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. 2012 Aug;13(8):616-23.
doi: 10.1631/jzus.B1201004.

Angiopoietin-1 preconditioning enhances survival and functional recovery of mesenchymal stem cell transplantation

Xian-bao Liu  1 , Han Chen, Hui-qiang Chen, Mei-fei Zhu, Xin-yang Hu, Ya-ping Wang, Zhi Jiang, Yin-chuan Xu, Mei-xiang Xiang, Jian-an Wang
Affiliations

Angiopoietin-1 preconditioning enhances survival and functional recovery of mesenchymal stem cell transplantation

Xian-bao Liu et al. J Zhejiang Univ Sci B. 2012 Aug.

Abstract

Objective: Mesenchymal stem cell (MSC) transplantation is a promising therapy for ischemic heart diseases. However, poor cell survival after transplantation greatly limits the therapeutic efficacy of MSCs. The purpose of this study was to investigate the protective effect of angiopoietin-1 (Ang1) preconditioning on MSC survival and subsequent heart function improvement after transplantation.

Methods: MSCs were cultured with or without 50 ng/ml Ang1 in complete medium for 24 h prior to experiments on cell survival and transplantation. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Hoechst staining were applied to evaluate MSC survival after serum deprivation in vitro, while cell survival in vivo was detected by terminal deoxynucleotidyl transferase biotin-dUPT nick end labeling (TUNEL) assay 24 and 72 h after transplantation. Heart function and infarct size were measured four weeks later by small animal echocardiography and Masson's trichrome staining, respectively.

Results: Ang1 preconditioning induced Akt phosphorylation and increased expression of Bcl-2 and the ratio of Bcl-2/Bax. In comparison with non-preconditioned MSCs, Ang1-preconditioned cell survival was significantly increased while the apoptotic rate decreased in vitro. However, the PI3K/Akt pathway inhibitor, LY294002, abrogated the protective effect of Ang1 preconditioning. After transplantation, the Ang1-preconditioned-MSC group showed a lower death rate, smaller infarct size, and better heart functional recovery compared to the non-preconditioned-MSC group.

Conclusions: Ang1 preconditioning enhances MSC survival, contributing to further improvement of heart function.

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Figures

Fig. 1
Fig. 1
Effect of Ang1 preconditioning on survival-related proteins MSCs were cultured with or without Ang1 for 24 h. Then, phospho-Akt (p-Akt), Akt, Bcl-2, and Bax were detected by Western blotting. GAPDH was used as the loading control. Densitometric analysis was applied for comparison of the relative expression levels of different proteins in AP-MSC and N-MSC which was arbitrarily presented as 1 (n=3). * P<0.05 compared with the N-MSC group. N-MSC: non-preconditioned mesenchymal stem cells; AP-MSC: Ang1-preconditioned mesenchymal stem cell
Fig. 2
Fig. 2
Effect of Ang1 on MSC survival in vitro and after transplantation (a) MSCs were cultured with or without Ang1 treatment for 24 h prior to serum deprivation for 48 h, and then MTT was applied to evaluate cell survival in vitro. (b) Hoechst staining was used for detection of apoptosis. The experimental protocol was the same as for MTT. (c) In vivo cell survival was determined by TUNEL assay after MSC engraftment into ischemic heart for 24 h (n=3). * P<0.05 compared with the control group. # P<0.05 compared with the N-MSC group. † P<0.05 compared with the AP-MSC group. CON: control (without serum deprivation). AP+LY: AP-MSC with LY294002
Fig. 3
Fig. 3
Effect of MSC transplantation on infarct size (a) Masson’s trichrome staining of MI, N-MSC, and AP-MSC groups. The blue area was defined as the infarcted region and scar formation, while red was defined as normal heart tissue. (b) Quantitative analysis of infarct size in the three groups (n=4). * P<0.05 compared with the MI group; # P<0.05 compared with the N-MSC group. MI: myocardial infarction without stem cell therapy
Fig. 4
Fig. 4
Effect of MSC transplantation on heart function (a) M-mode tracing images from Sham, MI, N-MSC, and AP-MSC groups. (b) Analysis of echocardiographic parameters (LVEDD, LVESD, LVFS, LVEF) according to M-mode images (n=8). * P<0.01 compared with the MI-only group. # P<0.01 compared with the N-MSC group. LVEDD: left ventricular end diastolic diameter; LVESD: left ventricular end systolic diameter; LVFS: left ventricular fractional shortening; LVEF: left ventricular ejection fraction
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