Endothelial Notch Signaling Regulates Neutrophil Transmigration Via Downregulating Endomucin During Hepatic Ischemia/Reperfusion Injury

BackgroundIn acute inflammatory response,leukocytes transmigrate across blood vessel wall in three consecutive steps.Firstly,leukocytes adhere to and roll on the surface of endothelial cells.Then,leukocytes interact with endothelial cells and adhere firmly.Finally,leukocytes transmigrate across endothelial cells and enter tissue parenchyma.In this process,recruitment and activation of leukocytes are regulated by a complex molecular network,in which chemokines,selectins,addressins and other adhesion molecules from endothelial cells are involved,but how they respond to inflammatory signals and coordinate their actions remains elusive.Hepatic ischemia/reperfusion injury（HIRI）commonly takes place during hepatic surgery and is one of the major causes of poor postoperative recovery of patients.The underlying mechanism of HIRI is complicated,among which,massive infiltration of neutrophils is thought to be a major cause of hepatic tissue damage.Therefore,blocking the infiltration of neutrophils may become a new therapeutic approach to prevent HIRI in the future.Also,HIRI is widely used as an ideal model to study the molecular mechanisms underlying acute inflammatory response.Notch signaling pathway is universally expressed in embryonic and adult tissues and is of great significance in the regulation of cell fate decision,proliferation and apoptosis.Our previous studies have indicated that Notch signaling in liver sinusoid endothelial cells（LSECs）plays an essential role in the HIRI process,the underlying mechanism of which lies in the changes of gene expression profile of LSECs brought up by the Notch signaling activation.This would lead to the changes of expression levels of molecules including adhesion molecules and secreted chemokines.These molecules are of great significance in terms of regulating neutrophil migration across endothelial cells.Can endothelial Notch signaling regulate neutrophil infiltration?What is its downstream molecular mechanism?Clarifying these issues would be of great help to further reveal the mechanisms of acute inflammation,such as HIRI,and can provide new targets for preventive and therapeutic strategy of HIRI.ObjectivesTo investigate the role of Notch signaling in LSECs during HIRI,and to elucidate the underlying mechanism of endothelial Notch signaling in regulating neutrophil trans-endothelial migration.Experimental procedure and methodsFirstly,wild-type mice were subjected to HIRI surgery.LSECs were isolated by immunomagnetic beads,and the expression of Notch signaling-related molecules was evaluated by quantitative reverse transcription-polymerase chain reaction（q RT-PCR）and western blotting（WB）.Immunohistochemical staining of clinical biopsies from patients was used to determine the expression of Notch receptor in situ.Secondly,by conditional over-expression of Notch intracellular domain（NICD）or conditional knockout of recombination signal-binding protein Jκ（RBP-J）,Notch signaling was specifically activated(NIC^{e CA})or inhibited(RBP-J^?E)in endothelial cells in mice,respectively.The mice were then subjected to HIRI surgery.Histological staining and serum liver function tests were used to evaluate tissue injury,and flow cytometry was used to observe the neutrophil infiltration.Thirdly,human umbilical vein endothelial cells（HUVECs）were subjected to oxygen glucose deprivation/re-oxygenation（OGD/Re-oxy）to mimic ischemia/reperfusion injury in vitro.Notch signaling was activated by Notch intracellular domain（NICD）overexpression using adenovirus,or inhibited byγ-secretase inhibitor.The effects of activation/inhibition of endothelial Notch signaling on cell adhesion and trans-endothelial migration were evaluated.Fourthly,gene set enrichment analysis（GSEA）was employed to further investigate the changes in the gene expression profile of endothelial cells after Notch signaling activation.Finally,HUVECs were infected with NICD overexpression adenovirus and human endomucin（EMCN）overexpression adenovirus and subjected to OGD/Re-oxy in vitro.The effect of EMCN overexpression was verified by cell adhesion assay and trans-endothelial migration assay.Results1.HIRI leads to Notch activation in endothelial cells.In wild type mice subjected to HIRI,m RNA levels of Notch1,Notch2 and Dll4 in LSECs were upregulated,and the protein level of NICD,which transmits Notch activation signaling into nucleus,also increased.NICD immunohistochemical staining of biopsies from patients accepting hepatectomy showed that the number of NICD-positive LSECs increased significantly after surgery.2.Endothelial Notch activation exacerbates HIRI.In comparison with the control,endothelial Notch activation exacerbated injury,which was characterized by increased necrotic area shown by HE staining,increased apoptosis of hepatocytes as shown by TUNEL assay,and increased serum AST and ALT levels.3.Endothelial Notch activation promotes neutrophil infiltration and inflammation in HIRI.After reperfusion,LSECs from mice with or without Notch activation were isolated.In the Notch activated group,flow cytometry revealed a significant increase in the number of neutrophils,and ELISA showed elevated tumor necrosis factor-α（TNF-α）level in serum.4.Notch activation downregulates EMCN in LSECs.Transcriptome data of LSECs from NIC^{e CA} and control mice were analyzed by GSEA.The results showed that the subset of genes involved in mediating leukocyte adhesion and infiltration was significantly enriched in the Notch activated mice,with a decrease of endomucin（EMCN）,an adhesion molecule inhibiting neutrophil infiltration.In HUVECs infected with NICD adenovirus,the expression of EMCN was significantly downregulated.The luciferase reporter assay indicated that Notch activation could significantly inhibit the transcriptional activity of the EMCN promoter.5.Notch activation promotes neutrophil adhesion and trans-endothelial migration via EMCN.In HUVECs insulted by OGD/Re-oxy in vitro,a significantly decreased EMCN level was shown after injury.Moreover,when HUVECs were infected with NICD overexpression adenovirus and human EMCN overexpression adenovirus simultaneously,both neutrophil adhesion and trans-endothelial migration were reduced as compared with the control group.6.Inhibition of Notch signaling upregulates EMCN expression and inhibited neutrophil adhesion and trans-endothelial migration.RBP-J^?E mice showed an increased expression of EMCN after HIRI,as compared with the control group.Inhibition of endothelial Notch signaling in vitro resulted in an increased EMCN expression level and inhibited neutrophil adhesion and trans-endothelial migration.ConclusionsNotch signaling is activated in LSECs during HIRI,which promotes the adhesion and infiltration of neutrophils to enhance inflammatory response and aggravate liver damage via transcriptional inhibition of EMCN expression in LSECs.