LECT2 Is A Functional Ligand Of Tie1 And Crucial For Liver Fibrosis Development And Therapy

Background and ObjectiveFibrotic liver injury is a world-wide disease which is an important part of the development of chronic liver diseases.There are no effective FDA approved treatment strategies for it yet.Therefore,it is important clinical value and social significance to study the mechanism of liver fibrosis,search for new molecular markers and therapeutic targets,improve the survival rate of patients through early intervention and individualized treatment.Leukocyte cell-derived chemotaxin 2(LECT2)is a secreted protein that mainly expressed by hepatocytes.Initially,it was originally identified as a chemotactic factor for neutrophils and stimulated the growth of chondrocytes and osteoblasts,and then it is gradually found that it could play the roles as a ligand.Tyrosine kinase receptor 1(Tie1)is a transmembrane receptor,which is mainly located on the membrane of vascular endothelial cells,and is crucial for angiogenesis and function maintenance.However,the ligand of Tie1 is still unknown by people for more than 20 years.This study revealed that LECT2 as a functional ligand bound directly to the Ig3 domain of Tie1.LECT2 interrupted the interaction of Tie1-Tie2,and inhibited angiogenesis during liver fibrosis processing.We will further explore the role and mechanism of LECT2 during liver fibrosis in order to provide a new target for clinical diagnosis,treatment and prognosis evaluation,and lay a theoretical foundation.Methods1.The expression of LECT2 and CD31 in liver tissue were measured by immunohistochemistry.2.The content of LECT2,ALT and AST in serum were measured by ELISA.3.The expression of collagen fibers in liver tissue was measured by Sirius red.4.The level of mRNA expression of some indexes related liver fibrosis were measured by RT-qPCR.5.Transwell and tube formation assays were used to measure the effects of rLECT2 and other related factors on the migration and and tube formation of EA.hy926 cells.6.Immunofluorescence was used to measure the expression of GFP,CD31 and collagen type Ⅳ in liver tissue,the co-localized expression of LECT2 and Tie1 in hepatic vascular endothelial cells,and the co-localized expression of LECT2 and Tie1 in HUVEC and EA.hy926 cells.7.The Tiel truncation were constructed and the CO-IP assay was used to detect the combination of LECT2 and Tie1.GST pull-down experiment verified that LECT2 bound directly to Tie1.8.The NanoBiTTM protein:protein interaction kit of Promega company was used to measure the influence of rLECT2 on the binding of Tie1-Tie2,Tie1-Tie1 and Tie2-Tie2.9.The effects of LECT2 on the content of total and phosphorylation of Tie1 and Tie2 were measured by western blot.10.Scanning electron microscope(SEM)was used to observe the fenestration of the endothelial cells in the liver sinusoids of mice in different states.Results1.LECT2 protein was highly expressed in the patients with liver cirrhosis,and the expression of LECT2 was positively correlated with the Child-Pugh classification of liver cirrhosis.2.Lect2 KO attenuated liver fibrosis and LECT2 overexpression worsened liver fibrosis.3.LECT2 inhibited vascular endothelial cell migration and tube formation in vitro and blocked angiogenesis in vivo.4.Lect2 KO inhibited capillarization of liver sinusoids.5.LECT2 bound directly to Ig3 domain of Tie 1.6.LECT2 dissociated Tie1/Tie2 heterodimers and promoted Tie2/Tie2 homodimers.7.LECT2 inhibited vascular endothelial cells functions through Tie1.8.AAV9-LECT2 shRNA attenuated liver fibrosisConclusion1.As a functional ligand,LECT2 can directly bind to the Ig3 domain of Tie1 and inhibit angiogenesis.2.Decreasing the expression of LECT2 can promote angiogenesis around fibrosis boundaries and inhibit the capillarization of hepatic sinusoidal endothelial cells,which attenuate liver fibrosis and provid a new target for the treatment of liver fibrosis.3.LECT2 protein was highly expressed in the patients with liver cirrhosis,and the expression of LECT2 was positively correlated with the Child-Pugh classification of liver cirrhosis.It prompts that the content of LECT2 in serum can be used as a marker for diagnosis of liver cirrhosis,judging the severity of liver cirrhosis and estimating prognosis.