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

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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.

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Authors and Affiliations

  1. Department of Clinical and Molecular Sciences, Università Politecnica delle Marche, Via Tronto 10/A, 60020, Ancona, Italy

    Fabiola Olivieri, Raffaella Lazzarini, Maria Rita Rippo, Silvia Di Nuzzo, Laura Graciotti, Lucia Babini & Antonio Domenico Procopio

  2. Center of Clinical Pathology and Innovative Therapy, IRCCS-INRCA, National Institute, Ancona, Italy

    Fabiola Olivieri, Raffaella Lazzarini, Rina Recchioni, Fiorella Marcheselli, Serena Mariotti & Antonio Domenico Procopio

  3. Dipartimento di Scienze Biomolecolari, Sezione di Biochimica e Biologia molecolare, Università degli Studi di Urbino “Carlo Bo”, Urbino, Italy

    Maria Cristina Albertini

  4. Dipartimento di Scienze di Base e Fondamenti, Università degli Studi di Urbino “Carlo Bo”, Urbino, Italy

    Giorgio Spada

  5. Scientific Direction, IRCCS-INRCA, National Institute, Ancona, Italy

    Angela Marie Abbatecola

  6. Cardiology Unit, IRCCS-INRCA, National Institute, Ancona, Italy

    Roberto Antonicelli

  7. Department of Experimental Pathology, “Alma Mater Studiorum” University of Bologna, Bologna, Italy

    Claudio Franceschi

  8. Centro Interdipartimentale Galvani “CIG”, Alma Mater Studiorum University of Bologna, Bologna, Italy

    Claudio Franceschi

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  1. Fabiola Olivieri
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  2. Raffaella Lazzarini
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  3. Rina Recchioni
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  9. Lucia Babini
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Correspondence to Fabiola Olivieri.

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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)

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)

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)

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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)

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Olivieri, F., Lazzarini, R., Recchioni, R. et al. MiR-146a as marker of senescence-associated pro-inflammatory status in cells involved in vascular remodelling. AGE 35, 1157–1172 (2013). https://doi.org/10.1007/s11357-012-9440-8

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