Fig 1.
BM-MSC therapy improves fibrosis and apoptosis in a rat model of MI.
(A) Representative images of Masson's trichrome staining of whole heart tissue at 4 weeks after treatment for each group. (B) Representative photomicrographs showing TUNEL assay in the peri-infarct region at 4 weeks after treatment for each group. Scale bar = 50 μm. (C) Results of quantitative analysis of collagen area as ratio of fibrotic area to whole heart area. (D) Results of quantitative analysis of apoptotic cells. Sham, surgical procedure with no induction of MI; Saline, saline treatment after induction of MI; Cell, cell treatment after induction of MI. All data are expressed as mean ± SD (n = 5 per group). *P < 0.05 vs. sham control group. †P < 0.05 vs. saline group.
Fig 2.
BM-MSC therapy regulates cardiac miRNAs in a rat model of MI.
MiRNA expression was measured in the peri-infarct region by real-time PCR using TaqMan probes at 3 days or 4 weeks after treatment. MiRNA-23a (A) and miRNA-92a (B) expression in response to treatment with BM-MSCs in comparison with saline or sham at 3 days or 4 weeks after treatment. All data are expressed as mean ± SD (n = 5 per group). *P < 0.05 vs. sham control group. †P < 0.05 vs. saline group at 3 days.
Fig 3.
BM-MSC-conditioned media contains paracrine factors when cells are exposed to hypoxia.
Paracrine factors were measured in the hypoxia-exposed BM-MSC-conditioned media by Magnetic Luminex assay. VEGF (A), MCP-1 (B), IL-6 (C), and ANG (D) expression in response to hypoxia exposure time in comparison with normoxia. N or H indicate normoxia or hypoxia, respectively. All data are expressed as mean ± SD (n = 5 per group). *P < 0.05 vs. normoxia for 48 h.
Fig 4.
Hypoxia-exposed BM-MSC-conditioned media reduces apoptosis upon exposure of cardiomyocytes to hypoxia for 48 h.
(A) Dot plots display the stages of apoptotic death of cardiomyocytes: Annexin−/PI− (Q3), viable cells; Annexin+/PI− (Q4), cells undergoing apoptosis; Annexin+/PI+ (Q2), cells that are in end-stage apoptosis or are already dead; Annexin−/PI+ (Q1), cells that are in necrosis. MSCs media indicates hypoxia-exposed BM-MSC-conditioned media. (B) Quantitative analysis of apoptotic cells (Q2+Q4). Hypoxia-exposed BM-MSC-conditioned media reduces hypoxia-induced miRNA expression in vitro. MiRNA expression was measured by real-time PCR using TaqMan probes. MiRNA-23a (C) and miRNA-92a (D) expression in response to treatment with hypoxia-exposed BM-MSC-conditioned media in comparison without hypoxia-exposed BM-MSC-conditioned media in hypoxia. All data are expressed as mean ± SD (n = 5 per group). *P < 0.05 vs. normoxia without hypoxia-exposed BM-MSC-conditioned media. &P < 0.05 vs. normoxia with hypoxia-exposed BM-MSC-conditioned media. †P < 0.05 vs. hypoxia without hypoxia-exposed BM-MSC-conditioned media.
Fig 5.
Inhibition of miRNA-23a and miRNA-92a prevents apoptosis of cardiomyocytes.
(A, B) Transfection efficiency of the miRNA-23a inhibitor (A) and miRNA-92a inhibitor (B) was determined by real-time PCR using TaqMan probes. Quantitative analysis of apoptotic cells in cardiomyocytes transfected either with miRNA-23a inhibitor (C) or miRNA-92a inhibiton (D). Apoptotic cells were measured by annexin V staining. All data are expressed as mean ± SD (n = 5 per group). *P < 0.05 vs. normoxia without miRNA inhibitor. &P < 0.05 vs. normoxia with miRNA inhibitor. †P < 0.05 vs. hypoxia without miRNA inhibitor.
Fig 6.
VEGF secreted from BM-MSCs regulates hypoxia-induced miRNA-23a and miRNA-92a expression in vitro.
The effects of VEGF secreted from BM-MSCs on hypoxia-induced miRNA-23a (A) and miRNA-92a (B) expression. Expression of the miRNA was determined by real-time PCR using TaqMan probes. (C) Quantitative analysis of apoptotic cells in cardiomyocytes-administered neutralizing antibodies against VEGF (VEGF ab). MSCs media indicates hypoxia-exposed BM-MSC-conditioned media. Apoptotic cells were measured by annexin V staining. All data are expressed as mean ± SD (n = 5 per group). *P < 0.05 vs. normoxia without VEGF ab. &P < 0.05 vs. normoxia with VEGF ab. †P < 0.05 vs. hypoxia without VEGF ab.
Fig 7.
Proposed mechanisms of the paracrine regulation of cardiac miRNAs by transplanted BM-MSCs.
Transplanted BM-MSCs release VEGF, which inhibits the MI-induced miRNA-23a and miRNA-92a, thereby blocking cardiomyocytes apoptosis.