Study On The Regulatory Mechanism Underlying Vegf Signaling In Cholangiocyte-to-hepatocyte Transdifferentiation Upon Extreme Liver Injury

As the largest digestive organ of human body,the liver is essential for digestion,metabolism,detoxification,and maintenance of homeostasis in the body.Liver diseases affect the health of thousands of people worldwide.The global burden of liver diseases is increasing year by year due to several health-modifying factors,such as increased life expectancy,sedentary lifestyles,and overnutrition.Investment in hepatology research continues to increase at home and abroad,and many significant achievements have been made in the pathogenesis and preventive treatment of common and rare liver diseases.However,the intractable high prevalence and persistent clinical resistance in patients with advanced liver diseases(e.g.,cirrhosis and hepatocellular carcinoma)suggests that there is still a long way to go in the research and treatment of liver diseases.Currently,liver transplantation is the only reliable treatment to prolong the lifetime of patients with endstage liver disease.However,the number of donor livers is insufficient to meet with the increasing demand,and allogeneic transplantation may produce severe rejection reactions.Therefore,promoting endogenous repair and regeneration of the liver may be a more direct and safer therapeutic strategy.Under different injury conditions,different cell sources are involved in the regeneration and repair of the damaged liver,based on which liver regeneration can be categorized into hepatocyte-mediated liver regeneration and biliary-mediated liver regeneration.Upon acute mechanical or chemical injuries,liver regeneration is mainly accomplished by the proliferation of residual healthy hepatocytes.Upon chronic liver injuries,accompanied by the reduced proliferative capacity of residual hepatocytes,liver regeneration is accomplished by the transdifferentiation of cholangiocytes.Biliarymediated liver regeneration was first found in zebrafish.Extreme loss of hepatocyte injuries in zebrafish could be achieved through the NTR-Mtz system,and liver regeneration was realized by the transdifferentiation of biliary epithelial cells(BECs).Zebrafish liver regeneration consists of three steps:(1)BECs dedifferentiation into bipotential progenitor cells(BPPCs);(2)BPPCs proliferation;(3)BPPCs redifferentiation.Severe damage of zebrafish hepatocytes induces the expression of the progenitor cell molecular markers foxa3,hhex,and sox9 b in BECs,as well as changes in BECs morphology,which signifies the dedifferentiation of BECs into BPPCs.Then,the BPPCs rapidly proliferate and subsequently re-differentiate into nascent hepatocytes and BECs.Many studies in mice have also demonstrated that when hepatocyte proliferation is pharmacologically or genetically inhibited after liver injury in mice,BECs will also transdifferentiate to replenish hepatocytes.Thus,biliary-mediated liver regeneration is conserved in zebrafish and mammals.In this study,we used a zebrafish model with severe liver injury and a mouse model with chronic liver injury to investigate the function and regulatory mechanisms of VEGF signaling in biliary-mediated liver regeneration.We performed single-cell RNA sequencing using the zebrafish model and found that the expression level of the VEGF ligand vegfaa was significantly upregulated in hepatic stellate cells at 0 hour after liver injury(i.e.,0 hour of liver regeneration,abbreviated as R0h),compared with that before the injury.However,other ligands of VEGF signaling,vegfab,vegfba,vegfbb,vegfc,and vegfd,were rarely detected in the liver area before and after liver injury,suggesting that vegfaa may play an important role in liver regeneration.Using the transgenic line Tg(hand2:GFP)which can label hepatic stellate cells,we further confirmed the upregulation of vegfaa in hepatic stellate cells with the help of both real-time quantitative PCR(q PCR)and fluorescence in situ hybridization(FISH).Similarly,the single-cell sequencing results show that the expression of VEGF receptor vegfr2/kdrl was upregulated in BECs after injury,whereas there was no significant difference in the expression level of the other receptors.FISH and q PCR further confirmed that vegfr2/kdrl is upregulated in BECs after liver injury,indicating that VEGFR2 may be the main receptor of VEGFA that regulates BEC-to-hepatocyte transdifferentiation during biliarymediated liver regeneration.In order to verify the function of VEGF signaling in biliarymediated liver regeneration,we used AV-951 to inhibit the activity of all VEGF receptors.AV-951 treatment resulted in a significant reduction in the size and fluorescence intensity of the regenerated liver,suggesting that VEGF signaling is important for liver regeneration.Then we investigated which step of biliary liver regeneration is mainly regulated