| Objective: The aim of this study was to elucidate the biological effects of low-concentration H2O2 on the angiogenic ability of human umbilical vein endothelial cells (HUVECs) in vitro by studying its effects on HUVECs proliferation, migration and tubule formation; and to explore the molecular mechanisms mediating the above effects. We aimed to reveal novel therapeutic targets for promoting angiogenesis after tissue transplantation, thus enhancing revascularization and improving graft survival.Methods:Part 1: To investigate the effects of low-concentration H2O2 on angiogenesis of HUVECs in vitro.? HUVECs were cultured and identified.? Effects of different concentrations of H2O2 on HUVECs viability were analyzed by flow cytometry, to determine the concentration of H2O2 used for subsequent experiments. ? Effects of 50 ?mol/L H2O2 on the proliferation, migration and tubule formation abilities of HUVECs were analyzed by CCK-8 assay,cells cratch wound healing assay and Matrigel tubule formation assay, respectively. The results of assays in this part were expressed as mean ± standard deviation (S.D). Comparison among multiple groups was performed by one-way analysis of variance (ANOVA) followed by the SNK post hoc test. The results were considered statistically significant when the P value was less than 0.05 (P<0.05).Part 2: Construction of ca-MEK5,ERK5 shRNA eukaryotic expression vectors and establishment of stable cell lines. ? ca-MEK5 eukaryotic expression vector construction. After construction of human ca-MEK5 overexpression lentiviral vector and transfection into 293T cells, flag expression was detected by western blot. After confirming the expression, lentiviruses were packaged and infected into HUVECs.? ERK5 shRNA eukaryotic expression vector construction. Three shRNA vectors were designed and constructed according to the human ERK5 gene sequence to package lentivirus and infect HUVECs. The target gene expression was detected by real-time PCR and western blot. The one with the highest interference efficiency was selected for subsequent experiments.? Stable cell line was screened and identified.Part 3: Study on the molecular mechanisms of low-concentration H2O2 in promoting angiogenesis of HUVECs in vitro. ?The effect of 50 ?mol/L H2O2 on the activation of ERK5 in HUVECs was analyzed by western blot. ? CCK-8 assay, cells cratch wound healing assay and Matrigel tubule formation assay were used to analyze the changes in proliferation, migration and tubule formation abilities of different cells stimulated by 50 ?mol/L H2O2.? VEGF mRNA levels in different cells were detected by real-time PCR. ? VEGF concentrations in different cell supernatants were assayed by ELISA. The results of assays in this part were expressed as (x±SD),while the statistical methods were the same as in Part 1.Results:Part 1: ? HUVECs grew adherently in a monolayer in 10% FBS and 1%Penicillin-Streptomycin-containing DMEM medium, which presented typical cobble stone morphology when the confluence approached 100% and were positive to the endothelial-specific marker CD31. ?After treating with different concentrations of H2O2 for 30 minutes, no marked apoptosis of cells was observed in the 25 ?mol/L and 50 ?mol/L groups compared to the control group. The apoptotic rate increased slightly for the 100 ?mol/L group,and increased significantly for the 200 ?mol/L group (P<0.05). The relatively high tolerable concentration (50 ?mol/L) for cells was finalized as the subsequent experimental concentration.? Compared to the control group, the proliferation, migration and tubule formation of HUVECs were enhanced significantly in the 50 ?mol/L H2O2 stimulated group. Catalase can eliminate the aforementioned pro-angiogenic effect of H2O2.?The p-ERK5 level in HUVECs of the 50 ?mol/L H2O2 stimulated group was significantly higher than the control group(P<0.05).Part 2: ? ca-MEK5 lentiviral vector was constructed successfully, and its HUVECs infection efficiency was about 60% at MOI 20. ? ERK5 shRNA lentiviral vector was constructed and screened successfully,while infection efficiency was about 70% at MOI 20.? Both vectors carried puromycin resistant and EGFP genes. The percentages of cells expressing GFP were all higher than 90% after 14 days of selective culture in puromycin-containing medium. In ca-MEK5 overexpression stable cell line, Flag was positive by western blot, and p-ERK5 increased significantly (P<0.05) while levels of ERK5 mRNA and ERK5 didn' t change. In ERK5 shRNA stable cell line, levels of ERK5 mRNA, ERK5 and p-ERK5 all decreased significantly (P<0.05) . No significant changes in total ERK5 or p-ERK5 level were found in the corresponding control cells.Part 3: ? Cell proliferation, migration and tubule formation (vs. corresponding control group):enhanced significantly in the 50 ?mol/L H2O2 stimulated HUVECs group, further enhanced in the ca-MEK5 transfection group (P<0.05), inhibited significantly in the ERK5 shRNA transfection group (P<0.05). ? The intracellular VEGF mRNA level (vs. corresponding controlgroup): elevated significantly in the 50 ?mol/L H2O2 stimulated HUVECs group (P<0.05),further increased in the ca-MEK5 transfection group(P<0.05), decresed significantly in the ERK5 shRNA transfection group (P>0.05).? The VEGF level in cell supernatants (vs.corresponding control group): elevated significantly in the 50 ?mol/L H2O2 stimulated HUVECs group (P<0.05), further increased in the ca-MEK5 transfection group (P<0.05), decresed significantly in the ERK5 shRNA transfection group (P>0.05).Conclusion: Low-concentration H2O2(50 ?mol/L) can promote the proliferation, migration and tubule formation of HUVECs. ERK5 is essential for mediating the aforementioned pro-angiogenic effects of H2O2. |
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