1Medizinische Hochschule Hannover Kardiologie und Angiologie Hannover, Deutschland; 2Fraunhofer ITEM Hannover, Deutschland; 3Medizinische Hochschule Hannover Institut für Molekulare und Translationale Therapiestrategien, OE-8886 Hannover, Deutschland; 4Medizinische Hochschule Hannover Core Unit Proteomics Hannover, Deutschland
Introduction: Senescent cells have been recently implicated as key players in cardiac and pulmonary fibrosis. The molecular mechanism driving senescent fibroblasts is unknown.
Methods: We produced senescent human fibroblasts (MRC5) in two senescence types: DNA-damage induced senescence through bleomycin (DS) and replicative senescence (RS), within multiple time points and in vitro conditions. We assessed known markers of senescence (p16, p21, LMNB-1, TNFRSF10C, SA-ß-galactosidase staining, γH2AX, nuclear and cell morphology), inflammation (IL-1ß, IL-6), as well as canonic features fibroblast activation (ACTA2, COL1A1, COL3A1, CTGF, MMP2, SMAD4, SMAD7, migration assay) on mRNA, protein and functional level. To decipher the mechanisms driving the secretion of extracellular matrix factors, we performed a LC-MS of senescent fibroblasts. To evaluate paracrine effects of the secretome, we have analyzed the supernatant of senescent fibroblasts.
Results: RS and DS had lower expression levels of canonic fibroblast activation markers than non-senescent fibroblasts, this feature being persistent at various timepoints and irresponsive to TGF-ß stimulation. However, senescent fibroblasts still secreted a plethora of collagen types and other extracellular matrix remodeling factors. The release of collagens from senescent cells could not be targeted through treatment with the current clinically approved medication for the direct treatment of fibrosis (nintedanib and pirfenidone). Bioinformatic analysis showed abnormalities in RNA-metabolism as a critical node behind this phenotype.
Conclusion: We have identified an atypical, autonomous and pro-fibrotic aspect of senescent fibroblasts. This phenotype cannot be targeted with contemporary anti-fibrotics. Therefore, therapeutics specifically targeting the senescence machinery are necessary to fully address fibrosis in heart failure.