Liver Sinusoidal Endothelial Cells Undergo Endothelial-Mesenchymal Transition In Liver Fibrosis Which Is Controlled By Notch Signaling

?Background?Liver fibrosis is characterized by abnormal colocalization of extracellular matrix?ECM?in the liver when being exposed to chronic injuries.Activated myofibroblasts are the main contributors of pathologic ECM.However,the cellular origin of myofibroblasts remains controvertial.Liver sinusoidal endothelial cells are highly differentiated endotheliums in the liver which are featured as numerous fenestration and lack of basement membrane.Although hepatic stellate cells?HSCs?,the specific pericytes of liver sinusoids,have been documented as dominant precursor of myofibroblasts,other studies showed that endothelial cell-derived myofibroblasts could contribute to organ fibrosis through endothial-mesenchymal transition?EnMT?.As the largest number of liver non-parenchymal cells,whether or not LSECs could undergo EnMT and participate in promoting liver fibrosis remains to be elucidated.Notch signaling plays a key role in vascular development,angiogenesis,vascular functions and organ homeostasis.Previous studies showed that notch signaling could promote EnMT,which was indispensable in heart developmenting process.We and other groups have also demonstrated that endothelial notch signaling modulated hepatic vascular architecture,angiocrine function of LSECs and vascular remodeling following acute liver injuries such as partial hepatectomy?PHx?.However,how does the endothelial notch signaling react to chronic liver injuries and whether or not LSECs derived notch signaling can regulate liver fibrosis through EnMT remains unkown.?Aims?To elucidate whether or not LSECs can transdifferentiate to myofibroblasts?MFs?-like cells through EnMT in liver fibrosis and to investigate the role of endothelial notch signaling during this process.?Methods?1.Murine LSECs were isolated,identified and in vitro cultured.EnMT phenotypes were evaluated during LSECs capillarization at length.2.Liver fibrosis model was created and colocalization of LSECs and mesenchymal markers was evaluated in the progression process of fibrosis by immunofluroresence.3.LSECs from different stages of fibrotic liver were isolated for detection of EnMT markers at mRNA level and protein level.4.Lineage tracing mouse model was established to further validate EnMT of LSECs in liver fibrosis through IF and flow cytometry.5.Quiescent and fibrotic HSCs/LSECs were isolated and subjected to transcriptome sequencing to evaluate the MFs features of fibrotic LSECs based on gene-expression data and to compare the similarities and differencies between HSCs derived MFs and LSECs derived MFs.6.Clinical cirrhotic liver samples were collected and were detected for EnMT phenomena by IF.7.The status of endothelial notch signaling during liver fibrosis progression was assessed at mRNA level and protein level through RT-PCR and western-blot.8.Endothelium specific notch activation/disruption transgenic mouse model were established to investigate the role of notch signaling in liver fibrosis and LSECs capillarization/EnMT.9.Inhibitor reagent targrting notch was administrated to in vitro-cultured LSECs to evaluate its effect on spontaneous EnMT of LSECs.10.YC-1,a kind of soluble guanylate cyclase?sGC?specific activator,has been previously proved to suppress LSECs capillarization.Treatment of in vitro-cultured LSECs as well as CCl4 mediated fibrosis model with YC-1 was performed to elucidate the relationship between capillarization and EnMT.?Results?1.We successfully isolated LSECs from mice by Magnetic Activated Cell Sorting?MACS?and verified that 99.7% isolated cells were VEGFR3⁺CD34^-.Isolated LSECs manifested homogeneous cellular morphology under microscope,possessed pronounced endocytosis capacity of Formaldehyde-treated Serum Albumin?FSA?and Acetyl Low Density Lipoprotein?acLDL?and could be observed with abundant fenestration under Scanning Electron Microscope?SEM?.During in vitro culturing,LSECs gradually grew as elongated spindle shape cells with distinguished upregulation of mesenchymal markers including alpha Smooth Muscle Actin?a-SMA?,Smooth Muscle 22 alpha?Sm22?,Type I Collagen?Col-1?and et al.2.We then established CCl₄ induced