Abstract
Numerous experimental and clinical studies have shown a beneficial effect of cardiosphere-derived cell (CDC) therapy on regeneration of injured myocardium. Paracrine signaling by CDC secreted exosomes is hypothesized to be the principal mediator of improved function, however the mechanism of exosome uptake, cell specificity, and information transfer has yet to be elucidated. Our aim was to define the role of physiologically secreted human CDC-derived exosomes on cardiac fibroblasts, endothelial cells and cardiomyocytes by employing a knockdown (KD) strategy against nSMase2 and Rab27a, two genes crucial in exosome secretion. Methods: Human CDCs (hCDCs) were grown from atrial tissue biopsy explant culture and characterized by flow cytometry and immunocytochemistry. CDC-derived exosomes were isolated and characterized by particle size, immunoblot analysis and electron microscopy. Protein concentration of exosome preparations was quantified by Bradford microassay. nSMase2 and Rab27a shRNAi lentivirus was generated and used to create stable CDC knockdown lines. Co-culture transwell in vitro assays were designed to assess target cell proliferation, migration, angiogenesis, and fibrotic gene expression. Results: hCDCs expressed CD105, CD90, GATA4 and Nkx2.5, and were CD45 and cardiac troponin T negative. hCDC-derived exosomes were immunoreactive for CD63 and HSP90 and negative for Cyctochrome c and EEA1. shRNAi KD of nSMase2 and Rab27a resulted in successful blockade of exosome release from hCDCs. HUVECs co-cultured with scrambled shRNAi CDCs versus shRNAi nSMase2 KD CDCs showed increased angiogenesis and migration without affecting proliferation. While TGF-β stimulation of human cardiac fibroblasts significantly increased collagen I (COLI) and collagen III (COLIII) gene expression, there was no significant decrease in COLI or COLIII after treatment with exosomes. Conclusions: Secretion of hCDC-derived exosomes was effectively inhibited by nSMase2 and Rab27a lentiviral KD. Exosome release contributed to the angiogenic and pro-migratory effects of hCDCs but did not play a role in the fibrotic gene expression of human cardiac fibroblasts, suggesting a role for physiological exosome secretion in stem cell-mediated cardiac repair.
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