Poliseno L, Pitto L, Simili M, Mariani L, Riccardi L, Ciucci A, Rizzo M, Evangelista M, Mercatanti A, Pandolfi PP, Rainaldi G (2008) The proto-oncogene LRF is under post-transcriptional control of MiR-20a: implications for senescence. PLoS One 2:2542. doi: 10.1371/journal.pone.0002542 CrossRefGoogle Scholar
  41. Rehman J, Li J, Orschell CM, March KL (2003) Peripheral blood "endothelial progenitor cells" are derived from monocyte/macrophages and secrete angiogenic growth factors. Circulation 4:1164–1169. doi: 10.1161/01.CIR.0000058702.69484.A0 CrossRefGoogle Scholar
  42. Saliques S, Zeller M, Lorin J, Lorgis L, Teyssier JR, Cottin Y, Rochette L, Vergely C (2010) Telomere length and cardiovascular disease. Arch Cardiovasc Dis 103:454–459. doi: 10.1016/j.acvd.2010.08.002 PubMedCrossRefGoogle Scholar
  43. Sikora E, Arendt T, Bennett M, Narita M (2011) Impact of cellular senescence signature on ageing research. Ageing Res Rev 10:146–152. doi: 10.1016/j.arr.2010.10.002 PubMedCrossRefGoogle Scholar
  44. Taganov KD, Boldin MP, Chang KJ, Baltimore D (2006) NF-kappaB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses. Proc Natl Acad Sci U S A 103:2481–2486. doi: 10.1073/pnas.0605298103 CrossRefGoogle Scholar
  45. Testa R, Olivieri F, Sirolla C, Spazzafumo L, Rippo MR, Marra M, Bonfigli AR, Ceriello A, Antonicelli R, Franceschi C, Castellucci C, Testa I, Procopio AD (2011) Leukocyte telomere length is associated with complications of type 2 diabetes mellitus. Diabet Med 28(11):1388–1394. doi: 10.1111/j.1464-5491.2011.03370.x PubMedCrossRefGoogle Scholar
  46. Unterluggauer H, Hütter E, Voglauer R, Grillari J, Vöth M, Bereiter-Hahn J, Jansen-Dürr P, Jendrach M (2007) Identification of cultivation-independent markers of human endothelial cell senescence in vitro. Biogerontology 8:383–397. doi: 10.1007/s10522-007-9082-x PubMedCrossRefGoogle Scholar
  47. Vasa-Nicotera M, Chen H, Tucci P, Yang AL, Saintigny G, Menghini R, Mahè C, Agostini M, Knight RA, Melino G, Federici M (2011) miR-146a is modulated in human endothelial cell with aging. Atherosclerosis 217:326–330. doi: 10.1016/j.atherosclerosis.2011.03.034 PubMedCrossRefGoogle Scholar
  48. Voghel G, Thorin-Trescases N, Farhat N, Nguyen A, Villeneuve L, Mamarbachi AM, Fortier A, Perrault LP, Carrier M, Thorin E (2007) Cellular senescence in endothelial cells from atherosclerotic patients is accelerated by oxidative stress associated with cardiovascular risk factors. Mech Ageing Dev 128(11–12):662–671. doi: 10.1016/j. mad.2007.09.006 PubMedCrossRefGoogle Scholar
  49. Wagner W, Horn P, Castoldi M, Diehlmann A, Bork S, Saffrich R, Benes V, Blake J, Pfister S, Eckstein V, Ho AD (2008) Replicative senescence of mesenchymal stem cells: a continuous and organized process. PLoS One 21:e2213. doi: 10.1371/journal.pone.0002213 CrossRefGoogle Scholar
  50. Wege H, Chui MS, Le HT, Tran JM, Zern MA (2003) SYBR Green real-time telomeric repeat amplification protocol for the rapid quantification of telomerase activity. Nucleic Acids Res 31:E3–3PubMedCrossRefGoogle Scholar
  51. Wong L, Lee K, Russell I, Chen C (2007) Endogenous controls for real-time quantitation of miRNA using TaqMan® microRNA assays. Applied Biosystems Application Note, Publication 127AP11-01, available at: www.appliedbiosystems.com

Copyright information

© American Aging Association 2012

Authors and Affiliations

  • Fabiola Olivieri
    • 1
    • 2
    Email author
  • Raffaella Lazzarini
    • 1
    • 2
  • Rina Recchioni
    • 2
  • Fiorella Marcheselli
    • 2
  • Maria Rita Rippo
    • 1
  • Silvia Di Nuzzo
    • 1
  • Maria Cristina Albertini
    • 3
  • Laura Graciotti
    • 1
  • Lucia Babini
    • 1
  • Serena Mariotti
    • 2
  • Giorgio Spada
    • 4
  • Angela Marie Abbatecola
    • 5
  • Roberto Antonicelli
    • 6
  • Claudio Franceschi
    • 7
    • 8
  • Antonio Domenico Procopio
    • 1
    • 2
  1. 1.Department of Clinical and Molecular SciencesUniversità Politecnica delle MarcheAnconaItaly
  2. 2.Center of Clinical Pathology and Innovative TherapyIRCCS-INRCA, National InstituteAnconaItaly
  3. 3.Dipartimento di Scienze Biomolecolari, Sezione di Biochimica e Biologia molecolareUniversità degli Studi di Urbino “Carlo Bo”UrbinoItaly
  4. 4.Dipartimento di Scienze di Base e FondamentiUniversità degli Studi di Urbino “Carlo Bo”UrbinoItaly
  5. 5.Scientific DirectionIRCCS-INRCA, National InstituteAnconaItaly
  6. 6.Cardiology UnitIRCCS-INRCA, National InstituteAnconaItaly
  7. 7.Department of Experimental Pathology“Alma Mater Studiorum” University of BolognaBolognaItaly
  8. 8.Centro Interdipartimentale Galvani “CIG”Alma Mater Studiorum University of BolognaBolognaItaly

Personalised recommendations