The Effection And Mechanism Of Ubiquitin-Like Protein FAT10 In Regulating Endothelial Cell Permeability

Background:Increased permeability of blood vessel wall is one of the important pathological changes in the development of cardiovascular diseases,such as vascular inflammation,atherosclerosis,stroke,etc.The pathological mechanism of increasing vascular permeability has not been fully revealed,which also limits the treatment of vascular permeability.The ubiquitin-like protein FAT10 is a newly discovered member of the ubiquitin-like protein family and has been found to play an important role in inflammation,tumor,autophagy,apoptosis,and DNA damage response.The regulation of FAT10 on the permeability of vascular endothelial cells is still unclear.Objective:To explore the role of FAT10 in regulating vascular endothelial cell permeability and its mechanism through in vivo and ex vivo cell experiments.Method:In vivo animal experiments:FAT10 knockout mice(FAT10^-/-)were constructed using Crispr/cas9 technology,and the subcutaneous inflammation model of mice was constructed by carrageenan.The total number and classification of leukocytes in the subcutaneous inflammation lavage fluid were measured to observe the vascular permeability.Histopathological changes were detected by histological Hemotoxylin&Eosin（HE）staining.The experimental mice were divided into four groups:control group（WT group）,FAT10 knockout group(FAT10^-/-group),normal+inflammatory stimulation group（WT+inflammatory group）,FAT10 knockout+inflammatory stimulation group(FAT10^-/-+inflammation group).In vitro cell experiments:Human umbilical vein endothelial cells（HUVECs）were selected as subjects,and induced by human recombinant interferon（IFN-γ）and tumor necrosis factor（TNF-α）to construct a cellular inflammatory model and treated with overexpression of FAT10adenovirus.Transfected with FAT10 plasmid and N-acetyl-l-cysteine（NAC）,the cells were divided into 12 groups:normal control group（con group）,solvent control group（Vehicle group）,TNF-αand IFN-γinduction group（TNF-α+IFN-γgroup）,overexpressing negative virus control group（ad-Ctrl group）,overexpressed FAT10group（ad-FAT10 group）,interference negative virus control group（sh-Ctrl group）,interfering the expression of FAT10 group（sh-FAT10 group）,transfected Flag plasmid group（Flag group）and its NAC treatment group（Flag+NAC group）,transfected FAT10 plasmid group（FAT10 group）and its NAC treatment group（FAT10+NAC group）,interference FAT10 expression and NAC treatment group（sh-FAT10+NAC group）.Western-Blot was used to detect protein expression,macrophage transwell migration assay to detect the changes in endothelial cell permeability before and after NAC treatment and the detection of changes in ROS in endothelial cells using confocal.Result:1.The results of the experiments in vivo showed that comparing with the control group,the total number of white blood cells in the back inflammatory balloon lavage fluid of the FAT10^-/-group decreased（1.775×10⁹/L vs 4.922×10⁹/L,P<0.05）.HE staining of the back inflammation showed that the infiltration of inflammatory cells in the subendothelial cells of FAT10^-/-mice was reduced,the formation of new and small blood vessels was also reduced.2.In vitro,the results showed that the protein expression level of FAT10increased in the TNF-α+IFN-γgroup,compared with the control group（P<0.05）.And in the macrophage migration experiment,compared with the control group,the number of macrophages migrated increased in the ad-FAT10 group（P<0.001）.In the ad-FAT10 group,the OD value of the eluate of gentian crystal violet staining was higher than that of the ad-Ctrl group、the sh-Ctrl group and the sh-FAT10 group（P<0.001）.3.Confocal microscopy results showed that compared with the Flag group,ROS increased significantly in the FAT10 group（P<0.001）,and ROS decreased in the sh-FAT10 group（P<0.01）.The transwell results of macrophages after NAC treatment showed that the number of macrophages penetrating in the FAT10 group was significantly increased compared with the Flag group（P<0.05）,and the number of macrophages decreased in the sh-FAT10 group（P<0.05）;and compared with the FAT10 group,the number of macrophages decreased in the FAT10+NAC group.Conclusion:Inflammatory factors can induce high expression of FAT10 in endothelial cells.FAT10 leads to the increase in the production of ROS and the endothelial cell permeability,which is beneficial to inflammatory cells and inflammatory substances exudation,aggravating and maintaining chronic inflammatory response.The results of this study provide the experimental basis for the further understanding of its mechanism.