M-CSF enhanced heart VEGF production in vitro. A: Cultured cardiomyocytes from neonatal mice were stimulated with M-CSF (100 ng/ml) for the indicated time periods. Culture medium was changed daily, and the supernatants were subjected to ELISA. M-CSF significantly enhanced VEGF production on days 2 and 3 (*P < 0.01). B: Cultured cardiomyocytes from neonatal mice expressed M-CSF-R. The shaded histogram indicates staining with M-CSF-R, and the blank histogram indicates background staining with control IgG. Similar results were obtained from two independent experiments.

M-CSF protects differentiated H9c2 cells from H₂O₂-induced cell death. A: H9c2 cardiomyocytes were cultured with the indicated amount of M-CSF and ATRA for the indicated time periods, and the culture medium was changed daily. H9c2 myotubes were cultured with the indicated amount of M-CSF for the indicated time periods. WST assay determined the cell number. B: H9c2 cardiomyocytes or H9c2 myotubes were cultured with the indicated amount of M-CSF for 24 hours and then stimulated with H₂O₂ (40 or 60 μmol/L) for 8 hours. The culture medium of H9c2 cardiomyocytes was supplemented with ATRA. WST assay determined the cell viability. M-CSF (100 and 1000 ng/ml) significantly protected the cells from H₂O₂-induced cell death (*P < 0.03). Similar results were obtained from three independent experiments.

M-CSF activated ERK, Akt, and up-regulated Bcl-xL expression in differentiated H9c2 cells. H9c2 cardiomyocytes (A and B) or H9c2 myotubes (C and D) were stimulated with M-CSF (100 ng/ml) for the indicated time periods, and then the cell lysates were blotted with antibodies specific for the activated form of ERK (phospho-ERK), Akt (phospho-Akt), Jak1 (phospho-Jak1), Stat1 (phospho-Stat1), Stat3 (phospho-Stat3), or phosphorylated Bad (phospho-Bad). The membranes were reblotted with antibodies to total ERK, Akt, Jak1, Stat1, Stat3, or Bad, respectively. Expression of M-CSF-R or Bcl-xL was confirmed by blotting the membrane with specific antibodies. Similar results were obtained from three independent experiments.

M-CSF increased VEGF production in differentiated H9c2 cells. A: H9c2 myoblasts cultured in DM (changed every 2 days) with daily supplementation of 10 nmol/L ATRA for 7 days were differentiated to H9c2 cardiomyocytes. The cells were stimulated with the indicated amount of M-CSF for indicated time periods. The culture medium was changed daily, and ELISA determined the VEGF level in the supernatant. M-CSF (100 ng/ml) increased VEGF production on days 2 and 3 (*P < 0.05). B: H9c2 myoblasts cultured in the same DM for 11 days were differentiated to H9c2 myotubes. Then the cells were stimulated with the indicated amount of M-CSF for the indicated time periods without medium change. M-CSF (100 ng/ml) significantly increased VEGF production on day 8 (*P < 0.03). Similar results were obtained from three independent experiments.

M-CSF pretreatment improved cardiac function after ischemic injury. M-CSF indicates goats intravenously injected with M-CSF daily for 3 days, whereas the control indicates goats injected with saline. The goat left anterior descending coronary artery was occluded for 30 minutes and then reperfused. A–C: Hemodynamic parameters before and during 30 minutes of left anterior descending coronary artery occlusion followed by 90 minutes of reperfusion are shown. Representative LVP records (A), representative positive dP/dt (B), and representative negative dP/dt (C) of control and M-CSF-treated goats. LVEDP, positive and negative dP/dt recovered in M-CSF-treated goats after reperfusion. D: Arrowheads indicate infarct areas. Compare arrows, which indicate wall contraction of nonischemic area at end systole, to arrowheads. In the infarct area, echocardiography shows dyskinetic wall movement in controls, whereas akinetic wall movement is shown in M-CSF-treated goats. Data are representative of three goats in each group.

