The Potential Mechanism Of Piezo1 Activation In Endothelial Cells In Portal Vein Ligation Induced Liver Regeneration

BackgroundSurgical treatment for common diseases in hepatobiliary and pancreatic surgery,such as primary liver cancer,hilar cholangiocarcinoma,and gallbladder cancer,may involve partial hepatectomy(PH).However,for patients with severe liver disease,liver failure caused by insufficient remaining liver volume and function is an important limiting factor for surgical treatment.To achieve the goal of safe surgery,portal vein ligation(PVL),portal vein embolization,or associating liver partition and portal vein ligation for staged hepatectomy(ALPPS)are often used in clinical practice to promote liver regeneration(LR).The precise decoding of LR mechanisms and regulatory methods has always been a focus and difficulty in the field of liver surgery research.Surgical procedures such as PH,PVL,portal vein embolization,and ALPPS,produce a common feature: dramatic changes in hemodynamics within the liver.More and more researchers believe that the hemodynamic changes after surgical operation have gradually become an important “trigger point and regulator” of LR.The increase of postoperative blood flow velocity and the dilatation of blood vessels caused by“relative congestion” in the liver produce shear force and tension changes on vascular endothelial cells(VEC)and liver sinusoid endothelial cells(LSEC),which is the key to start and regulate LR.Mechanosensing cation channels are a class of mechanosensing proteins that can directly sense mechanical stimuli and transduce mechano-chemical signals through calcium ion flow.Piezo1 is an important mechanical sensing cation channel,which is involved in the maintenance of various homeostasis of the body.In Budd-Chiari syndrome,due to severe hepatic blood flow obstruction,blood stasis occurs in the liver.The activation of Piezo1 in promotes the production of a large number of inflammatory factors,further promotes the formation of neutrophil extracellular traps,promotes the formation of microthrombosis in microvessels,and accelerates the development of cirrhosis.After surgical intervention,changes in portal vein blood flow cause residual liver to be in a relatively “open source and throttling” state.Blood stasis in this liver is temporary,and the activation level,time,and biological effects of Piezo1 are inevitably different from the sustained blood flow obstruction caused by Budd-Chiari syndrome.It is currently unclear whether the activation of Piezo1 in VEC is involved in the LR process and its mechanism.Objective1.Explore the hemodynamic changes and molecular mechanisms of LR induced by portal vein ligation.2.Potential mechanisms of interaction between blood flow,VEC,Piezo1 and hepatocytes.3.To explore the role of Piezo1 and EGFR in LR.Methods1.Selection and construction of animal models: A 70% PVL model of Sprague-Dawley(SD)rats was used to explore the expression changes of Ki67 and Cyclin D1 in regenerated liver.The 70% PVL model of SD rats was used to investigate the effects of EGFR inhibitor gefitinib and Piezo1 agonist Yoda1 on LR.2.Observation of microvascular hemodynamic changes during LR in rats with SD after PVL: A platform for ultrasound localization microscope(ULM)detection was constructed to evaluate the changes of microblood flow during LR after PVL.3.Transcriptome sequencing : To explore the changes of transcription level in liver tissue at 0h,12 h,24h,48 h,and 72 h after PVL in rats and VEC after Yoda1 activated Piezo1.4.Analysis and utilization of public databases : https://www.proteinatlas.org/,https://livercellatlas.org,GEPIA,https://bis.zju.edu.cn/HCL/ http://liveratlas-vilarin holab.med.yale.edu and GEO databases fully mine the expression of Piezo1.5.The conditioned medium co-culture model of VEC and hepatocyte in vitro was constructed to explore the regulation and mechanism of Piezo1 activation in VEC on hepatocyte proliferation and epithelial-mesenchymal transition(EMT).Lentivirus-loaded sh RNA infected VEC(HUVEC and SK-Hep1 cells)to construct VEC cell lines with stable knockdown of Piezo1 gene,and further explored the role of Piezo1 activation in promoting hepatocyte proliferation and EMT.6.Immunofluorescence,immunohistochemistry,q-RT-PCR,WB,ELISA,etc.were used to evaluate the expression of the target gene at the transcriptional or protein level.Results1.By constructing a stable rat 70 % PVL model,LR was successfully induced without significant damage to liver function.From the distribution of proliferating hepatocytes,it was found that the distribution of proliferating hepatocytes was dynamically changed from portal vein(PV)area to central vein(CV)area during the LR process.At the same time,transcriptome sequencing analysis showed that complex molecular events occurred during PVL induced LR,mainly metabolic inhibition(including lipid metabolism,cholesterol metabolism,drug metabolism and amino acid metabolism)at the initial stage,followed by obvious cell proliferation related molecular events(including DNA replication,mismatch repair,mitosis,etc.),accompanied by obvious EMT.At the peak of cell proliferation,some factors that inhibit cell proliferation and participate in extracellular matrix remodeling were expressed.2.The ULM detection platform was successfully built,and the structure of microvessels in the liver was successfully evaluated by ULM.Furthermore,the ULM technique was successfully used to evaluate the hemodynamic changes of microvessels in the regenerated liver in the 70 % PVL model of SD rats.The main manifestations were increased microvascular density,accelerated blood flow velocity,and increased perfusion volume in a short period of time after surgery,the average microvascular diameter increased on the 14 th day after surgery3.From multiple public databases,it can be found that the expression level of Piezo1 in VEC in the liver is higher than that in hepatocytes and bile duct cells.The expression level of Piezo1 in LSEC was much higher than that of other mechanosensing cation channels.4.Through systematic in vitro studies,we found that the conditioned medium produced by Piezo1 activated by Yoda1 in vascular endothelial cells can rapidly promote hepatocyte proliferation and EMT.The detailed mechanism is that Yoda1 activates Piezo1 in VEC and causes extracellular calcium influx.After activating PKCα,it activates ERK1/2 in the MAPK family signaling pathway,thereby promoting the transcriptional expression and secretion of ERBB family ligands HBEGF,EREG and AREG.5.We demonstrated that VEC-derived AREG and EREG promote hepatocyte proliferation and EMT by activating EGFR and ERK1/2 in hepatocytes through recombinant AREG and EREG,EGFR-specific inhibitors..6.Through the public database and rat liver tissue immunofluorescence staining,it was found that the distribution of EGFR in liver tissue was uneven,showing higher expression in PV area and lower expression in CV area.Further in vivo experiments demonstrated that Piezo1 and EGFR were involved in PVL-induced LR process and early proliferative hepatocyte distribution.ConclusionThrough experimental studies on PVL induced LR and a series of in vivo and in vitro experiments,our study explores the potential mechanism of Piezo1 activation in VEC participating in LR.The final conclusion of this article is as follows: 1.The LR induced by portal vein ligation exhibits spatiotemporal changes from PV area to CV area,accompanied by cell proliferation and EMT.2.Ultrasound localization microscopy technology can effectively detect hemodynamic changes in liver microvasculature,which can be used as a technique for LR evaluation.The activation of Piezo1 in VEC is mainly regulated by the PKC/ERK1/2 signaling pathway to regulate the expression of HBEGF,EREG,and AREG.4.VEC derived EREG and AREG promote hepatocyte proliferation and partial EMT through EGFR and ERK1/2.5.Piezo1 and EGFR participate in PVL induced LR.