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

Huili Li,Ashok Pandurangan,Yunliang Cui,Shuliu Zhang,He Huang,Yugang Chen,Weiwei Li

doi:10.1155/2021/9130650

Abstract

Purposes This study mainly explored the mechanism of capillary leakage caused by hypoxia-inducible factor-1α 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 (TNFα) and HIF-1α inhibitor 2-methoxyestradiol (2ME2) to act on HUVEC, and at the same time constructed siRNA-transfected HUVEC to interfere with the expression of HIF-1α. 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- 1α, 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-1α 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 TNFα, and detected the effect of 7 hypoxia response element plasmids on the transcription activity of MMP-9 promoter. Results Under the action of TNFα, the permeability of monolayer endothelial cells increased, and the concentration of MMP-9 in the cell supernatant increased. 2ME2 and HIF-1α-siRNA transfection can improve the above situation (P < 0.05). 2ME2 and HIF-1α-siRNA transfection can inhibit the high expression of HIF-1α and MMP-9 caused by TNFα, thereby increasing the expression of claudin-5 and ZO-1 (P < 0.05). 2ME2 and HIF-1α-siRNA transfection can reduce the inhibition of TNFα 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-1α interacts with MMP-9 at the transcription level. Conclusion This study shows that HIF-1α 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.

Highlights

Vascular leakage is the main reaction of tissue damage [1]. e destruction of the endothelial barrier and vascular leakage are characteristics of a variety of life-threatening diseases, including sepsis, acute respiratory distress syndrome (ARDS), and COVID-19, which have an important impact on the morbidity and mortality of critically ill patients [2,3,4]
It has been reported that HIF-1α promotes tumor chemotherapy resistance by recruiting GDF15induced tumor-associated macrophages in colorectal cancer [6]; silencing long noncoding RNA NEAT1 stimulates HIF1α/NF-kappaB by competitively binding miR-33a-5p to inhibit the occurrence of infantile hemangioma [7]; under hypoxic conditions, miR-375 impairs the invasion ability of liver cancer cells through targeted regulation of HIF-1α [8]; in the process of IL-1β-induced chondrocyte degeneration, FBW7 regulates the HIF-1α/VEGF pathway [9]; miR-210 regulates the inflammatory response of exudative otitis media by inhibiting the expression of HIF-1α [10]; extracellular vesicles from healthy cells activate and improve the cell function of progeria stem cells and stem cells themselves through miR-302b and HIF-1α [11]
After drug action as grouped, we washed with PBS for 3 times, seeded 100 ul PBS which contains FITC-Dextran40 (FD40, dextran labeled with fluorescein isothiocyanate) 100 mg/L in the upper transwell chamber, filled the lower chamber with 600 ul PBS for 1hours, collected the PBS in the lower chamber, measured the intensity of FITC fluorescence with a fluorescence spectrometer, established a standard curve between fluorescence intensity and FD40 concentration, and used FD40 concentration to reflect the permeability of monolayer endothelial cells

Summary

Introduction

Vascular leakage is the main reaction of tissue damage [1]. e destruction of the endothelial barrier and vascular leakage are characteristics of a variety of life-threatening diseases, including sepsis, acute respiratory distress syndrome (ARDS), and COVID-19, which have an important impact on the morbidity and mortality of critically ill patients [2,3,4]. E destruction of the endothelial barrier and vascular leakage are characteristics of a variety of life-threatening diseases, including sepsis, acute respiratory distress syndrome (ARDS), and COVID-19, which have an important impact on the morbidity and mortality of critically ill patients [2,3,4]. Despite the serious adverse clinical results associated with vascular leakage, there is currently no therapy to reverse the destruction of the endothelial barrier [5]. HIF-1 activates the transcription of many genes, including genes involved in energy metabolism, angiogenesis, and apoptosis and genes generating other protein products that can increase oxygen transfer or promote. Journal of Oncology metabolism to adapt to hypoxia, as a main regulator in the homeostatic response to cells and the whole body hypoxia. It has been reported that HIF-1α promotes tumor chemotherapy resistance by recruiting GDF15induced tumor-associated macrophages in colorectal cancer [6]; silencing long noncoding RNA NEAT1 stimulates HIF1α/NF-kappaB by competitively binding miR-33a-5p to inhibit the occurrence of infantile hemangioma [7]; under hypoxic conditions, miR-375 impairs the invasion ability of liver cancer cells through targeted regulation of HIF-1α [8]; in the process of IL-1β-induced chondrocyte degeneration, FBW7 regulates the HIF-1α/VEGF pathway [9]; miR-210 regulates the inflammatory response of exudative otitis media by inhibiting the expression of HIF-1α [10]; extracellular vesicles from healthy cells activate and improve the cell function of progeria stem cells and stem cells themselves through miR-302b and HIF-1α [11]

Methods

Results

Conclusion