Study on the Mechanism of Capillary Leakage Caused by Hypoxia-Inducible Factor-1α through Inducing High Expression of Matrix Metalloproteinase-9

Purposes. This study mainly explored the mechanism of capillary leakage caused by hypoxia-inducible factor-1 alpha through inducing high expression of matrix metalloproteinase-9. Method. We established a monolayer endothelial cell model by culturing human umbilical vein endothelial cells (HUVEC) in vitro, used tumor necrosis factor (TNF alpha) and HIF-1 alpha inhibitor 2-methoxyestradiol (2ME2) to act on HUVEC, and at the same time constructed siRNA-transfected HUVEC to interfere with the expression of HIF-1 alpha. The permeability of monolayer endothelial cells was measured by transwell chamber method, the concentration of MMP-9 in the supernatant was measured by ELISA method, the expression of key molecules related to permeability (HIF-1 alpha, MMP-9, claudin-5, and ZO-1) was measured by RT-PCR and Western blot method, and the localization and expression of claudin-5 and ZO-1 were measured by immunofluorescence method. We searched for 7 HIF-1 alpha hypoxia response elements within 4000 bp before the transcription start site in the MMP-9 promoter region, constructed the MMP-9 promoter-luciferase reporter gene recombinant plasmid, transfected and stimulated HUVEC with TNF alpha, and detected the effect of 7 hypoxia response element plasmids on the transcription activity of MMP-9 promoter. Results. Under the action of TNF alpha, the permeability of monolayer endothelial cells increased, and the concentration of MMP-9 in the cell supernatant increased. 2ME2 and HIF-1 alpha-siRNA transfection can improve the above situation (P<0.05). 2ME2 and HIF-1 alpha-siRNA transfection can inhibit the high expression of HIF-1 alpha and MMP-9 caused by TNF alpha, thereby increasing the expression of claudin-5 and ZO-1 (P<0.05). 2ME2 and HIF-1 alpha-siRNA transfection can reduce the inhibition of TNF alpha on the expression of cell membrane protein claudin-5 and tight junction protein ZO-1. Element 1, element 5, and element 7 are the sites where HIF-1 alpha interacts with MMP-9 at the transcription level. Conclusion. This study shows that HIF-1 alpha can increase the permeability of monolayer epithelial cells by inducing the high expression of MMP-9, leading to capillary leakage. Its target is at the -3798 bp, -1878 bp, and -1489 bp points of the transcription initiation site in the MMP-9 promoter region.