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

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, , 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, , 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 separate experiments, , and one-way ANOVA followed by SNK post hoc test). (g) VEGF secretion assessed by ELISA. Cells were treated with PBS or H₂O₂ (50 μmol/L) for 30 min; then the medium was changed to fresh normal medium. After 12 h or 24 h culture, the cell supernatant was collected, and the concentration of VEGF was determined by a human VEGF ELISA kit (mean ± SD of six separate experiments, , and one-way ANOVA followed by SNK post hoc test).

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