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. 2014 Jun 10;9(6):e99756.
doi: 10.1371/journal.pone.0099756. eCollection 2014.

Exendin-4 pretreated adipose derived stem cells are resistant to oxidative stress and improve cardiac performance via enhanced adhesion in the infarcted heart

Jianfeng Liu  1 , Haibin Wang  2 , Yan Wang  2 , Yujing Yin  3 , Liman Wang  2 , Zhiqiang Liu  2 , Junjie Yang  4 , Yundai Chen  4 , Changyong Wang  2
Affiliations

Exendin-4 pretreated adipose derived stem cells are resistant to oxidative stress and improve cardiac performance via enhanced adhesion in the infarcted heart

Jianfeng Liu et al. PLoS One. .

Abstract

Reactive oxygen species (ROS), which were largely generated after myocardial ischemia, severely impaired the adhesion and survival of transplanted stem cells. In this study, we aimed to determine whether Exendin-4 pretreatment could improve the adhesion and therapeutic efficacy of transplanted adipose derived stem cells (ADSCs) in ischemic myocardium. In vitro, H2O2 was used to provide ROS environments, in which ADSCs pretreated with Exendin-4 were incubated. ADSCs without pretreatment were used as control. Then, cell adhesion and viability were analyzed with time. Compared with control ADSCs, Exendin-4 treatment significantly increased the adhesion of ADSCs in ROS environment, while reduced intracellular ROS and cell injury as determined by dihydroethidium (DHE) staining live/Dead staining, lactate dehydrogenase-release assay and MTT assay. Western Blotting demonstrated that ROS significantly decreased the expression of adhesion-related integrins and integrin-related focal adhesion proteins, which were significantly reversed by Exendin-4 pretreatment and followed by decreases in caspase-3, indicating that Exendin-4 may facilitate cell survival through enhanced adhesion. In vivo, myocardial infarction (MI) was induced by the left anterior descending artery ligation in SD rats. Autologous ADSCs with or without Exendin-4 pretreatment were injected into the border area of infarcted hearts, respectively. Multi-techniques were used to assess the beneficial effects after transplantation. Longitudinal bioluminescence imaging and histological staining revealed that Exendin-4 pretreatment enhanced the survival and differentiation of engrafted ADSCs in ischemic myocardium, accompanied with significant benefits in cardiac function, matrix remodeling, and angiogenesis compared with non-pretreated ADSCs 4 weeks post-transplantation. In conclusion, transplantation of Exendin-4 pretreated ADSCs significantly improved cardiac performance and can be an innovative approach in the clinical perspective.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Exendin-4 restores ROS-induced attenuation of ADSCs adhesion, increases their viability and promotes ADSCs proliferation at 30, 60, 120 min after pretreatment.
(A) The effect of Exendin-4 on ADSCs adhesion in the presence of H2O2. Scale bars = 200 µm. (B) The effects of Exendin-4 on scavenging intracellular ROS of ADSCs which were treated with H2O2 with or without Exendin-4. ROS of ADSCs were detected using DHE reagent. Scale bars = 100 µm. (C) Live/dead staining showed that the effects of Exendin-4 pretreatment on ADSCs viability against H2O2. Scale bars = 100 µm. (D) Quantification of adhesive ADSCs. (E) Quantification of intracellular ROS. (F) Quantification of viable ADSCs. (G) Quantitative analysis of LDH release in the cell supernatant. (H) Caspase-3 activity determined by using Caspase-3 ELISA kit. (I) MTT assay was performed to analyze the effect of Exendin-4 on viability of ADSCs after H2O2 injury for 6 h. Statistical differences (p<0.05) are indicated from Control group(*) and ADSCs+ H2O2 group (#).
Figure 2
Figure 2. Exendin-4 attenuates integrin-related adhesion protein impairment induced by H2O2.
(A and B) mRNA expression levels of integrin β1 and αV in ADSCs with or without Exendin-4 under H2O2 injury by qRT-PCR analysis. (C-G) Representative western blotting of p-FAK, p-Src, paxillin, vinculin, talin and caspase-3 expression levels in ADSCs with or without Exendin-4 under H2O2 injury. ADSCs were pretreated with 50 nM Exendin-4 for 24 h and then treated with or without 30 µM H2O2 for 12 h. *p<0.05.
Figure 3
Figure 3. Survival and engraftment of transplanted ADSCs.
(A) Representative images of in vivo BLI at days 1, 7, 14 and 28 (n = 12/group). The BLI signal decreased gradually from day 1 to day 28 after cells injection in both groups. But the signals in the Ex-ADSCs group were significantly higher than those in the ADSCs group. (B) Quantitative analysis of serial BLI signal showed a moderate signal was still observed in the Ex-ADSCs group at day 28 after transplantation. (C) Confocal laser microscopic images of ADSCs (fluc-mRFP), cardiomyocytes (cTnI, red florescence) and DAPI (blue fluorescence) at 2 weeks after transplantation (n = 4/group). Scale bars = 50 µm (D) Quantitative analysis of the ratio of fluc and mRFP double-positive cells. *p<0.05.
Figure 4
Figure 4. Echocardiographic Assessment of Cardiac Function (n = 16/group).
(A) Representative M-mode echocardiograms in each group at day 28 after myocardial infarction. (B and C) Quantitative analysis of ejection fraction (B) and fractional shortening (C) by echocardiography. (D and E) Quantitative analysis of hemodynamic parameters LVEDP (D) and dP/dtmax (E). Statistical differences (p<0.05) are indicated from the sham (*), PBS (#), and ADSCs ($).
Figure 5
Figure 5. Confocal laser microscopic images of immunofluorescence analysis of differentiation of transplanted ADSCs in vivo (n = 12/group).
(A and B) Representative images of differentiated cardiomyocytes-like cells using anti-cardiac troponin-I (green, cTnI) revealed significant augmentation of enhanced mRFP (red)/cTnI double positive cardiomyocyte-like cells (white arrow) in Ex-ADSCs group (B) compared with ADSCs group (A). (C) Quantitative analysis of the ratio of differentiated cardiomyocytes-like cells. (D and E) Representative images of differentiated vessel specific cells using anti-α-SMA (green) revealed significant enhancement of mRFP (red)/α-SMA double positive vessel-specific cells (white arrow) in Ex-ADSCs group (E) compared with ADSCs group(D). (F) Quantitative analysis of the ratio of differentiated vessel specific cells. *p<0.05. Inset shows the corresponding boxed area magnified.
Figure 6
Figure 6. Transplantation of Exendin-4 pretreated ADSCs decrease apoptosis, fibrosis, and promote angiogenesis.
(A) Representative TUNEL staining images and quantitative analysis in the peri-infarcted and remote zone of heart sections from each group (n = 4/group). Apoptotic nuclei were identified as TUNEL positive (green). Total nuclei were counterstained by DAPI (blue). Apoptotic cells nuclei were considered as apoptotic cardiomyocytes (white arrow). Scale bars = 50 µm. (B) Representative images and quantitative analysis of fibrotic area in different groups by Masson's trichrome staining (n = 12/group). Red represented viable myocardium, blue represented fibrosis. Scale bars = 200 µm. (C) Representative images and quantitative analysis of vessels intensity using anti-vWF antibody at the border zone of MI in each group by immunohistochemistry (n = 12/group). Scale bars = 100 µm. Statistical differences (p<0.05) are indicated from PBS (*) and ADSCs (#).
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