The Role Of Hepatocyte-derived VEGFA In Liver Regeneration And Non-alcoholic Fatty Liver Disease

Part ?Hepatocyte Control Liver Sinusoidal Endothelial Cell Revascularization of Regenerating Liver through Gata3/Ramp2-PEDF/VEGFA CascadeBackground and ObjectivePartial hepatectomy or liver resection is a type of surgery designed to remove cancerous tumors from the liver.The regenerative capacity to regain mass after tissue loss has revolutionized the treatment or cure of many patients with liver tumor.Most HCC patients diagnosed at the intermediate and advanced stages,when the insufficient functional liver limited the effective regeneration.Hepatic failure may develop after liver resection.Revolutionization in effective regeneration after hepatectomy can expand the indications for liver resection,reduce the risk of post hepatectomy liver failure(PHLF),and improve the safety of surgery.Liver regeneration is a complex process involving all resided cells in the liver.In the early stage of liver regeneration,hepatocyte proliferates to form cell clusters,non-parenchymal cells such as liver sinusoidal endothelial cells(LSEC)and hepatic stellate cells(HSC)subsequently follow.LSEC proliferate and migrate into hepatocyte clusters to form blood vessels,providing metabolic conditions for new hepatocytes,while HSC secrete extracellular matrix to reconstruct hepatic microstructure.Our previous studies implied that hepatocyte promote LSEC proliferation by down-regulating pigment epithelium-derived factor(PEDF)and up-regulating vascular endothelial growth factor-A(VEGFA)during liver regeneration.On this basis,this project intends to identify how hepatocytes regulate LSEC revascularization in regenerating liver,and explore the potential intervention.Method1.Transcriptome sequencing of hepatocytes from sham-operated(Con),hepatectomized wild type(Blank)or Met?hep mice on day 1 after PHx was performed to identify the angiogenesis-related regulators,which are possibly involved in regulating LSECs proliferation in regenerative liver.Then,these differential genes were confirmed by PCR assay.2.Lentivirus delivery-mediated up-or down-regulating above differential genes in AML-12 cells,coincubation the delivered AML-12 with SVEC-40 or primary LSECs,identification the key regulators in hepatocytes controlling LSECs proliferation.3.Based on gene-edited mice that specifically overexpress or knockout above genes in hepatocytes by combination of CRISPR-Cas9 with adeno-associated virus,the functional genes were identified and verified in two models,partial hepatectomy and associating liver partition and portal vein ligation for staged hepatectomy(ALPPS)models.4.Exploring the mechanism and the possible intervention.5.The feasibility of related pathways intervention was explored in patient-derived hepatocyte organoid systems,which co-incubated with HUVECs.Result1.Down-regulation of Gata3 and up-regulation of Ramp2 in hepatocyte after PHx contribute to LSECs regeneration.2.The data from hepatectomized Gata3hep and Ramp2?hep mice demonstrated that the valve of Gata3 and Ramp2 in hepatocyte sparks LSEC proliferation via an equilibrium flow control of PEDF and VEGFA.Gata3 as a negative regulator of regeneration,while Ramp2 as a positive regulator,both coordination contributes to LSECs proliferation in regenerative liver.3.Gata3hep-and Ramp2?hep-ALPPS models also demonstrated that Gata3 or Ramp2 affect just LSECs but not hepatocyte proliferation.4.Recombinant VEGFAi64 or PEDF neutralized antibodies treatment partially relived inhibition of Gata3hep-or Ramp2?hep on LSECs proliferation during the late stage of liver regeneration.6.Patients-derived hepatocyte organoids showed the differential expression of GATA3 among 28 individual para-cancer tissues of HCC,which partially characterized the remnant liver.According to the mRNA levels of GA TA3,these samples-derived organoids were divided into three groups:low,moderate,and high GATA3 expression(GATA3high,GATA3mecha or GATA3low).Conditioned medium from GATA3high organoids relatively promoted HUVEC proliferation and tube formation,compared with that from GATA3media or GATA3low.However,this promotion was selectively abolished by GATA3 inhibitor K-7174 or ?-PEDF.ConclusionGata3 and Ramp2 like a valve in hepatocyte control an equilibrium flow of VEGFA and PEDF to spatiotemporally spark LSEC proliferation and revascularization of regenerating liver.Part ?Hepatocyte-derived VEGFA Accelerates Non-alcoholic Fatty Liver Disease-Hepatocellular Carcinoma Transition by Activating Hepatic Stellate CellsBackground and ObjectiveNon-alcoholic fatty liver disease(NAFLD)has become a major healthcare burden worldwide.Vascular endothelial growth factor A(VEGFA)is involved in angiogenesis and fibrosis in chronic liver disease.We aimed to investigate the role of hepatocyte-derived VEGFA and its potential as a therapeutic target in the progression of NAFKD-HCC transitionMethodsHepatocyte-specific dismption of VEGFA(Vegfa?hep)and WT mice were received western diet/carbon tetrachloride(WD/CC14)to establish the NAFLD-HCC model.Peri-tumor samples of patients with NAFLD-HCC,HBV-HCC and hepatic hemangioma were collected.The levels and cellular distribution of VEGFA during NAFLD progression were analyzed in murine model and patient tissues.Hepatocytes were isolated from patients and established hepatic-organoids to examine the role of VEGFA in activating LX2 cells.ResultsElevation of VEGFA was observed in WD/CC14-WT mice during NAFLD progression,and this elevation mainly distributed in hepatocytes.WD/CC14-Vegfa?hep mice exhibited less fibrosis and tumor burden rather than hepatic steatosis.Hepatocyte-derived VEGFA promoted fibrosis by activating hepatic stellate cells(HSCs).Importantly,this correlation of VEGFA with hepatic fibrosis were observed in patients with NAFLD.In vitro experiments confirmed that the conditional medium from NAFLD patients-derived organoids stimulated HSC activation,and this stimulation could be blocked by ?-VEGFA treatment.ConclusionOur findings indicated a novel link between fibrosis and hepatocyte-derived VEGFA during NAFLD progression,and provided an evidence for VEGFA inhibition as a therapeutic strategy for NAFLD.