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Exosomes Secreted From GATA-4 Overexpressing Mesenchymal Stem Cells Serve as a Reservoir of Anti-Apoptotic microRNAs for Cardioprotection

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Exosomes Secreted From GATA-4 Overexpressing Mesenchymal Stem Cells Serve as a Reservoir of Anti-Apoptotic microRNAs for Cardioprotection

Bin Yu et al. Int J Cardiol.

Abstract

Background: Exosomes play an important role in intercellular signaling and exert regulatory function by carrying bioactive molecules. This study investigated (1) the cardioprotective capabilities of exosomes derived from mesenchymal stem cells (MSCs) overexpressing GATA-4 (MSC(GATA-4)) and (2) its underlying regulatory mechanisms for expression of target proteins in recipient cells.

Methods and results: Exosomes were isolated and purified from MSC(GATA-4) (Exo(GATA-4)) and control MSCs (Exo(Null)). Cell injury was investigated in primary cultured rat neonatal cardiomyocytes (CM) and in the rat heart. Exosomes contributed to increased CM survival, reduced CM apoptosis, and preserved mitochondrial membrane potential in CM cultured under a hypoxic environment. Direct intramyocardial transplantation of exosomes at the border of an ischemic region following ligation of the left anterior descending coronary artery significantly restored cardiac contractile function and reduced infarct size. Real-time PCR revealed that several anti-apoptotic miRs were highly expressed in Exo(GATA-4). Rapid internalization of Exo(GATA-4) by CM was documented using time-lapse imaging. Subsequent expression of these miRs, particularly miR-19a was higher in CM and in the myocardium treated with Exo(GATA-4) compared to those treated with Exo(Null). The enhanced protective effects observed in CM were diminished by the inhibition of miR-19a. The expression level of PTEN, a predicted target of miR-19a, was reduced in CM treated with Exo(GATA-4), which resulted in the activation of the Akt and ERK signaling pathways.

Conclusions: Exo(GATA-4) upon transplantation in the damaged tissue mediate protection by releasing multiple miRs responsible for activation of the cell survival signaling pathway.

Keywords: Bone marrow stem cells; Cardioprotection; Exosomes; GATA-4; MiR transfer; Target proteins.

Figures

Figure 1
Figure 1
Characterization of MSCs derived from bone marrow and MSCs transduced with GATA-4 (MSCGATA-4). A, Second passage of basal bone marrow stem cells. B and C, Immunostaining of GATA-4 in MSCGATA-4 (B) and MSCNull (C). GFP was expressed in both MSCNull and MSCGATA-4, but GATA-4 was only expressed in MSCGATA-4. D, Real-time PCR results show that the expression of miR-19a and miR-451 was up-regulated in MSCGATA-4 compared with MSCNull. E, Expression of miR-19 was down-regulated in MSCs that were transfected with GATA-4-siRNA (MSCsi-GATA-4) compared with MSCs transfected with the negative control siRNA (MSCsi-NC). *, p<0.05 vs MSCNull and #, p<0.05 vs MSCsi-NC.
Figure 2
Figure 2
Cardioprotective effect of exosomes. A, Ultrastructural features of exosomes derived from MSCs. B, Western blotting analyses revealed that exosomes highly expressed HSP70, CD63, and CD9 compared to MSCs; C-F, Exosomes protect CM against ischemic injury induced by exposure of CM to hypoxia for 48 hours. Panel C: Exosomes increased CM survival in a concentration-dependent manner. D, CM morphology in different treatments. Bright dots: dead or dying cells. E, LDH release from CM. F, The number of survived CM. *, p<0.05 vs medium control; #, p<0.05 vs ExoNull treatment.
Figure 3
Figure 3
Exosomes significantly reduced CM apoptosis and maintained ΔΨm after CM exposure to hypoxia for 24 hours. A, Representative images of TUNEL staining in CM and positive cell quantification data. B, Representative JC-1 fluorescence imaging of mitochondria and quantitative data of the ratio of J-aggregate to monomer fluorescence. Green and red fluorescence indicate monomeric and J-aggregate mitochondria, respectively. *, p<0.05 vs medium control, and # p<0.05 vs ExoNull, respectively.
Figure 4
Figure 4
ExoGATA-4 improves cardiac function and reduces infarction size after transplantation for 4 weeks. A, Representative echocardiography images. B, Parameters of cardiac function calculated from M-mode echocardiograms. C, Heart sections stained with Masson-Trichome (C1) and quantitative data of LV thickness and fibrosis (C2). #, p < 0.05 vs sham animals; *, p < 0.05 vs ligation control; , p < 0.05 vs ExoNull treatment, respectively.
Figure 4
Figure 4
ExoGATA-4 improves cardiac function and reduces infarction size after transplantation for 4 weeks. A, Representative echocardiography images. B, Parameters of cardiac function calculated from M-mode echocardiograms. C, Heart sections stained with Masson-Trichome (C1) and quantitative data of LV thickness and fibrosis (C2). #, p < 0.05 vs sham animals; *, p < 0.05 vs ligation control; , p < 0.05 vs ExoNull treatment, respectively.
Figure 5
Figure 5
The expression of miRs and the role of miRs in ExoGATA-4 mediated cardioprotection. A, Real-time PCR expressed as the fold change of miR-19a and miR-451 in exosomes. #, p<0.05 vs ExoNull. B and C, The expression of miR-19a in MSC (B) and in exosomes (C) following transfection with si-miR-19a. *, p<0.05 vs scramble si-miR transfected. D~F, The effect of exosomes obtained from MSCGATA-4 transfected with si-miR-19a on LDH release (D), TUNEL-positive cells (E), and ΔΨm (F). *, p<0.05 vs scramble si-miR transfected, respectively.
Figure 6
Figure 6
The internalization of exosomes and the effect of internalized exosomes on the expression of miRs in CM. A, Time-lapse images following the addition of ExoPKH-26 to CM culture. B, Immunostaining shows the red fluorescence (ExoPKH26) was inside α-actinin-positive cells. C-E, Expression of miRs in CM after exposure to hypoxia for 24 hours and treatment with exosomes. *, p<0.05 vs normal control; #, p<0.05 vs hypoxia, and , p<0.05 vs ExoNull treated, respectively.
Figure 7
Figure 7
Exosome distribution in the myocardium and their effect on the expression of miRs 2 days post-transplantation. A, ExoPKH-26 was mainly located in the ischemic boarder area (white arrows). B, Real-time PCR of miR-19a in normal and ischemic border myocardium. *, p<0.05 vs normal control; #, p<0.05 vs medium treated; and , p < 0.05 vs ExoNull treated, respectively.
Figure 8
Figure 8
Figure 8. Exosomes regulate PTEN, BIM, p-ERK and p-Akt signaling in CM. A, TargetScan shows the 3’ UTR of PTEN and BIM (BCL2L11) containing the conserved miR-19a binding site. B, Western blotting analyses of PTEN and BIM, and corresponding semi-quantitative data in CM. C, Western blotting analyses of p-ERK and p-Akt, and corresponding semi-quantitative data in CM. *, p<0.05 vs medium control, and #, p<0.05 vs ExoNull treated, respectively.

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