Introduction: Recent evidence implicated senescent cells as drivers of cardiac fibrosis. The molecular machinery driving senescent fibroblasts has not been explored.
Methods: We generated senescent human fibroblasts (MRC5) via two methods: DNA-damage induced senescence via alkylating agent bleomycin (DS) and replicative senescence through repeated mitosis (RS). The cells were examined at multiple time points and in vitro environments. We measured known markers of senescence (CDKN2A, CDKN1A, LMNB-1, DCR1, SA-ß-galactosidase staining, phosphorylated-γH2AX, cellular morphology), inflammation (IL-1B, IL-6), as well as conventional markers of fibroblast activation (αSMA, COL1A1, COL3A1, CCN2, MMP2, the SMAD pathway) on mRNA (qPCR), protein (Western Blot, Liquid chromatography–mass spectrometry) and functional level (migration assay). For an unbiased analysis of the senescent associated secretory phenotype (SASP) containing extracellular matrix factors, we performed a LC-MS of the cell lysate and supernatant of senescent fibroblasts.
Results: RS and DS upregulated inflammation markers (IL-1B, IL-6, p<0.05, t-test) but had repressed levels of standard fibroblast activation markers consistently in multiple timepoints and irrespective of TGF-ß stimulation (COL1A1, COL3A1, CCN2, MMP2, the SMAD2, SMAD7, p<0.05, t-test). However, senescent fibroblasts retained a rich secretory phenotype that included multiple collagen types and other extracellular matrix remodeling factors. The SASP could not be inhibited through treatment with the current clinically approved anti-fibrotics (nintedanib and pirfenidone, p>0.05). Bioinformatic analysis implicated strongly deregulated RNA-metabolism as the principal mechanism driving this non-canonical pro-fibrotic phenotype.
Conclusion: In addition to a pro-inflammatory phenotype, we have identified an unconventional pro-fibrotic feature of senescent fibroblasts. Senescence-associated fibrosis cannot be inhibited with contemporary anti-fibrotics. Therefore, new drugs specifically targeting senescent cells are needed to comprehensively inhibit fibrosis in heart failure.