M-CSF increased heart VEGF production in vivo. Mice were injected intramuscularly with M-CSF (200 μg/kg) or PBS (control) for 3 consecutive days (n = 5 per group). A: Quantitative RT-PCR determined the VEGF mRNA expression. M-CSF treatment significantly increased the VEGF mRNA expression in hearts (*P < 0.05). B: Conventional RT-PCR determined the M-CSF receptor (M-CSF-R) expression (top), and β-actin expression (bottom). C: The hearts were washed, homogenized in PBS, and centrifuged. ELISA determined the VEGF level in the supernatants containing 10 mg/ml protein. M-CSF significantly increased the VEGF level. M-CSF + carrageenan + anti-CD11b Ab indicates mice injected with carrageenin (1 mg) on days 1 and 4, with anti-CD11b monoclonal antibody (0.5 mg) on days 3 and 5, and with M-CSF on days 3, 4, and 5. On day 6, the hearts were isolated. This treatment did not affect the VEGF level (*P < 0.05). Similar results were obtained from two independent experiments.

The role of M-CSF-induced Akt and ERK activation in VEGF production and cell protection in differentiated H9c2 cells. A: H9c2 cardiomyocytes were cultured with M-CSF and 10 μmol/L LY294002 for the indicated time periods. The culture medium was changed daily and ELISA determined the VEGF level. B: H9c2 cardiomyocytes were incubated with 30 μmol/L PD98059 (PD) or 10 μmol/L LY294002 (Ly) for 30 minutes, then stimulated with M-CSF (100 ng/ml) and inhibitors, and analyzed as described in Figure 5. C: Differentiated H9c2 cells were stimulated with indicated amount of M-CSF with PD98059 (30 μmol/L) or LY294002 (10 μmol/L) for 24 hours. Then the cells were stimulated with H₂O₂ (40 μmol/L) for 8 hours, and WST assay determined the dead cells. M-CSF (0 ng/ml) in each group is considered as 100%, and relative cell death rates in each group are shown. *P < 0.03 compared with 0 ng/ml M-CSF in each group. Similar results were obtained from three independent experiments. D: H9c2 cardiomyocytes were incubated with reduced concentrations of PD98059 (6 μmol/L) or LY294002 (2 μmol/L). Left: H9c2 cardiomyocytes were treated with PD98059 or LY294002 for 30 minutes, stimulated with M-CSF (100 ng/ml) and inhibitors, and then analyzed as described in Figure 5. Right: H9c2 cardiomyocytes were treated with PD98059, LY294002, or without inhibitors (control) with (100 ng/ml) or without (0 ng/ml) M-CSF for 24 hours. Then the cells were stimulated with H₂O₂ (40 μmol/L) for 8 hours, and dead cells were assessed by WST assay. In each inhibitor group, dead cells at 0 ng/ml M-CSF are considered as 100%, and relative cell death rates at 100 to 0 ng/ml M-CSF in each inhibitor group are shown. *P < 0.02 compared with 0 ng/ml M-CSF in each group.

M-CSF promotes angiogenesis in goat heart after myocardial infarction. The goat left anterior descending coronary artery was permanently ligated, and the goats were sacrificed on day 14. M-CSF indicates goats intravenously injected with M-CSF shortly after the coronary artery ligation daily until day 13. Controls were injected with saline. Paraffin sections were stained with H&E (A–C, E–G, I, J, L, and M), Masson’s elastic stain (D and H), and anti-factor VIII-related antigen antibody (K and N). A and E: Left anterior descending coronary artery ligation induced myocardial infarction. Arrowheads indicate cardiomyocytes in ischemic lesions. (B, C, F, and G) Microscopic observations indicated the cardiomyocytes in the ischemic lesions were dead. D and H: The green staining indicates fibrosis or scars in hearts. I, J, L, and M: The microvessels in ischemic lesions. K and N: The microvessels in ischemic lesions were immunohistochemically stained with anti-factor VIII-related antigen antibody. O: M-CSF significantly increased microvessel density in ischemic lesions (*P < 0.01, n = 3 per group). The images represent one of three goats in each group. Scale bars: 200 μm (B and F); 20 μm (C, G, J, K, M, and N); 100 μm (I and L).