by VEGF signaling.FISH and q PCR data showed that the expression of progenitor cell markers foxa3,hhex,and sox9 b was significantly down-regulated in BECs after injury and the diameter of BECs could not be enlarged in the AV-951-treated group,indicating defective BEC dedifferentiation upon VEGF inhibition.Through Ed U proliferation staining,in situ hybridization,and antibody staining,we found that the proliferation and redifferentiation of progenitor cells after AV-951 treatment were also inhibited.The above results suggest that inhibition of VEGF signaling causes defects in the initiation of biliary-mediated liver regeneration so that subsequent liver regeneration cannot proceed smoothly.Given the specific upregulation of kdrl expression in BPPCs,we applied another chemical,SU5416,which specifically inhibits the VEGFR2 activity.SU5416 treatment causes liver regeneration defects,which is similar to the effects observed with AV-951 treatment,indicating that VEGFR2 serves as the primary transducer of VEGF signaling that governs biliary-mediated liver regeneration.The above experiments are all based on pharmacological inhibition of VEGF signaling,we next verified the function of VEGF signaling based on endogenous inhibition through genetic means.We engineered a heat-shock-induced transgenic line Tg(hsp:dnvegfaa)expressing a dominant-negative form of vegfaa(dnvegfaa),which could effectively block VEGFA signaling.Upon liver injury,competitive binding of VEGF ligand leads to defective liver regeneration and prevents the initiation of the process of cholangiocyte dedifferentiation.Next,we knocked out the VEGF receptor vegfr2/kdrl.kdrl mutant had relatively normal liver development,but liver regeneration failed to proceed normally due to a defective dedifferentiation process.To further validate that vegfr2/kdrl upregulation in BECs is important for biliary-mediated liver regeneration,we constructed a Tet-Onbased transgenic line,Tg(krt18:Tet3G;Tre3G:dnkdrl-P2A-Ds Red)that expresses a dominant-negative form of kdrl(dnkdrl)specifically in BECs.It was found that overexpression of dnkdrl in BECs also caused dedifferentiation defects,resulting in the defects of BECs transdifferentiating to new hepatocytes,indicating that VEGFR2 serves as the primary transducer of VEGF signaling that governs biliary-mediated liver regeneration.To explore the molecular mechanism of VEGF signaling regulating biliary-mediated liver regeneration,we performed differential analysis of m RNA and protein and found that VEGF signaling mediates the initiation of biliary-mediated liver regeneration through the PI3K-m TORC1 axis.We identified and validated that phosphorylated ribosomal S6(p-RS6),an indicator protein for m TORC1 signaling,was significantly upregulated in BECs at R0 h and R8 h.At the same time,using small molecule drugs to inhibit PI3K-m TORC1 resulted in defective BECs dedifferentiation.Pharmacological or genetic inhibition of VEGF signaling both resulted in reduced p-RS6,suggesting that VEGF signaling is required for activation of m TORC1 signaling after liver injury.Subsequently,we used an activator of PI3 K and found that activation of PI3 K rescued the downregulated of p-RS6 expression caused by inhibition of VEGF,confirming that the PI3K-m TORC1 axis acts downstream of VEGF signaling.Further experiments showed that PI3 K activation could rescue the liver regeneration defects upon VEGF inhibition,including disrupted BEC dedifferentiation,compromised BPPC proliferation,and reduced hepatocyte regeneration.These data suggest that VEGF signaling controls the initiation of biliary-mediated liver regeneration through the PI3K-m TORC1 axis.Using a mouse model of chronic liver injury,we verified the conservation of VEGF signaling mediated biliary-derived liver regeneration in mammals.By using Ctnnb1flox/floxtd TomatoLSL mice,AAV8-TBG-Cre injection could induce the conditional knockout of Ctnnb1 specifically in hepatocytes.After CDE-induced BEC-to-hepatocyte transdifferentiation,the newly BEC-derived hepatocytes are marked as Ctnnb1+ and td Tomato-.Based on this model,AV-951 injection significantly reduced the generation of BEC-derived Ctnnb1+/td Tomato-hepatocytes,confirming that VEGF signaling is required for biliary-mediated liver regeneration in mice.In summary,VEGF signaling regulates BEC dedifferentiation and BPPC proliferation by activating the PI3K-m TORC1 axis during biliary-mediated liver regeneration.Given that patients with end-stage liver disease exhibit a significant increase in BEC coupled with impaired hepatocyte proliferation,promoting biliarymediated liver regeneration may be an alternative way to accelerate liver repair and regeneration.Controlling the activation of VEGF signaling in BECs may enable this novel therapeutic paradigm and therefore may provide a theoretical basis for the treatment of end-stage liver disease.