Transdifferentiation Of Tie1 Positive Hepatocytes In Liver Fibrosis

BackgroundAs the largest vital organ in body,the liver participates in various activities in life,including metabolism of nutrients,blood circulation and detoxification.Because of its abundant blood supply and strong detoxification capacity,the liver is more likely to be attacked by a variety of toxic and harmful factors.In the early stage of injury or when the injury is gentle,the fibrosis process can be reversed through self-regulation.If the injury is severe or undergoes multiple repeated injuries,irreversible fibrosis will occur and the normal structure and function of the liver will be affected.Severe fibrosis can lead to liver failure.If it continues to develop,it can lead to cirrhosis and even liver cancer.Liver fibrosis has seriously affected human life and health over decades.Due to the complexity of fibrosis and the limitations of current medical methods,the treatment of liver fibrosis has not yet achieved satisfactory results.The number of people who die of this disease is growing with time.Therefore,the study on liver injury,post-injury regeneration and restoration are the hotspot of current research.and it is also the entry point for the development of follow-up treatment programs.The self-regenerating of damaged liver tissue depends on the type of injury it suffered.The process of regeneration following hepatectomy involves the proliferation and hypertrophy of the remaining hepatocytes.When the liver is chronically damaged by toxic substances,one of the mainly sources of cells in the process of regeneration is to dedifferentiate into the state of stem cells with have self-renewal capacity and multiple differentiation potential through existing mature cells,and then further differentiate into other epithelial cells,and help restore liver function.Tie1 is a member of the tyrosine kinase family and generally functions as a heterodimer by forming a heterodimer with Tie2.Tie1 ligands have not yet been clearly identified,so most studies have focused on targeting Tie2.Our latest research has found that LECT2 can be one of the ligands of Tie1.In mouse model of carbon tetrachloride-induced hepatic fibrosis,we studied LECT2/Tie1 signaling pathways that affected liver fibrosis through regulation of vascular endothelial cells(unpublished).The study of Tie1 has long been focused on endothelial cells,which interact with vascular endothelial growth factor through Tie2 and participate in the formation and stability of the vascular system.At present,there is only a very small amount of research involving the relationship between liver injury and Tie1.Whether Tie1 can express in hepatocytes and what other functions of Tie1 in liver have not been reported yet.METHODS1.Observation of the expression of Tie1 in the carbon tetrachloride-induced liver fibrosis injury model.Normal C57 male mice were given carbon tetrachloride twice a week by intraperitoneal injection.On the basis of successful fibrosis model,the expression of Tie1 was observed by immunohistochemistry and immunofluorescence techniques.2.Study the type of Tie1-positive cells.For Tie1-positive cell populations that appear during fibrosis,combined their morphological and localization characteristics with the co-localization results of Tiel and other hepatic intracellular markers to identify Its cell types.And study the relationship between Tie1 expression and liver injury in primary cells.3.Further study on whether there are dynamic changes of Tie1-positive cells during fibrosis.By verifying whether Tie1-positive cells express stem cell markers at the same time to confirm whether there is dedifferentiation in Tie1-positive cell population in fibrosis liver tissue.Comparing the expression levels of other cell types in primary hepatocytes before and after carbon tetrachloride injury to infer whether there is a potential to transdifferentiation of Tie1-positive cells.RESULTS1.Successful establishment of carbon tetrachloride-induced liver fibrosis model in mice,in which we found the specific expression of Tie1.It is the first time in the fibrotic liver tissue that we found in the specific expression of Tie1,and part of these special cell population not only have a special location around the wound area,but also have dynamic change during the damage progresses.2.Tie1-positive cells that appeared in fibrosis are hepatocytes.Co-expression of ALB and Tie1 in fibrotic liver tissue was detected by immunofluorescence co-localization technique.It was confirmed that all Tie1 positive cells were hepatocytes,and their expression was closely related to whether they were damaged.The detection of primary hepatocytes showed that carbon tetrachloride injury can cause a significant upregulation of Tie1 expression.3.Confirmation of transdifferentiation potential of Tie1-positive Cells.Confirmation of the co-localization between Tie1 and Axin2,which has been proved as a stem cell marker of liver.And further detection showed the increased expression of a series of stem cell-related molecules in carbon tetrachloride-stimulated hepatocytes.By comparing the levels of mRNA expression of angiogenesis-related molecules in primary hepatocytes before and after stimulation with carbon tetrachloride,it was demonstrated that the target cell population may has the potential to differentiate from stem cells into vascular endothelial cells.4.Construction of transgenic mice for further research.Tie1-e(2A-Cre)1,Rosa26(ACTB-tdTomato-EGFP),Alb-CreERT2,and Rosa26(loxp-stop-loxp-DsRed)transgenic mice were initially constructed.Using genetic lineage tracing technology in subsequent experiments can clarify the origin and destination of Tie 1-positive hepatocytes in hepatic fibrosis and the mechanism of its transdifferentiation.CONCLUSIONS1.Tie1 has special expression in liver fibrosis tissue.2.The cells that specifically express Tie1 are hepatocytes.3.Tie1-positive cell population has a potential to transdifferentiation during liver fibrosisINNOVATIONSIt is the first time to find the presence of Tie1-positive hepatocyte populations in fibrotic liver tissue.The study have demonstrated that this group of cells associated with injury has the potential to transdifferentiate into the vascular(sinus)endothelium during fibrosis.Through the follow-up genetic lineage tracing technology,we can better verify the origin and destination of Tie1-positive hepatocytes in the process of hepatic fibrosis and study on its molecular regulation mechanism.Further find the new targets for treatment of liver fibrosis.