Extracellular Signal-Regulated Kinase 5 is Required for Low-Concentration H₂O₂-Induced Angiogenesis of Human Umbilical Vein Endothelial Cells

Figures

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  Figure 1. Effects of different concentrations of H2O2 on HUVEC viability. (a–e) Scatter plots of flow cytometric results with annexin V (horizontal axis) and 7-AAD (vertical axis). Cells were labeled with annexin V and 7-AAD after a 30 min incubation with different concentrations of H2O2. The conditions were as follows: (a) 0 μmol/L H2O2 (control), (b) 25 μmol/L H2O2, (c) 50 μmol/L H2O2, (d) 100 μmol/L H2O2, and (e) 200 μmol/L H2O2. Early apoptotic cells were defined as annexin V positive/7-AAD negative. (f) Quantitative analysis of cell apoptosis with different concentrations of H2O2 (mean ± SD of six separate experiments, ∗p < 0.05 versus control, and one-way ANOVA followed by SNK post hoc test).

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  Figure 2. Low concentrations of H2O2 promote HUVEC proliferation, migration, and tubule formation. (a) Cell proliferation assessed by CCK-8 assays. HUVECs were treated with PBS, H2O2 (50 μmol/L), or catalase (5 U/ml) and H2O2 (50 μmol/L) for 30 min. Then, 10 μl CCK-8 was added. The OD at 450 nm was measured 2.5 h later (mean ± SD of six separate experiments, ∗p < 0.05 versus control, and one-way ANOVA followed by SNK post hoc test). (b, c) Cell migration assessed by scratch wound assays. HUVECs were seeded at confluence and cultured overnight. Following treatment with PBS, H2O2 (50 μmol/L), or catalase (5 U/ml) and H2O2 (50 μmol/L) for 30 min, a scratch was made. The wounded HUVECs monolayers were then incubated with medium containing 2% FBS for 24 h. Photographs were taken at 0 h and 24 h at fixed locations along the scratch, and the closure of the wound area was quantified (mean ± SD of six separate experiments, ∗p < 0.05 versus control, and one-way ANOVA followed by SNK post hoc test). (d, e, f) In vitro tubule formation. Tubule formation was assayed on Matrigel. Cells were pretreated with PBS, H2O2 (50 μmol/L), or catalase (5 U/ml) and H2O2 (50 μmol/L) for 30 min and then seeded onto…

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  Figure 3. Low-concentration H2O2 induced ERK5 activation in HUVECs. (a) ERK5, p-ERK5, MEF2C, p-MEF2C, ERK1/2, and p-ERK1/2 protein levels were measured by western blotting. Following treatment with PBS, H2O2 (50 μmol/L), or catalase (5 U/ml) and H2O2 (50 μmol/L) for 30 min, cells were lysed, and the lysates were resolved by 10% or 6% SDS-PAGE. (b, c, d, e, f, g) Densitometric quantification of ERK5, p-ERK5, MEF2C, p-MEF2C, ERK1/2, and p-ERK1/2 expression (mean ± SD of three separate experiments, ∗p < 0.05 versus control, and one-way ANOVA followed by SNK post hoc test).

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  Figure 4. Construction of high or low p-ERK5 HUVEC populations. (a) Images of lentivirus-transfected HUVECs. (b) ERK5, p-ERK5, MEF2C, p-MEF2C, ERK1/2, and p-ERK1/2 protein levels were determined by western blotting. Different cell populations were lysed, and the lysates were resolved by 10% or 6% SDS-PAGE. (c, d, e, f, g, h) Densitometric quantification of ERK5, p-ERK5, MEF2C, p-MEF2C, ERK1/2, and p-ERK1/2 expression (mean ± SD of three separate experiments, ∗p < 0.05, and one-way ANOVA followed by SNK post hoc test).

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  Figure 5. Activated ERK5 is essential for low-concentration H2O2-induced HUVEC angiogenesis. (a) Cell proliferation assessed by CCK-8 assays. Cells were treated with PBS or H2O2 (50 μmol/L) for 30 min. Then, 10 μl CCK-8 was added. The OD value at 450 nm was measured 2.5 h later (mean ± SD of six separate experiments, ∗p < 0.05, and one-way ANOVA followed by SNK post hoc test). (b, c) Cell migration assessed by scratch wound assays. Cells were seeded at confluence and cultured overnight. Following treatment with PBS or H2O2 (50 μmol/L) for 30 min, a scratch was made. The wounded cell monolayers were then incubated with medium containing 2% FBS for 12 h. Photographs were taken at 0 h and 12 h at fixed locations along the scratch, and closure of the wound was quantified (mean ± SD of six separate experiments, ∗p < 0.05, and one-way ANOVA followed by SNK post hoc test). (d, e, f) In vitro tubule formation. Tubule formation was assayed on Matrigel. Cells were pretreated with PBS or H2O2 (50 μmol/L) for 30 min and then seeded onto Matrigel-coated wells in normal medium and cultured for 4 to 8 h. Tubule formation was analyzed by measuring branch length and counting tubule number (mean ± SD of six…