Senescence and atherosclerosis: characterization of a replicative senescence model in vascular smooth muscle cells

Cellular senescence is characterized by growth arrest, senescence-associated secretory phenotype (SASP), and oxidative stress. Accumulation of senescent vascular smooth muscle cells (SMCs) contributes to aging and cardiovascular disease. Senescent SMCs are present in atherosclerotic plaques and contribute to their instability. We aimed at establishing the molecular signatures of replicative senescence (RS) in SMCs. Human aortic SMCs were serially passaged to represent different stages of RS and used from 5th to 7th passages (young cells) and from 15th to 17th passages (old cells). We measured SA-β-gal activity (a marker of senescence), genes, proteins, and long non-coding RNA (lncRNAs) expression by qPCR and western blot analysis, morphological and nuclear changes by immunofluorescence, and cell proliferation by cell counting. More than 40% of old cells stained positive for SA-b-gal compared to 10% of young cells and showed an increased b-galactosidase-1 expression. Old cells have a diminished proliferation rate (doubling time of 42 hours compared to 29 hours in young cells), a migratory activity reduced by 50%, a downregulation of contractile markers (a-actin, calponin), but increased cell cycle inhibitors (p21/p16) expression. Senescent/old cells showed a flattened appearance and enlarged and irregular nuclei. The expression levels of LMNB1 and HMGB1 were downregulated in old cells, indicating an altered nuclear membrane. Old cells showed an increased expression of SASP molecules (e.g.NF-kb, IL1b, MMP-1,-2,-3), and of “Related glycolysis inhibitor and calcium channel regulator” (RRAD), recently associated in cellular senescence as a negative regulator. Also, a set of lncRNAs (PURPL, SENEBLOC, NEAT1, MIR31HG and ANRIL) was modulated in old cells. The modification of markers specific for the contractile state and migratory activity, together with low-level proliferation in old SMCs, could contribute significantly to inefficient plaque repair and instability. The detection of novel genes/lncRNAs deregulated in senescent SMCs could be helpful for future studies on potential anti-aging factors.