BM-MSC-conditioned media contains paracrine factors when cells are exposed to hypoxia.

<p>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.</p

Inhibition of miRNA-23a and miRNA-92a prevents apoptosis of cardiomyocytes.

<p>(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.</p

BM-MSC therapy improves fibrosis and apoptosis in a rat model of MI.

<p>(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.</p

Hypoxia-exposed BM-MSC-conditioned media reduces apoptosis upon exposure of cardiomyocytes to hypoxia for 48 h.

<p>(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.</p

VEGF secreted from BM-MSCs regulates hypoxia-induced miRNA-23a and miRNA-92a expression in vitro.

<p>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.</p

Anti-Obesity Effects of Granulocyte-Colony Stimulating Factor in Otsuka-Long-Evans-Tokushima Fatty Rats

<div><p>Granulocyte-colony stimulating factor (G-CSF) has molecular structures and intracellular signaling pathways that are similar to those of leptin and ciliary neurotropic factor (CNTF). It also has immune-modulatory properties. Given that leptin and CNTF play important roles in energy homeostasis and that obesity is an inflammatory condition in adipose tissue, we hypothesized that G-CSF could also play a role in energy homeostasis. We treated 12 38-week-old male Otsuka-Long-Evans-Tokushima fatty rats (OLETF, diabetic) and 12 age-matched male Long-Evans-Tokushima rats (LETO, healthy) with 200 µg/day G-CSF or saline for 5 consecutive days. Body weight reduction was greater in G-CSF-treated OLETF (G-CSF/OLETF) than saline-treated OLETF (saline/OLETF) following 8 weeks of treatment (−6.9±1.6% vs. −3.1±2.2%, <i>p</i><0.05). G-CSF treatment had no effect on body weight in LETO or on food intake in either OLETF or LETO. Body fat in G-CSF/OLETF was more reduced than in saline/OLETF (−32.2±3.1% vs. −20.8±6.2%, <i>p</i><0.05). Energy expenditure was higher in G-CSF/OLETF from 4 weeks after the treatments than in saline/OLETF. Serum levels of cholesterol, triglyceride, interleukin-6 and tumor necrosis factor-α were lower in G-CSF/OLETF than in saline/OLETF. Uncoupling protein-1 (UCP-1) expression in brown adipose tissue (BAT) was higher in G-CSF/OLETF than in saline/OLETF, but was unaffected in LETO. Immunofluorescence staining and PCR results revealed that G-CSF receptors were expressed in BAT. In vitro experiments using brown adipocyte primary culture revealed that G-CSF enhanced UCP-1 expression from mature brown adipocytes via p38 mitogen-activated protein kinase pathway. In conclusion, G-CSF treatment reduced body weight and increased energy expenditure in a diabetic model, and enhanced UCP-1 expression and decreased inflammatory cytokine levels may be associated with the effects of G-CSF treatment.</p></div

UCP-1 and G-CSFR expression in BAT.

<p>(A) UCP-1 mRNA levels are higher in the G-CSF-treated OLETF than those in the saline-treated OLETF. UCP-1 mRNA levels are unaffected by G-CSF treatment in LETO; (B) UCP-1 protein levels are higher in the G-CSF-treated OLETF than those in the saline-treated OLETF. UCP-1 protein levels are unaffected by G-CSF treatment in LETO; (C) RT-PCR results show the presence of G-CSFR in BAT; (D), (E) and (F) Immunofluorescence staining revealed that G-CSFR was presented in the islands of brown adipocytes (white arrows) (D, immunofluorescence staining for G-CSFR, 200; E, DAPI, 200; F, merged, 200). * p<0.05. - The data were presented as the mean ± S.E.M. - Measurements of UCP-1 mRNA levels were duplicated, and the mRNA levels were normalized against those of the saline-treated LETO rats. - UCP-1 protein levels were normalized against those of the saline-treated LETO rats. UCP-1, uncoupling protein-1; G-CSFR, G-CSF receptor; BAT, brown adipose tissue.</p

Changes in body composition.

<p>The fat mass reduction was significantly larger in G-CSF-treated OLETF than that in saline-treated OLETF, while was not different between G-CSF-treated LETO and saline-treated LETO. The lean body mass differences were not significantly different in all animal groups. * p<0.05; - The data were shown as the mean ± S.E.M. - Body composition was measured 1 week before (pre-treatment) and 7 weeks after (post-treatment) G-CSF or saline treatment. - Percentage differences are shown as the difference between the pre-treatment value and post-treatment value divided by the pre-treatment value. G-CSF, granulocyte colony-stimulating factor; OLETF, Otsuka Long-Evans Tokushima Fatty rats; LETO, Long-Evans Tokushima Otsuka rats.</p

Body weight and blood glucose levels before G-CSF or saline treatment.

<p>(A) Body weights of the OLETF rats peaked at 22 weeks of age, then gradually decreased and were not significantly different with those of the LETO rats around 34 weeks of age; (B) Pre-treatment body weights at 38 weeks of age were not significantly different in all animal groups; (C) Blood glucose levels after 8 hours of fasting indicate the OLETF rats showed an overt diabetic phenotype after 30 weeks of age. - The data was presented as the mean ± S.E.M. OLETF, Otsuka Long-Evans Tokushima Fatty rats; LETO, Long-Evans Tokushima Otsuka rats; NS, not significant.</p

Changes in body weight and food intake after G-CSF or saline treatment.

<p>(A) Body weight difference from the pre-treatment body weight. Gradual decrease in body weight was shown in all animal groups, but the body weight reduction was significantly larger in G-CSF/OLETF than those in Saline/OLETF. The body weight reduction was not significantly different between G-CSF/LETO and Saline/LETO; (B) The percentage of body weight reduction in 8 weeks after the treatments was higher in G-CSF/OLETF than those in the other animal group; (C) The food intake was not significantly different between the G-CSF-treated groups and saline-treated groups of LETO and OLETF. * p<0.05 vs. saline/OLETF; † p<0.05 vs. other groups; - The data are shown as the mean ± S.E.M. G-CSF/OLETF, G-CSF-treated OLEFT; Saline/OLETF, saline-treated OLETF; G-CSF/LETO, G-CSF-treated LETO; Saline/LETO, saline-treated LETO; G-CSF, granulocyte colony-stimulating factor; OLETF, Otsuka Long-Evans Tokushima Fatty rats; LETO, Long-Evans Tokushima Otsuka rats.</p