Overexpression of adrenomedullin protects mesenchymal stem cells against hypoxia and serum deprivation‑induced apoptosis via the Akt/GSK3β and Bcl‑2 signaling pathways

Abstract

The poor survival rate of transplanted mesenchymal stem cells (MSCs) within the ischemic heart limits their therapeutic potential for cardiac repair. Adrenomedullin (ADM) has been identified as a potent apoptotic inhibitor. The present study aimed to investigate the protective effects of ADM on MSCs against hypoxia and serum deprivation (H/SD)‑induced apoptosis, and to determine the potential underlying mechanisms. In the present study, a recombinant adenovirus expressing the ADM gene was established and was infected into MSCs. The infection rate was determined via microscopic detection of green fluorescence and flow cytometric analysis. The mRNA expression levels of ADM were detected by reverse transcription‑polymerase chain reaction. In addition, a model of H/SD was generated. The MSCs were randomly separated into six groups: Control, enhanced green fluorescent protein (EGFP)‑Adv, EGFP‑ADM, H/SD, EGFP‑Adv + H/SD and EGFP‑ADM + H/SD. Cell viability and proliferation were determined using the Cell Counting kit‑8 assay. Apoptosis was assessed by terminal deoxynucleotidyl transferase‑mediated‑dUTP nick‑end labeling assay and flow cytometric analysis using Annexin V‑phycoerythrin/7‑aminoactinomycin D staining. The protein expression levels of total protein kinase B (Akt), phosphorylated (p)‑Akt, total glycogen synthase kinase (GSK)3β, p‑GSK3β, B‑cell lymphoma 2 (Bcl‑2), Bcl‑2‑associated X protein (Bax), caspase‑3 and cleaved caspase‑3 were detected by western blot analysis. The results indicated that ADM overexpression could improve MSC proliferation and viability, and protect MSCs against H/SD‑induced apoptosis. In addition, ADM overexpression increased Akt and GSK3β phosphorylation, and Bcl‑2/Bax ratio, and decreased the activation of caspase‑3. These results suggested that ADM protects MSCs against H/SD‑induced apoptosis, which may be mediated via the Akt/GSK3β and Bcl‑2 signaling pathways.

Figure 1

Characterization and differentiation of cultured MSCs. (A and B) Flow cytometric analysis of adherent, spindle-shaped MSCs (passage 3 or 4). Most cultured MSCs expressed CD29. However, the majority of MSCs were CD34- and CD45-negative. (C) Adiopogenic differentiation, as determined by Oil Red O staining. MSCs developed some lipid droplets (magnification, ×400). (D) Osteogenic differentiation, as determined using von Kossa staining (magnification, ×400). Mineralized matrix was formed in MSCs. CD, cluster of differentiation; FITC, fluorescein isothiocyanate; MSCs, mesenchymal stem cells; PE, phycoerythrin.

Figure 2

Infection of MSCs with adenovirus vectors. Fluorescence microscopy confirmed transduction efficiency in MSCs 48 h post-transduction with (A) EGFP-Adv or (B) EGFP-ADM (magnification, ×100). (C) Representative reverse transcription-polymerase chain reaction results confirm ADM gene abundance following EGFP-ADM infection 24, 48 or 72 h post-transduction compared with in the control or EGFP-Adv groups. (D) Detection of transduction efficiency by flow cytometry. Adv, adenovirus; ADM, adrenomedullin; EGFP, enhanced green fluorescent protein; MSCs, mesenchymal stem cells.

Figure 3

Effects of ADM on mesenchymal stem cell (A) proliferation and (B) viability. Data are presented as the means ± standard deviation of three independent experiments. ^*P<0.05 vs. the control group; ^#P<0.05 vs. the H/SD group. Adv, adenovirus; ADM, adrenomedullin; EGFP, enhanced green fluorescent protein; H/SD, hypoxia and serum deprivation; OD, optical denisty.

Figure 4

Apoptosis of MSCs induced by H/SD was measured by TUNEL staining. (A) Representative TUNEL staining of MSCs. Nuclear staining of red fluorescence was considered a positive apoptotic signal. DAPI served as a normal nuclear stain. The merge column presents an overlay of TUNEL and DAPI staining (magnification, ×100). (B) Quantitative analysis of TUNEL staining. Data are presented as the means ± standard deviation of three independent experiments. ^*P<0.05 vs. the control group; ^#P<0.05 vs. the H/SD group. Adv, adenovirus; ADM, adrenomedullin; EGFP, enhanced green fluorescent protein; H/SD, hypoxia and serum deprivation; MSCs, mesenchymal stem cells; TUNEL, terminal deoxynucleotidyl transferase-mediated-dUTP nick-end labeling

Figure 5

Apoptosis of mesenchymal stem cells was assessed by Annexin V/7-AAD double staining and FACS. (A) Representative FACS plots. (B) Quantitative analysis of the percentage of early-stage apoptotic cells (Annexin V⁺/AAD⁻). Data are presented as the means ± standard deviation from three independent experiments. ^*P<0.05 vs. the control group; ^#P<0.05 vs. the H/SD group. 7-AAD, 7-aminoactinomycin D; Adv, adenovirus; ADM, adrenomedullin; EGFP, enhanced green fluorescent protein; FACS, fluorescence-activated cell sorting; H/SD, hypoxia and serum deprivation; PE, phycoerythrin.

Figure 6

Effects of ADM on caspase-3 activition. (A) Representative western blot analyses of procaspase-3 and cleaved caspase-3 in the different groups. (B) Semi-quantitative analysis of the expression of cleaved caspase-3 was estimated as a fold-change relative to procaspase-3. Data are presented as the means ± standard deviation from three independent experiments. ^*P<0.05 vs. the control group; ^#P<0.05 vs. the H/SD group. Adv, adenovirus; ADM, adrenomedullin; EGFP, enhanced green fluorescent protein; H/SD, hypoxia and serum deprivation.

Figure 7

ADM activates the Akt/GSK3β pathway in the apoptosis of mesenchymal stem cells. (A) Representative western blot analyses of p-Akt and Akt. (B) Semi-quantitative analysis of the expression of p-Akt was estimated as a fold-change relative to Akt. (C) Representative western blot analyses of p-GSK3β and GSK3β. (D) Semi-quantitative analysis of the expression of p-GSK3β was estimated as a fold-change relative to GSK3β. Data are presented as the means ± standard deviation from three independent experiments. ^*P<0.05 vs. the control group; ^#P<0.05 vs. the H/SD group. Adv, adenovirus; ADM, adrenomedullin; Akt, protein kinase B; EGFP, enhanced green fluorescent protein; GSK3β, glycogen synthase kinase3β; H/SD, hypoxia and serum deprivation; p, phosphorylated; t, total.

Figure 8

Effects of ADM on Bcl-2 and Bax expression. (A) Representative western blot analyses of Bcl-2, Bax and GADPH expression in the different groups. (B) Semi-quantitative analysis was used to calculate the relative ratio of Bcl-2/Bax. Data are presented as the means ± standard deviation from three independent experiments. ^*P<0.05 vs. the control group; ^#P<0.05 vs. the H/SD group. Adv, adenovirus; ADM, adrenomedullin; Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2; EGFP, enhanced green fluorescent protein; H/SD, hypoxia and serum deprivation.