To investigate the role of HIF-1α / VEGF / Notch signaling pathways in hypoxia-induced angiogenesis of human umbilical vein endothelial cells (HUVEC) .

HUVEC was induced by normoxic and hypoxia mimetic agent-Cobalt dichloride (CoCl₂, 200 μmol/ L) , followed by inhibitor of Notch signaling pathway DAPT (30 μmol/L, 24 h) , and activator of Notch signaling pathway JAG-1 (30 μmol/L, 24 h) . The effect of hypoxia on the angiogenesis of HUVEC cells was investigated by in vitro tubule formation assay. The effect of hypoxia on the angiogenesis ability of HUVEC was observed through in vitro tubule formation experiments.The mRNA and protein expression level of hypoxia inducible factor-1α (HIF-1α) , vascular endothelial growth factor (VEGF) , matrix metalloprotein-9 (MMP-9) and molecules of Notch1 signaling pathway (Notch1, Dell4 and Jagged1) were detected by RT-PCR and Western blot respectively. The effects of hypoxia, DAPT, and JAG-1 on HUVEC migration were investigated by Transwell migration assay and wound healing assay. The effects of hypoxia and Notch1 on HUVEC proliferation were measured by MTT assay. Factorial design variance was used to analyze the interaction of DAPT and JAG-1 on HUVEC migration ability, migration distance, tubule formation ability, and cell proliferation under hypoxic conditions. The t test was used to compare the two groups.

Compared with the normoxia treated cells, the total length of tubules in CoCl₂-treated cells was significantly higher [ (8.18±0.62) mm vs (15.43±1.32) mm, P < 0.05]. Compared with the normoxia treated cells, the mRNA of HIF-1α, VEGF, MMP-9, Notch1, Dell4 and Jagged1 in CoCl₂-treated cell were significantly up-regulated, (1.01±0.03 vs 4.43±0.35, 1.02±0.03 vs 3.55±0.28, 0.98±0.04 vs 3.24±0.25, 1.01±0.03 vs 3.22±0.25, 0.99±0.02 vs 2.89±0.22, 1.02±0.04 vs 2.43±0.19) , as well as the protein expressions increased (0.98±0.01 vs 3.13±0.24, 0.98±0.02 vs 2.67±0.21, 0.97±0.03 vs 2.45±0.19, 1.01±0.03 vs 2.44±0.19, 1.00±0.04 vs 2.30±0.18, 1.03±0.05 vs 2.27±0.18) (all P < 0.05) Transwell migration experiments showed that compared with the normoxic treated cells, the number of HUVEC migrating cells treated by CoCl₂ was significantly increased (P < 0.05) . Wound healing experiments showed that compared with the normoxic-treated cells, the migration distance of HUVEC treated by CoCl₂ was significantly increased (P < 0.05) . Compared with the hypoxia group, the migration ability of HUVEC treated by CoCl₂ + DAPT was significantly reduced, while the migration of HUVEC treated by CoCl₂+ JAG-1 was significantly increased (P < 0.05) . Tubular formation and MTT assay showed that compared with the hypoxic group, the tubular formation ability and cell proliferation of HUVEC treated by CoCl₂ + DAPT were significantly reduced, while the tubular formation and cell proliferation of HUVEC treated by CoCl₂ + JAG-1 were significantly increased (P < 0.05) . Factorial design analysis of variance showed that hypoxia and JAG-1 had a synergistic effect on the interaction of migrating cell number, tubule formation, and cell proliferation ability (P < 0.05) .

Hypoxia could significantly induced the angiogenesis, migration and proliferation of HUVEC cells by activating HIF-1α/VEGF/Notch1 signaling pathway.