liver fibrosis in mice and found that LSECs capillarization emerged in the early phase of liver damage before which fibrosis could be observed.The colocalization of LSECs and MFs markers?a-SMA,Sm22 and Col-1?was discovered in the area around fibrous scar using confocal laser scanning microscope.This kind of colocalization existed in the quiescent liver sections and was boosted immediately since liver injury started.The same phenomenon was also confirmed in bile duct ligation model.3.LSECs from different periods of liver fibrosis were isolated and were detected for MFs markers at mRNA level and protein level.In accord with previous histological findings,the MFs markers could be detected in quiescent LSECs with small amount,and were substantially upregulated in their fibrotic counterparts during liver fibrosis.4.Cdh5^CreROSA-YFP^stop/flox mice were bred to build CCl₄ or BDL induced liver fibrosis to further confirm the EnMT of LSECs thourgh lineage tracing study.Using confocal laser scanning microscope,we found that a small amount of YFP+LSECs were colocalized with MFs markers in quiescent oil or sham controls and a massive amount in CCl₄ or BDL sections.After isolating LSECs from these Cdh5^CreROSA-YFP^stop/flox mice,we analyzed the proportion of MFs-markers positive LSECs at single cell level.Strikingky,we found that LSECs could be seemed as quiescent precursors of endothelial MFs in the liver and could be quickly instigated to undergo EnMT by hepatic injurious factors.5.Transcriptom sequencing and further Principal Component Analysis?PCA?of quiescent and fibrotic HSCs/LSECs revealed that LSECs derived MFs through EnMT process and activated HSCs derived MFs were actually two differente subpopulations based on their gene expression profiles.Nevertheless,mesenchymal transited LSECs and activated HSCs shared similar MFs related activation signatures in liver fibrosis.6.IF detection of clinical cirrhotic liver sections demonstrated that EnMT existed in patients with cirrhosis.And LSECs EnMT was more likely to be correlated with sinusoidal fibrosis.7.Notch signaling was depressed in the early phase of liver damage but was gradually augmented during liver fibrosis progression as showed by Hes1 and Hey1 evaluation.It implied that notch activation might play a role in LSECs EnMT.8.Liver fibrosis,LSECs capillarization and LSECs EnMT were aggravated in Cdh5^CreERT ROSA26-N1ICD^stop/flox mice,which indicated that forced activation of endothelial notch signaling might facilitate LSECs' EnMT and subsequent liver fibrosis progression.Furthermore,LSECs capillarization and LSECs EnMT,but not liver fibrosis,were ameliorated in Cdh5^CreERTRBP-j^flox/flox mice,which implied that interuption of endothelial notch signaling might serve as an effective strategy for antogonizing LSECs' deteriorative transition in liver fibrosis.9.In vitro administration of notch inhibitor demonstrated that blocking notch could rescue spontaneously EnMT of cultured LSECs as manifested by blunted mesenchymal mophorlogy transition and decreased expression of MFs markers.10.In order to investigate the relationship between EnMT and capillarization,YC-1,the specific sGC activator which has been documented to restore the differentiated state of LSECs,was used to study whether restraining of capillarization could hold back LSECs EnMT.As a result,YC-1 remarkably maintained the initial epithelial morphology of LSECs and inhibated mesenchymal markers expression in vitro.In vivo study also verified that YC-1 treatment not only restricted LSECs capillarization during fibrosis,but also alleviated fibrogenesis and liver damage in mice.This implied that LSECs EnMT and capillarization might actutally be the same pathological process but were merely defined from different point of view,and that targeting LSECs EnMT might be an effective strategy for treating liver fibrosis.?Conclusion?1.As a kind of potential precursors of MFs,LSECs can transdifferentiate to MFs though EnMT during liver fibrosis pregression.2.Notch signaling controls LSECs EnMT in liver fibrosis.3.LSECs EnMT and capillarization may be the same pathological processes which are defined from different point of view.Targeting LSECs EnMT might be an effective strategy for treating liver fibrosis.