Yonsei Med J.  2018 Aug;59(6):736-745. 10.3349/ymj.2018.59.6.736.

Extracellular Vesicles Derived from Hypoxic Human Mesenchymal Stem Cells Attenuate GSK3β Expression via miRNA-26a in an Ischemia-Reperfusion Injury Model

• Park H
• Park H
• Mun D
• Kang J
• Kim H
• Kim M
• Cui S
• Lee SH
• Joung B

• KMID: 2415532
• DOI: http://doi.org/10.3349/ymj.2018.59.6.736

Abstract

PURPOSE
Bioactive molecules critical to intracellular signaling are contained in extracellular vesicles (EVs) and have cardioprotective effects in ischemia/reperfusion (IR) injured hearts. This study investigated the mechanism of the cardioprotective effects of EVs derived from hypoxia-preconditioned human mesenchymal stem cells (MSCs).
MATERIALS AND METHODS
EV solutions (0.4 µg/µL) derived from normoxia-preconditioned MSCs (EVNM) and hypoxia-preconditioned MSCs (EVHM) were delivered in a rat IR injury model. Successful EV delivery was confirmed by the detection of PKH26 staining in hearts from EV-treated rats.
RESULTS
EVHM significantly reduced infarct size (24±2% vs. 8±1%, p < 0.001), and diminished arrhythmias by recovering electrical conduction, INa current, and Cx43 expression. EVHM also reversed reductions in Wnt1 and β-catenin levels and increases in GSK3β induced after IR injury. miRNA-26a was significantly increased in EVHM, compared with EVNM, in real-time PCR. Finally, in in vitro experiments, hypoxia-induced increases in GSK3β expression were significantly reduced by the overexpression of miRNA-26a.
CONCLUSION
EVHM reduced IR injury by suppressing GSK3β expression via miRNA-26a and increased Cx43 expression. These findings suggest that the beneficial effect of EVHM is related with Wnt signaling pathway.

Keyword

Extracellular vesicles; ischemia reperfusion; arrhythmia; GSK3β; miRNA-26a

MeSH Terms

Animals
Arrhythmias, Cardiac
Connexin 43
Extracellular Vesicles*
Heart
Humans*
In Vitro Techniques
Mesenchymal Stromal Cells*
Rats
Real-Time Polymerase Chain Reaction
Reperfusion Injury*
Wnt Signaling Pathway
Connexin 43

Figure

• 

  Fig. 1 Isolation, characterization, and delivery of EVs. (A) Isolation of EV-rich fractions from human MSC media using a standard protocol of serial differential centrifugation and ultracentrifugation steps. (B) Transmission electron microscopy image showing typical EVs. (C) Nanoparticle tracking analysis of EVs showing the number and size distributions of particles. (D) Western blot of HSP70, Annexin, integrin, and CD63. (E and F) PKH26-labeled IR+EVHM was detected in the H9C2 cells (E) and heart tissue after leg vein injection (F); white arrows, PKH26. EV, extracellular vesicle; MSC, mesenchymal stem cell; IR, ischemia/reperfusion; EVHM, hypoxia-preconditioned extracellular vesicles.

• 

  Fig. 2 Systemic injection of EVHM decreases IR injury and arrhythmias in a rat IR injury model. (A and B) Comparison of 2,3,5-triphenyltetrazolium chloride (TTC) staining and reduction in the infarct size by the EVs (n=6 per group). (C) Typical examples of an EKG in the IR (n=10) and IR+EV (n=8) groups. (D) Typical examples of VT and VF in the IR group (n=10). (E) Comparison of spontaneous VT or VF. All data are presented as the mean±SEM; *p<0.001. EV, extracellular vesicle; IR, ischemia/reperfusion; VT, ventricular tachycardia; VF, ventricular fibrillation; EVHM, hypoxia-preconditioned extracellular vesicles; EVNM, normoxia-preconditioned extracellular vesicles.

• 

  Fig. 3 Electrophysiologic effects of EVHM after IR injury. (A) Sample traces of Vm from the three groups (cycle length=300 ms). (B) Representative activation (left panels) and action potential duration maps (right panels) from the three groups. (C) Conduction velocity vector maps. EVHM, hypoxia-preconditioned extracellular vesicles; EVNM, normoxia-preconditioned extracellular vesicles; IR, ischemia/reperfusion; LV, left ventricle, RV, right ventricle.

• 

  Fig. 4 EVHM recovers the sodium current (INa) after IR injury. Representative traces of whole-cell currents recorded from adult rat cardiomyocytes during the control, IR, and IR+EVHM conditions (A). Current/voltage relationship of INa under the three conditions (B). The protocol is indicated in the inset, and the number of cells recorded was five in each group. EVHM, hypoxia-preconditioned extracellular vesicles; IR, ischemia/reperfusion.

• 

  Fig. 5 EVs decrease and increase Wnt signaling proteins after IR injury and elevation of miRNA-26a in EVHM and the reversal of miRNA-26a by EVHM after IR injury. (A and B) Expression level of Wnt signaling proteins (e.g., Wnt1, GSK3β, p-GSK3β, β-catenin, and p-β-catenin) (n=6 per group). All data are presented as the mean±SEM; *p<0.05, **p<0.001. (C) Reversal of miRNA-26a after IR injury by EVHM (n=6 per group). Data are presented as the mean±SEM; **p<0.001. EV, extracellular vesicle; EVHM, hypoxia-preconditioned extracellular vesicles; EVNM, normoxia-preconditioned extracellular vesicles; IR, ischemia/reperfusion.

• 

  Fig. 6 Confocal microscope image of H9C2 cells showing GSK3β and p-β-catenin staining in various conditions. (A) Hypoxia-preconditioned extracellular vesicles (EVHM), compared with EVNM (normoxic), significantly prevented ischemia/reperfusion injury-induced increases in GSK3β and decreases in p-β-catenin. These effects were abrogated by anti-miRNA-26a. (B) Quantitative analysis of fluorescence signals in H9C2 cells stained for GSK3β and p-β-catenin.

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