The stem cell adjuvant with Exendin-4 repairs the heart after myocardial infarction via STAT3 activation

Abstract

The poor survival of cells in ischaemic myocardium is a major obstacle for stem cell therapy. Exendin-4 holds the potential of cardioprotective effect based on its pleiotropic activity. This study investigated whether Exendin-4 in conjunction with adipose-derived stem cells (ADSCs) could improve the stem cell survival and contribute to myocardial repairs after infarction. Myocardial infarction (MI) was induced by the left anterior descending artery ligation in adult male Sprague-Dawley rats. ADSCs carrying double-fusion reporter gene [firefly luciferase and monomeric red fluorescent protein (fluc-mRFP)] were quickly injected into border zone of MI in rats treated with or without Exendin-4. Exendin-4 enhanced the survival of transplanted ADSCs, as demonstrated by the longitudinal in vivo bioluminescence imaging. Moreover, ADSCs adjuvant with Exendin-4 decreased oxidative stress, apoptosis and fibrosis. They also improved myocardial viability and cardiac function and increased the differentiation rates of ADSCs into cardiomyocytes and vascular smooth muscle cells in vivo. Then, ADSCs were exposed to hydrogen peroxide/serum deprivation (H(2)O(2)/SD) to mimic the ischaemic environment in vitro. Results showed that Exendin-4 decreased the apoptosis and enhanced the paracrine effect of ADSCs. In addition, Exendin-4 activated signal transducers and activators of transcription 3 (STAT3) through the phosphorylation of Akt and ERK1/2. Furthermore, Exendin-4 increased the anti-apoptotic protein Bcl-2, but decreased the pro-apoptotic protein Bax of ADSCs. In conclusion, Exendin-4 could improve the survival and therapeutic efficacy of transplanted ADSCs through STAT3 activation via the phosphorylation of Akt and ERK1/2. This study suggests the potential application of Exendin-4 for stem cell-based heart regeneration.

Keywords: Exendin-4; STAT3; adipose-derived stem cell; myocardial infarction; paracrine.

Fig. 1

Myocardial reactive oxygen species generation and apoptosis at 1 week after myocardial infarction. (A) Dihydroethidium (DHE) staining showed a least red fluorescence intensity in combination-treated group compared with the other groups. (B) Quantification analysis for DHE staining (n = 6). (C) Representative images of TUNEL staining for groups. (D) Quantification analysis for TUNEL staining (n = 6); scale bars = 50 μm. *P < 0.05 versus PBS group; #P < 0.05 versus Exendin-4 group; $P < 0.05 versus adipose-derived stem cell group.

Fig. 2

The therapeutic efficacy of adipose-derived stem cells (ADSCs) adjuvant with exendin-4 for myocardial infarction. (A) Representative BLI images of rat and quantitative analysis from ADSCs group and Exendin-4+ ADSCs group. *P < 0.05 versus ADSCs group; n = 12. (B) Confocal laser microscopic images and quantitative analysis of the ratio of mRFP/DAPI cells at 4 weeks after transplantation; scale bars = 50 μm. *P < 0.05; n = 6. (C) Echocardiography showed that EF and FS were significantly improved in rats treated with Exendin-4+ ADSCs group, n = 12. (D) Representative images of Micro-PET in infarcted hearts among groups and quantitative analysis, n = 5. (E) Representative images of Masson trichrome staining and statistical results of infarct size and fibrotic area, n = 24. *P < 0.05 versus PBS group; #P < 0.05 versus Exendin-4 group; $P < 0.05 versus ADSCs group.

Fig. 3

Confocal laser microscopic images for adipose-derived stem cells (ADSCs) differentiation in the peri-infarct area. (A) Representative immunofluorescence images for cTnT+/mRFP+ cardiomyocytes. (B) Quantification analysis of cardiomyocyte differentiation of ADSCs. n = 12. (C) Representative immunofluorescence images for α-SMA+/mRFP+ vascular smooth muscle cells. (D) Quantification analysis of vascular smooth muscle cells differentiation of ADSCs. n = 12; scale bars = 30 μm. *P < 0.05 versus ADSCs group.

Fig. 4

Adipose-derived stem cells (ADSCs) adjuvant with Exendin-4 activated the Akt, ERK1/2 and STAT3 in ischaemic myocardium. (A) Representative western blotting of p-Akt, Akt, p-ERK, ERK, p-STAT3, STAT3 and GAPDH in ischaemic myocardium. (B–D) The relative optical density ratio of p-Akt, p-ERK, p-STAT3 (n = 6). *P < 0.05 versus PBS group; #P < 0.05 versus Exendin-4 group; $P < 0.05 versus ADSCs group.

Fig. 5

Exendin-4 prevented H₂O₂/SD- induced apoptosis of adipose-derived stem cells (ADSCs) and activated Akt, ERK and STAT3. (A) In vitro BLI determined that Exendin-4 at 5 nM promoted the attenuated viability of ADSCs exposed to H₂O₂/SD (n = 6). (B) Apoptosis was evaluated with flow cytometry analysis. (C) The quantitative analysis of apoptotic index (apoptotic cells/total cells) in ADSCs (n = 6). (D) Caspase-3 activity determined by using Caspase-3 ELISA kit (n = 6). (E) Representative blots of p-Akt, Akt, p-ERK, ERK, p-STAT3, STAT3 in ADSCs exposed to H₂O₂/SD and quantitative analysis of p-Akt, p-ERK, p-STAT3. β-actin is used as internal parameter (n = 6). (F) Representative blots of Bcl-2 and Bax in ADSCs exposed to H₂O₂/SD and quantitative analysis of Bcl-2 and Bax (n = 6); *P < 0.05.

Fig. 6

Exendin-4 increased paracrine factor release from adipose-derived stem cells (ADSCs). (A) Quantitative real-time PCR analysis of gene expression of VEGF, bFGF, HGF, IGF-1 in ADSCs with or without Exendin-4 treatment. (B) ELISA detection of VEGF, bFGF, HGF, IGF-1 levels in the ADSCs-conditioned medium with or without Exendin-4 treatment (n = 6); *P < 0.05 versus control.