Hypoxia inducible factor-2 alpha regulating hypoxia-induced angiogenesis via angiopoietin-2 pathway

Abstract

Objective To investigate the mechanisms of hypoxia inducible factor-2 alpha (HIF-2a) regulating human umbilical vein endothelial cells (HUVECs) under hypoxic conditions. Methods The experimental study was adopted. (1) HUVECs in logarithmic growth phase were taken: HUVECs without any disposals as control group, HUVECs with shRNA transfection control as shRNA control group, HUVECs with HIF-2α shRNA transfection as HIF-2α shRNA group and HUVECs with HIF-2α shRNA transfection then added rh-Ang-2 as HIF-2α + rh-Ang-2 group. (2) Western blot testing: the expressions of Ang-2 and HIF-2α proteins in HUVECs were cultured under hypoxia conditions at 0, 2, 4, 8, 12, 16, 20 hours, and the levels of which were detected in the control group, shRNA control group and HIF-2α shRNA group. (3) Enzyme-linked immunosorbent assay(ELISA): the level of Ang-2 protein in supernatant of HUVECs was detected in the control group, shRNA control group and HIF-2α shRNA group. (4)The amounts of endothelial cell tubes in HUVECs among the 4 groups were detected by tube formation experimental testing.(5)Transwell method was performed to detect the amounts of cells migration in HUVECs and hepatoma cells SMMC-7721 migration intervened by supernatant of HUVECs among the 4 groups. Measurement data with normal distribution were presented as ±s, repeated measurement data were analyzed by the repeated measures ANOVA, comparison among groups and pairwise comparison were conducted respectively by the one-way ANOVA and Dunnett's test. Results (1)Western blot test: the expression levels of Ang-2 and HIF-2α proteins in HUVECs under hypoxia conditions at 0, 2, 4, 8, 12, 16, 20 hours were 0.110±0.011, 0.120±0.020, 0.210±0.070, 0.410±0.100, 0.520±0.090, 0.790±0.130 1.010±0.220 and 0.180±0.090, 0.410±0.070, 0.470±0.110, 0.470±0.070, 0.580±0.120, 0.690±0.140, 0.920±0.130, respectively, and which were increased after culturing under hypoxia conditions and had an ascending tendency as the hypoxia time extended, with statistically significant differences (F=403.550, 3 265.587, P<0.05). The expression levels of Ang-2 and HIF-2α proteins in the control group, shRNA control group and HIF-2α shRNA group were 1.030±0.180, 1.070±0.120, 0.210±0.070, and 0.940±0.110, 0.930±0.190, 0.170±0.021, respectively, showing statistically significant differences (F=290.242, 26.688, P<0.05). (2) The results of ELISA: the expression levels of Ang-2 in the control group, shRNA control group and HIF-2α shRNA group were (433.2±9.7)ng/L, (438.3±2.6)ng/L, (114.6±4.2)ng/L, with a statistically significant difference(F=2 642.180, P<0.05). (3) The results of tube formation experiments: the number of endothelial cell tubes in the control group, shRNA control group, HIF-2α shRNA group and HIF-2α + rh-Ang-2 group were 48.3±2.5, 47.4±3.1, 19.7±1.5 and 38.3±2.1, respectively, with a statistically significant difference (F=148.196, P<0.05). (4) The results of Transwell method: ① the number of HUVECs migration in the control group, shRNA control group, HIF-2α shRNA group and HIF-2α + rh-Ang-2 group were 140.3±3.5, 142.7±2.1, 42.7±3.1 and 78.1±4.2, respectively, showing a statistically significant differences (F=212.205, P<0.05). ②The results of Transwell method: the number of SMMC-7721 cells migration after intervening using four different supernatant in the control group, shRNA control group, HIF-2α shRNA group and HIF-2α + rh-Ang-2 group were 106.7±5.5, 102.7±6.6, 63.0±3.3 and 96.7±2.1, respectively, showing a statistically significant difference (F=55.122, P<0.05). Conclusion HIF-2a could not only affect HUVECs formation but also promote SMMC-7721 cells migration via regulating Ang-2 expression. Key words: Liver neoplasms; Hypoxia inducible factor-2α; Angiopoietin-2; Angiogenesis; Migration