Mesenchymal stem cells overexpressing adrenomedullin improve heart function through antifibrotic action in rats experiencing heart failure

Abstract

Previous studies of the authors have indicated that the transplantation of mesenchymal stem cells (MSCs) can attenuate cardiac fibrosis through the secretion of antifibrotic factors, such as adrenomedullin (ADM). Therefore, the authors addressed the hypothesis that ADM overexpression could enhance the antifibrotic effect of MSCs transplantation in a rat model of heart failure. The results of the present study demonstrated that, compared with the group that received the GFP‑MSCs, the transplantation of ADM‑MSCs significantly improved heart function and decreased the percentage of fibrotic area and the expression of matrix metalloproteinase 2. In addition, fluorescence microscopy indicated that the survival of transplanted MSCs also increased significantly in the ADM‑MSCs‑treated group. Furthermore, the expression of fibrosis‑related genes, such as ADM and hepatocyte growth factor, were significantly influenced in the ADM‑MSCs‑treated group. Based on these findings, it may be concluded that, compared with MSCs, MSCs overexpressing ADM can further improve heart function in rats experiencing heart failure through enhanced antifibrotic activity.

Figure 1.

Transfection of MSCs. (A) Transfected MSCs observed by light and fluorescent microscopy (multiplicity of infection, 1:200; magnification, ×40). (B) Transduction efficiency determined by fluorescence analysis. (C) Western blot analysis of ADM protein in ADM-MSCs and GFP-MSCs. (D) Quantitative analysis of western blot results. ^‡P<0.01 vs. the GFP-MSCs. Data are presented as the mean ± standard error of the mean. MSCs, mesenchymal stem cells; ADM, adrenomedullin; GRP, green fluorescent protein.

Figure 2.

Representative echocardiography images for all four experimental groups. GFP, green fluorescent protein; ADM, adrenomedullin; MSCs, mesenchymal stem cells.

Figure 3.

Expression of ADM. (A) The expression of AMD was measured by western blotting. (B) Densitometric analysis was used to calculate the relative ratio of ADM/β-actin. Data are presented as the mean ± standard error of the mean. **P<0.01 vs. the GFP-MSCs-treated group, ^ΔP<0.05 vs. the Medium-treated group, ^†P<0.05 vs. the Sham group, ^‡P<0.01 vs. the Sham group. MSCs, mesenchymal stem cells; ADM, adrenomedullin; GRP, green fluorescent protein.

Figure 4.

Survival of transplanted MSCs. (A) Representative frozen myocardial sections. (B) Data are presented as the mean ± standard error of the mean. *P<0.05 vs. the GFP-MSCs-treated group. MSCs, mesenchymal stem cells; ADM, adrenomedullin; GRP, green fluorescent protein.

Figure 5.

Effect of cell transplantation on cardiac fibrosis. (A) Representative myocardial sections stained with Masson's trichrome staining of four groups (magnification, ×200): (a) Sham group, (b) medium-treated group, (c) GRP-MSCs-treated group and the (d) ADM-MSCs-treated group. (B) Quantitative analysis of collagen fraction. *P<0.05 vs. the GFP-MSCs-treated group, ^ΔΔP<0.01 vs. the Medium-treated group, ^‡P<0.01 vs. the Sham group. MSCs, mesenchymal stem cells; ADM, adrenomedullin; GRP, green fluorescent protein.

Figure 6.

Reverse transcription-quantitative polymerase chain reaction analysis of collagen I and collagen III mRNA levels. *P<0.05 vs. the GFP-MSCs-treated group, ^ΔP<0.05 vs. the Medium-treated group, ^‡P<0.01 vs. the Sham group. MSCs, mesenchymal stem cells; ADM, adrenomedullin; GRP, green fluorescent protein.

Figure 7.

(A) RT-qPCR analysis of mRNA levels. (B and C) Western blot analysis of protein levels. *P<0.05, **P<0.01 vs. the GFP-MSCs-treated group; ^ΔP<0.05, ^∆∆P<0.01 vs. the Medium-treated group; ^†P<0.05, ^‡P<0.01 vs. the Sham group. MMP, matrix metalloproteinase; HGF, hepatocyte growth factor; TNF-β, tumor necrosis factor-β; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.