Senescence-based predicted potency of extracellular vesicles determines their rejuvenating cardiac effects in an ageing rodent model

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación CIBERCV Background Cardiac ageing is characterized by increased cell senescence, hypertrophy and diastolic dysfunction, which can frequently lead to heart failure with preserved ejection fraction. Still, this is an unmet medical need for treatment development. Cardiosphere-derived cells (CDCs) have demonstrated efficacy in old animals with cardiac dysfunction, however variability of effect among different CDC-donors is observed. Purpose To identify if the in vitro anti-senescent potential of CDC-secreted extracellular vesicles (CDC-EVs) predicts the in vivo anti-senescent effect in a model of cardiac ageing. Methods CDCs were derived from cardiac biopsies of 18 patients (age range 0 - 81 years old, both sexes included) who underwent cardiac surgery for other reasons. CDC-secreted extracellular vesicles (CDC-EVs) from the 18 donors were purified and used in vitro and in vivo experiments. Their in vitro anti-senescent potential (at genetic, secretory and cellular level) was evaluated over cardiac stromal cells from two human patients. CDC-EVs with the most anti-senescent potential and with the least anti-senescent potential were classified as potent (P-EVs) and non-potent (NP-EVs), respectively. Cardiac anti-senescent effect of P-EVs and NP-EVs were then tested in vivo in rats with induced cardiac aging. 3-months old SD-rats (30% female) received 3-months of daily intraperitoneal injections of saline (healthy control) or D-galactose. Animals in the D-Gal group were randomly allocated to receive intraperitoneal injection of saline (sham control, n=12), P-EVs (n=7) or NP-EVs (n=6) and followed-up for one month. Results Variations in the extent of the anti-senescent potential were observed in vitro among the CDC-EVs from the different donors. While most CDC-EVs significantly reduced cellular senescence and increased IL-6 secretion of human cardiac stromal cells, most CDC-EVs decreased the expression of senescence-related genes (p21, p16, p53, TGF-b). In vivo, D-gal induced cardiac hypertrophy (2.5 mg/g vs. 2.2 mg/g, p<0.01). P-EVs, but not NP-EVs, prevented this hypertrophy (2.4 vs. 2.9 mg/gr, p=0.05). This finding was in parallel to the levels of galactosidase-beta 1 expression in cardiac tissue, which were increased in sham vs healthy (1.22 vs. 0.78, p=0.04) and in sham vs. P-EVs (1.22 vs. 0.73, p=0.03) but levels in NP-EVs were not significantly different to the sham group (0.86, p=0.1). Conclusions The anti-senescent in vitro effect of CDC-EVs is correlated to their anti-senescent and anti-hypertrophic effect in an in vivo model of cardiac ageing. This study shows the relevance of exploring senescence-related markers as determinant of therapeutic potency of the tested product and confirms the potential utility of CDC-EVs as a therapy for cardiac ageing-related pathologies.