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

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, 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, 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 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, versus control, and one-way ANOVA followed by SNK post hoc test).

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