BACKGROUND Cardiosphere-derived cells (CDCs) mediate therapeutic regeneration in patients after myocardial infarction and are undergoing further clinical testing for cardiomyopathy. The beneficial effects of CDCs are mediated by the secretion of exosomes and possibly other extracellular membrane vesicles (EMV). OBJECTIVES We investigated the effect of cardiosphere-derived EMVs (CSp-EMV) on fibroblasts in vitro and tested whether priming with CSp-EMV could confer salutary properties on fibroblasts in vivo. METHODS CSp-EMVs were isolated from serum-free media conditioned for 3 days by cardiospheres. Dermal fibroblasts were primed with CSp-EMV for 24 h followed by exosomal micro-ribonucleic acid (miRNA) profiling. In vivo, we injected CSp-EMV-primed or -unprimed dermal fibroblasts (or CSp-EMV) in a chronic rat model of myocardial infarction and defined the functional and structural consequences. RESULTS CSp-EMV amplified their own biological signals: exposure of “inert” fibroblasts to CSp-EMV rendered the fibroblasts therapeutic. Intramyocardially-injected CSp-EMV-primed (but not unprimed) fibroblasts increased global pump function and vessel density while reducing scar mass. CSp-EMV priming caused fibroblasts to secrete much higher levels of stromal-cell derived factor 1 and vascular endothelial growth factor, and dramatically changed the microRNA profile of fibroblast-secreted EMVs in vitro. The priming was followed by significant angiogenic and cardioprotective effects. CONCLUSIONS CSp-EMVs alter fibroblast phenotype and secretome in a salutary positive-feedback loop. The phenotypic conversion of inert cells to therapeutically-active cells reveals a novel mechanism for amplification of exosome bioactivity.
Background: Multicellular self-assembling cardiospheres (CSps) exert regenerative and anti-fibrotic effects via paracrine mechanisms. CSp-derived cells are known to secrete exosomes which mediate most or all of the beneficial therapeutic effects. Objective: We evaluated the regenerative capacity of CSp-secreted exosomes in a model of chronic myocardial infarction (MI). We also determined whether CSp-exosomes could convert the phenotype of therapeutically-inert cells. Methods: Exosomes were isolated from CSp-conditioned media by adding a precipitation solution followed by centrifugation. One month post-MI, Wistar Kyoto rats (n=46) with permanent LAD ligation were injected intramyocardially with: a) human dermal fibroblasts (DFs), b) CSp exosomes, c) DFs primed with CSp-exosomes, d) CSps only or e) vehicle. Functional and histological analyses were performed 4 weeks after therapy. Mechanisms were also probed in vitro . Results: In vivo, CSp-exosomes and CSps equally increased ejection fraction (EF= 45±1% [CSp-exo], 44±2% [CSps], 33±1% [placebo] and 35±2% [DFs]) and reduced scar mass (48±8mg [CSp-exo], 45±4mg [CSps], 96±12mg [placebo] and 90±6mg [DFS]; both p<0.01 by one way ANOVA). DFs that had been incubated with CSp-exosomes for 24 hours in culture conferred enhanced benefits compared to unprimed DFs (EF= 41±1% [primed-DFs]; p=0.05 vs unprimed DFs; scar mass= 49±5mg [primed-DFs]; p<0.01 vs unprimed DFs). Confocal imaging revealed internalization of fluorescently-labeled CSp-exosomes in exosome-primed DFs. In vitro , exosome-primed DFs increased tube formation by HUVECs and inhibited cardiomyocyte apoptosis. Immunohistochemistry showed increased vessel density in all groups compared to vehicle or unprimed DFs. Conclusions: Administration of CSp-exosomes recapitulates the regenerative potential and functional benefits of CSps themselves. The surprising ability of CSp-exosomes to confer therapeutic efficacy on inert DFs may represent an unanticipated amplification mechanism for exosome-mediated benefits.
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