Mineralocorticoid receptor deficiency improves the therapeutic effects of mesenchymal stem cells for myocardial infarction via enhanced cell survival

Figures

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  Figure 1. Flow cytometric analysis of surface antigen expression on MSCs. The rat MSCs were positive for CD29,CD44, and CD90 but negative for myeloid/hematopoietic cell‐specific antigens of CD34 and CD45

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  Figure 2. Transfection of MSCs with AAV vector. (A) Representative photomicrograph of MSCs transfected with AAV‐NC‐siRNA/GFP, scale bar: 100 μm. (B) FACS analysis showing the transfection efficiency of GFP transfected MSCs. (C) qRT‐PCR analysis of MR mRNA expression in MSCs 48 h after transfection with AAV‐MR‐siRNA/GFP. (D) AAV‐siRNA/GFP transfection did not affect cell viability examined with CCK‐8 assay. All data were collected from three independent experiments. *P < 0.05 vs control and NC

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  Figure 3. Effects of MR depletion on MSCs’ survival in vitro. (A) MSCs were exposed to hypoxia for 6 h after transfection with AAV‐ siRNA/GFP, cell injury was determined by flow cytometry after staining with Annexin V/7‐AAD. (B) Quantitative analysis of apoptotic cells by FACS analysis with Annexin V/7‐AAD staining. Data were collected from three independent experiments. *P < 0.05 vs NC, # P < 0.05 vs NC+hypoxia

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  Figure 4. Effects of MR depletion on MSCs’ survival in vivo. (A) Representative images of GFP + MSCs in infarcted myocardium. (B) Quantitative analysis of MSCs survival 4 days after MI. (C) Real‐time PCR was used to detect the male Sry gene in female rat hearts 4 days after transplantation (n = 3). The β‐actin gene was used as internal control. *P < 0.05 vs NC

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  Figure 5. Effects of MR depletion on ROS production and expression of apoptotic proteins induced by hypoxia. (A) Representative photomicrographs of intracellular ROS in MSCs visualized by fluorescent microscopy. Red fluorescent intensity indicated ROS accumulation. (B) Quantitative analysis of red fluorescent intensity in individual group. Data were collected from three independent experiments. (C) Apoptotic proteins determined by immunoblotting in MSCs subjected to hypoxia or normoxia. (D) Quantitative analysis of Bcl‐2/Bax ratio using data from three independent experiments. *P < 0.05 vs NC, # P < 0.05 vs NC+hypoxia

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  Figure 6. Effects of MR depletion on infarct size 4 weeks after cell transplantation. (A) Representative images of Masson staining from histological sections. (B) Representative images of myocyte islands in scar tissue from Masson staining histological sections. (C) Quantitative analysis of infarct size in various treatment groups (n = 5). (D) Quantitative analysis of myocyte islands in scar tissure (n = 5). *P < 0.05 vs PBS, # P < 0.05 vs NC

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  Figure 7. Effects of MR depletion on angiogenesis in the peri‐infarct area 4 weeks after cell transplantation. (A) Representative photomicrographs showing capillary density in different experimental groups based on CD31 immunostaining. Scale bar: 100 μm. (B) Quantitative analysis of the numbers of capillary vessels in various treatment groups (n = 5). (C) Representative electrophoresis graph of vascular endothelial growth factor by western blot. (D) Quantitative analysis for vascular endothelial growth factor (n = 5). *P < 0.05 vs PBS, # P < 0.05 vs NC