Production of bioactive recombinant human myeloid‐derived growth factor in Escherichia coli and its mechanism on vascular endothelial cell proliferation

Figures

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  Figure 1. Expression and purification of the recombinant human MYDGF and target protein. A, Schematic representation of expression vector pET31b‐rhMYDGF. Lane M molecular weight standards: Lanes 2, 4, 6 and 8 represent the uninduced BL21 (DE3)/pET31b‐rhMYDGF in different batches: Lanes 1, 3, 5, 7 and 9 represent the induced BL21 (DE3)/pET31b‐rhMYDGF in different batches. B, Schematic representation of nickel column eluted sample, Lane M: molecular weight marker. Lane 1: sample; Lane 2: unbound sample; Lanes 3, 4, 5, 6 and 7: eluted with 50, 100, 150, 200 and 300 mmol/L imidazole, respectively. C, Schematic representation of gel filtration chromatography eluted sample, Lane M: molecular weight marker: Lane 1 and 2: target protein sample eluted after gel filtration chromatography. D, Expression of the His‐rhMYDGF protein analysed by Western blotting. Lane 1, uninduced; Lanes 2, 3, 4 and 5: after induction for 20, 16, 12 and 8 h at 20°C

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  Figure 2. Identification of rhMYDGF by HPLC and mass spectrometry. A, HPLC analysis of the purified rhMYDGF. The purity of rhMYDGF eluted from a heparin affinity column was further evaluated by HPLC analysis using a C18 column. As seen from the chromatogram, the y‐axis indicates the absorbance (280 nm, AU), while the x‐axis showed elution time (min). The main peak was observed at 7.20 min. The purity of purified rhMYDGF was more than 95%. B, Mass spectrometry analysis of the rhMYDGF. The peptide mixture of rhMYDGF was separated by the C4 reversed‐phase column to identify its molecular weight. The y‐axis indicates the relative abundance. While the x‐axis showed molecular weight, the main peak was observed at 17 032 Da

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  Figure 3. Mitogenic and proliferation activity analysis of different concentrations of rhMYDGF on HCAECs in vitro. A, Cells were plated into 96‐well plates and incubated for 24 h, and then serum‐starved for 24 h. The rhMYDGF and VEGF were added for 48 h, after which CCK8 incorporation was measured, as described in ‘Materials and methods’ section. Five independent experiments were performed. B, BrdU incorporation in HCAECs after stimulation for 16 h with different concentrations of VEGF and rhMYDGF. Five independent experiments were performed. C, Cells were plated into 96‐well plates and incubated for 24 h, and then serum‐starved for 24 h. The rhMYDGF and VEGF were added for 48 h, and cell viability was measured by MTT assay, as described in ‘Materials and methods’ section. *P < .05, **P < .01 and ***P < .001 vs baseline (two independent‐sample t test). The number of cells in the control group represents the 100%

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  Figure 4. rhMYDGF promotes HCAEC proliferation and is unstimulated or stimulated with rhMYDGF (100 ng/mL unless otherwise indicated), PD98059 (PD, 20 µmol/L) or Stattic (1 nmol/L), as indicated. A, Representative immunoblots showing phosphor (p)‐MAPK1/3 (Y204/T202), MAPK1/3, p‐STAT3 (S727) and p‐STAT3 (Y705) expression in HCAECs. B, Representative immunoblots showing p‐MAPK1/3, MAPK1/3, p‐STAT3 (S727) and p‐STAT3 (Y705) expression in HCAECs in the presence of MAPK1/3 inhibitor (PD98059). C, Representative immunoblots showing p‐MAPK1/3, MAPK1/3, p‐STAT3 (S727) and p‐STAT3 (Y705) expression in HCAECs in the presence of STAT3 inhibitor (Stattic). D‐F, Western blot analysis was performed to examine the protein levels of p‐MAPK1/3 and p‐STAT3 by ImageJ. *P < .05, **P < .01 and ***P < .001 vs baseline (two independent‐sample t test)

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  Figure 5. rhMYDGF promotes HCAEC proliferation. HCAECs were stimulated with rhMYDGF (100 ng/mL unless otherwise indicated) in the absence or presence of PD98059 (PD, 20 µmol/L) or Stattic (1 nmol/L), as indicated. A, Representative immunoblots showing cyclin D1 and GAPDH expression in HCAECs. B, Representative immunoblots showing cyclin D1 and GAPDH expression in HCAECs stimulated for 12 h. C‐D, Western blot analysis was performed to examine the protein levels of cyclin D1. *P < .05, **P < .01 and ***P < .001 vs baseline (two independent‐sample t test)

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  Figure 6. Mitogenic and proliferation activity analyses of HCAECs with different concentrations of rhMYDGF in the absence or presence of MAPK1/3 and STAT3‐specific inhibitors PD98059 (20 μmol/L) and Stattic (1 nmol/L) in vitro.A,HCAECs were treated with increasing concentrations of rhMYDGF in the absence or presence of MAPK1/3‐specific inhibitors PD98059. B, HCAECs were treated with increasing concentrations of rhMYDGF in the absence or presence of STAT3‐specific inhibitors Stattic. *P < .05, **P < .01 and ***P < .001 vs control (two independent‐sample t test)

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  Figure 7. The effect of rhMYDGF on cells cycle regulation and apoptosis. A, Flow cytometry analysis showing changes in cell cycle phases after treatment with rhMYDGF (100 ng/mL) in the absence or presence of PD98059 and Stattic. B, Proportion of each phase in the cell cycle of different groups, *P < .05, **P < .01 and ***P < .001 vs vehicle. C, Flow cytometry analysis showing that pre‐incubation with rhMYDGF (100 ng/mL) for 24 h inhibited H2O2 (200 μmol/L)‐induced apoptosis in HCAECs in the presence or absence of PD98059 (20 μmol/L) and Stattic (1 nmol/L). A total of 100 000 cells were analysed in each group. The right upper quadrant indicates the late apoptotic cells; the right lower quadrant indicates the early apoptotic cells, and the left upper quadrant indicates necrotic cells

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  Figure 8. A working model of the potential mechanism of rhMYDGF‐mediated proliferation on HCAECs