Abstract 17615: MSC Exosome Mediated Myocardial Repair is Inhibited by Knockdown of Igf-1 Signaling Pathway Through CRISPR/Cas9 Genome Editing
Introduction: Mesenchymal stem cell (MSC) repairs infarcted heart through paracrine mechanism. We sought to determine whether MSC - derived exosomes could enhance myocardial repair and how exosome mediated cardiac repair is regulated.
Methods and Results: MSC was prepared from bone marrow of 2-week old rats and exosome was isolated from MSC conditioned media by ultracentrifugation. MSC - derived exosome was examined by electron microscopy, and the expression of exosome marker CD63 was analyzed by Western blot and flow cytometry. PKH26 -labeled exosomes were delivered into the rat myocardium by direct intramyocardial injection at the time of initial induction of myocardial infarction. In a rat myocardial infarction model, we found exosomes had similar effect as MSC in promoting survival, angiogenesis, improving cardiac function (EF and FS), and inhibiting cardiac fibrosis. To determine how exosome mediated cardiac repair is regulated, we constructed a lentivirus expressing CRISPR-Cas9 and a CRISPR guide RNA targeting IGF-1. The lentivirus particles were transfected into MSC. This CRISPR gene editing approach reduced expression of IGF-1 and pAkt (the downstream kinase of IGF-1 signaling pathway) in MSC as assessed by Western blot and immunostaining. Importantly, the CRISPR/Cas9 based knockdown IGF-1 approach impaired the ability of MSC and exosomes to repair injured heart in a rat myocardial infarction model. These findings were confirmed using an in vitro cardiac injury model. We treated cardiomyocytes H9C2 with H2O2 for 24-96 hours. H2O2 treatment led to H9C2 mitochondrial dysfunction at 48 h as assessed by Rh123, and apoptosis at 96 h as assessed by Death ELISA and Annexin V. Exosome-derived from wild type MSC but not from IGF-1 knockout MSC prevented H2O2-induced H9C2 dysfunction.
Conclusion: This study demonstrates that MSC - derived exosome enhances cardiac repair, and CRISPR/Cas9 based gene editing approach confirmed that IGF-1 signaling pathway plays an important role in regulating exosome’s effect.
Author Disclosures: Y. Li: None. L. Shao: None. Y. Zhang: None. Z. Zhang: None. J. Wang: None. B. Lan: None. L. Zhang: None. B. Zhu: None. C. Han: None. C. Liang: None. X. Pan: None. Y. Geng: None. Z. Shen: None. X. Yu: None.
- © 2016 by American Heart Association, Inc.