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

Abstract

Background. The aim of this study was to assess the effects of low concentrations of H₂O₂ on angiogenesis of human umbilical vein endothelial cells (HUVECs) in vitro and explore the underlying mechanisms. Methods. HUVECs were cultured and stimulated with different concentrations of H₂O₂. Flow cytometric analysis was used to select an optimal concentration of H₂O₂ for the following experiments. Cell proliferation, migration, and tubule formation were evaluated by Cell Counting Kit-8 (CCK-8) assays, scratch wound assays, and Matrigel tubule formation assays, respectively. For gain and loss of function studies, constitutively active MEK5 (CA-MEK5) and ERK5 shRNA lentiviruses were used to activate or knock down extracellular signal-regulated kinase 5 (ERK5). Results. We found that low concentrations of H₂O₂ promoted HUVECs proliferation, migration, and tubule formation. ERK5 in HUVECs was significantly activated by H₂O₂. Enhanced ERK5 activity significantly amplified the proangiogenic effects of H₂O₂; in contrast, ERK5 knock-down abrogated the effects of H₂O₂. Conclusions. Our results confirmed that low concentrations of H₂O₂ promoted HUVECs angiogenesis in vitro, and ERK5 is an essential mediator of this process. Therefore, ERK5 may be a potential therapeutic target for promoting angiogenesis and improving graft survival.

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).

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 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, ^∗p < 0.05 versus control, and one-way ANOVA followed by SNK post hoc test).

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).

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).

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 separate experiments, ^∗p < 0.05, 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, ^∗p < 0.05, and one-way ANOVA followed by SNK post hoc test).