Antisenescence in vitro potency of extracellular vesicles secreted by cardiosphere-derived cells predicts the antihypertrophic effect in a rat model of cardiac ageing
Authors: Gomez-Cid L , Cervera-Negueruela M , Campo-Fonseca A , Suarez-Sancho S , Pinto A , Gil-Jaurena J M , Fernandez-Santos M E , Fernandez-Aviles F , Grigorian L 
Published: October 2021

Abstract

Background
Cardiac ageing is characterized by hypertrophy and diastolic dysfunction and related with heart failure with preserved ejection fraction, an unmet medical need for treatments development. Cardiosphere-derived cells (CDCs) have demonstrated glimpses of efficacy in old animals with cardiac dysfunction, however variability of effect among different CDC-donors is observed.

Purpose
To identify markers of in vitro potency of CDCs and to test it in an in vivo model of cardiac ageing.

Methods
CDCs were derived from cardiac biopsies of forty patients (age range 0 - 81 years old, 16 females) who underwent cardiac surgery for other reasons. CDC-secreted extracellular vesicles (CDC-EVs) were purified and used in in vitro and in vivo experiments. Since anti-ageing effect was targeted in vivo, in vitro potency of CDC-EVs was checked in terms of their anti-senescence bioactivity. A panel of six parameters (genetic, secretory and cellular) related with cellular senescence were analysed in vitro for all 40-donors CDC-EVs and those with most favourable and unfavourable profiles were classified as potent (P-EVs) and non-potent (NP-EVs), respectively. P-EVs and NP-EVs were then tested in vivo in 25 rats with D-galactose-induced cardiac ageing and 7-healthy controls in a randomized blinded study.

Results
In vitro, despite variations in the extent of the observed effect, all 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). Based on the observed profiles as P-EVs were selected CDC-EVs from 75- and 14-years old donors and as NP-EVs from 73-years old. In vivo, 3-months old SD-rats (30% female) after 3-months of daily intraperitoneal injections of saline (healthy control) or D-Gal, were randomly allocated (D-Gal group) to receive intraperitoneal injection of saline (sham control, n=12), P-EVs (n=7), NP-EVs (n=6) and followed-up for one month. While sham control group developed myocardial hypertrophy compared to healthy control animals (heart/body weight 2.7 vs. 2.2 mg/gr, p=0.03), this effect was prevented by P-EVs but not by NP-EVs (2.5 vs. 2.9 mg/gr, p=0.02). This finding was in parallel to the levels of serum antioxidants which were increased in sham vs healthy (p=0.02) and in sham vs. P-EVs (p=0.01) but levels in NP-EVs were similar to the sham group.

Conclusions
Anti-senescence in vitro effect of CDC-EVs is correlated to their therapeutic efficacy in vivo model of cardiac ageing. The relevance of this study is double: identification of senescence-related markers as determinant of therapeutic potency of the tested product and confirming the potential utility of CDC-EVs as therapy for cardiac ageing-related pathologies.

Funding Acknowledgement
Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Instituto de Salud Carlos III, Ministerio de Ciencia e Innovaciόn,CIBERCV, Spain