Objectives: Approach to mechanism of hypoxia-induced factor 1alpha expression in endothelial cells of human umbilical vein and its induction of apoptosis
Methods: In vitro models, and such techniques as transmission electron microscopy, flow cytometry, RT-PCR and Western blot, were applied to investigate the transcription and protein expression of HIF-1alpha mRNA in ECV 304 cells and the action of HIF-1alpha on cell cycle blocking, proliferation inhibition and induction of apoptosis. Cells were divided into two groups: normal oxygen and hypoxic for various time periods (2, 4, 8, 12, 24 and 48 h).
Results: We observed that the expression level of HIF-1alpha mRNA and its protein were correlated with the degree of hypoxia. The expression of HIF-1alpha mRNA notably increased after 4, 8, 12, 24 and 48 h of hypoxia, particularly at 24 and 48 h with a gray scale of (71 +/- 1.81) and (70 +/- 2.02) respectively, differing significantly from the control group (P < 0.01), The protein expression of HIF-1alpha also increased 4, 8, 12, 24 and 48 h after hypoxia, particularly at 24 and 48 h, with gray scale (83 +/- 0.15) and (98 +/- 0.10) respectively (P < 0.01), indicating an increase of HIF-1alpha protein expression significantly different from the other groups (P < 0.01). In the control group, there was slight transcription and protein expression of HIF-1alpha at 0 h after hypoxia. Meanwhile, each phase of the cell cycle was detected to have increased expression of HIF-1alpha in response to oxygen-deficient treatment. At times of 24 and 48 h, the number percentage of cells in G1 significantly increased with values of 66.335 +/- 2.144 and 58.890 +/- 5.128; however, the number cells in S phase decreased to 36.215 +/- 1.582 and 39.826 +/- 5.097, significantly different compared to the control group at 0 h with values of 43.903 +/- 6.506 and 60.571 +/- 24.026-(P < 0.05). When the cell cycle was blocked in the G2/S phase, the cell apoptosis ratio significantly increased by 9.24 +/- 1.828 and 30.735 +/- 11.38, compared to each control group-(P < 0.01).
Conclusions: By induction of hypoxia, the cell cycle was dramatically blocked in G1/S phase, and the expression of HIF-1alpha also increased Therefore, sustained expression of HIF-1alpha can inhibit cell hyperplasia, and the apoptosis promotion of injured cells as well as provide protection of endothelial cells.