Extracorporeal Cardiac Shock Wave-Induced Exosome Derived From Endothelial Colony-Forming Cells Carrying miR-140-3p Alleviate Cardiomyocyte Hypoxia/Reoxygenation Injury via the PTEN/PI3K/AKT Pathway.

Dan Yang,Mingqiang Wang,Hongbo Cai,Tao Guo,Yunke Shi,Zhao Hu,Xingyu Cao,Yiming Ma,Hongyan Cai

doi:10.3389/fcell.2021.779936

Abstract

Background: Stem cell-derived exosomes have great potential in the treatment of myocardial ischemia–reperfusion injury (IRI). Extracorporeal cardiac shock waves (ECSW) as effective therapy, in part, could activate the function of exosomes. In this study, we explored the effect of ECSW-induced exosome derived from endothelial colony-forming cells on cardiomyocyte hypoxia/reoxygenation (H/R) injury and its underlying mechanisms. Methods: The exosomes were extracted and purified from the supernatant of endothelial colony-forming cells (ECFCs-exo). ECFCs-exo treated with shock wave (SW-exo) or without shock wave (CON-exo) were performed with high-throughput sequencing of the miRNA. H9c2 cells were incubated with SW-exo or CON-exo after H/R injury. The cell viability, cell apoptosis, oxidative stress level, and inflammatory factor were assessed. qRT-PCR was used to detect the expression levels of miRNA and mRNA in cells and exosomes. The PTEN/PI3K/AKT pathway-related proteins were detected by Western blotting, respectively. Results: Exosomes secreted by ECFCs could be taken up by H9c2 cells. Administration of SW-exo to H9c2 cells after H/R injury could significantly improve cell viability, inhibit cell apoptosis, and downregulate oxidative stress level (p < 0.01), with an increase in Bcl-2 protein and a decrease in Bax, cleaved caspase-3, and NF-κB protein (p < 0.05). Notably, miR-140-3p was found to be highly enriched both in ECFCs and ECFCs-exo treated with ECSW (p < 0.05) and served as a critical mediator. SW-exo increased miR-140-3p expression but decreased PTEN expression in H9c2 cells with enhanced phosphorylation of the PI3K/AKT signaling pathway. These cardioprotective effects of SW-exo on H/R injury were blunted by the miR-140-3p inhibitor. Dual-luciferase assay verified that miR-140-3p could directly target the 3′UTR of PTEN mRNA and exert a negative regulatory effect. Conclusion: This study has shown the potential of ECSW as an effective stimulation for the exosomes derived from ECFCs in vitro. SW-exo exerted a stronger therapeutic effect on H/R injury in H9c2 cells possibly via delivering exosomal miR-140-3p, which might be a novel promising strategy for the myocardial IRI.

Highlights

Acute myocardial infarction (AMI) is a serious killer threatening human health and life with a high rate of morbidity and mortality worldwide (Reed et al, 2017)
Fluorescence microscopy showed that endothelial colony-forming cells (ECFCs) were double-stained attributed to the ability of Dil-Ac-LDL uptake and FITC-UEA I binding (Figure 1B)
The obtained cells were identified by cell morphology, cell surface marker detection, and cell angiogenic function, which were akin to endothelial colony-forming cells

Summary

Introduction

Acute myocardial infarction (AMI) is a serious killer threatening human health and life with a high rate of morbidity and mortality worldwide (Reed et al, 2017). Clinical studies suggested that patients with myocardial IRI were closely related to poor prognosis owing to their aggravated progression of heart failure and worsening ventricular remodeling (Stone et al, 2016). In this regard, some promising approaches to multitargeted cardioprotective therapy were proposed in order to reduce myocardial IRI (Davidson et al, 2019). Stem cell-derived exosomes have great potential in the treatment of myocardial ischemia–reperfusion injury (IRI). We explored the effect of ECSW-induced exosome derived from endothelial colonyforming cells on cardiomyocyte hypoxia/reoxygenation (H/R) injury and its underlying mechanisms

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Discussion

Conclusion