Structural Remodeling Of Peribiliary Hepatic Artery Accelerates The Progression Of Biliary Atresia Mediated By Notch3 Signaling

Background:Biliary atresia(BA)is a multifactorial cholangiopathy characterized by progressive sclerosing of the intra-and extra-hepatic bile ducts.The biliary tree is nourished by a specific blood supply-branches of the hepatic artery strictly run in parallel.Although a series of evidence suggest the existence of arteriopathy and ischemia in BA,the specific mechanism of the angiogenesis-mediated biliary epithelial injury remains largely undefined.Given the vital role of the Notch signaling pathway for the perinatal vascular development,this study intends to explore how the Notch signaling pathway and hepatic artery structural changes affect the pathogenesis of BA from a new perspective of vascular factors.Material and Methods:Patients and animal models Liver biopsies and clinical data were obtained from infants with cholestasis enrolled in a retrospective study evaluated at Wuhan Union Hospital with informed consent from children's guardians.Three groups of subjects were enrolled according to their diagnosis: BA,intrahepatic cholestasis(as diseased controls and named Non-BA),and age-matched non-cholestasis controls(Non-CC,take specimens at resection margin of the completely excised benign hepatic tumors).The wild-type(WT)and Notch3 gene knockout(Notch3-/-)BALB/c mice were respectively purchased from HUBEI provincial center for disease control and prevention and Chongqing Gaosheng bio-pharmaceutical co.LTD.Newborn mice were injected with rhesus rotavirus(RRV)or MEM to induce experimental BA or controls.Interventions consisted of adenovirus(Ade)vectors,Notch3-Ab and DAPT.The Ade vector was used to overexpress NICD3 in vivo.The next day after RRV injection,we overactivated the Notch3 signaling by intraperitoneal injection of Ade-NICD3.Then we inhibited Notch3-Ab/DAPT.Interventions repeated twice.We set a time gradient for antibody-blocking and a concentration for DAPT inhibition.Besides,Ade-NICD3 was injected to the normal newborn mice to observe the effects of overexpressed Notch3 signaling as a single factor.Hepatic artery doppler ultrasound,intraoperative indocyanine green angiography(ICG),and HSST sign observation in infants with BA Doppler ultrasound examinations were performed in Wuhan union hospital.Our subjects were infants with obstructive jaundice(14BA vs 14Non-BA).The hepatic artery diameter(HAD),Portal Vein diameter(PVD),Hepatic artery resistance index(HARI)and Hepatic acceleration time(HAT)were measured to verify the changes of structure and blood flow of the main hepatic artery.We photographed the images of the liver surface at first to identify if there was classical HSST sign during the laparoscopic cholangiography.To evaluate the overall perfusion of the liver,we introduced ICG angiography.Visualization of the ICG fluorescence was provided by the laparoscopic system.The dynamic images were documented during a continuous observation of ten minutes.Observation of hepatic subcapsular vascular plexus(HSVP)in mouse models The liver of the BA model was exposed and placed on the platform.The HSVP on the surface of the mouse liver was observed under a bright upright metallurgical microscope.Immunolocalization and Multiplexed Immunofluorescence Immunolocalization of ?-SMA and HE staining were used to highlight the structure of the hepatic artery.Immunolocalization of HIF-1? indicated the location and severity of hypoxia in tissue.The paraffin slides were made from specimens including the liver parenchyma for human and liver parenchyma/hilar tissue for mice.Structured arterioles were randomly selected to measure their inner diameters(ID)and arterial walls(AW).Multiplexed IF was performed by staining of 4-?m thick paraffin sections of liver tissue with standard,primary antibodies sequentially and paired with a unique fluorochrome followed by staining with DAPI per published protocols.Antibodies/fluorescent dyes in this study were,in order: anti-CK19/Green,anti-Notch3/Cyan,anti-HIF-1?/Yellow,anti-SMMHC/Red,anti-Vimentin/Magenta.With a Vectra full-spectrum imaging system,we got pictures with high SNR that allowing for a quantitative analysis by In Form image analysis software.Western blot and real time-PCR Total RNA was extracted by TRIzol.Reverse transcription reactions were conducted using the Prime Script RT reagent kit with g DNA Eraser.RT-PCR on target genes was carried out with SYBR Premix Ex Taq TM.For immunoblots,all procedures were done as previously described.Isolation of lymphocyte and vascular smooth muscle cells(VSMCs)from mouse liver Anti-CD11 c antibody and anti-NKG2 D antibody were used to mark DCs and activated NK cells.Mononuclear leucocytes in the liver were concentrated using the lymphocyte separation medium and then collected for flow cytometry.Vessels in the Glisson system were extracted and cut into pieces.After digestion by collagenase II,the vascular tissue turned into cell clusters.Then SMCs were cultured for observations under a phase-contrast microscope or for IF staining to evaluate their phenotypes.Results:1.Hepatic arterial remodeling in BA The statistical differences in widened HAD and PVD along with the increased HARI in infants with BA were in line with the previous reports.According to the results of ICG angiography,the whole surface of the liver fluoresced within 5 seconds after ICG injection in Non-BA group.Interestingly,there were two distinct phenomena among infants with BA: about 1/3(4/11)cases showed a rapid "glowing" within a few seconds,while the other 2/3(7/11)reached a comparative blue-fluorescence intensity until 2 minutes later after the ICG injection,indicating a slower hepatic arterial perfusion.In the RRV-induced mouse models,the structure of small blood vessels composed of monolayer smooth muscle cell or monolayer endothelial cell along with the small bile ducts were rapidly destroyed in Day3 after RRV infection;a large number of immune cells gathered around the portal vein.As BA continued to advance,HIF-1? increased and located in the portal area.Ten days after RRV/MEM injection,we observed large arteries and main bile ducts in the hilar tissue in NC group;while only remnants of small bile ducts and arteries with an increased AW/ID ratio were observed in RRV group.The change of arterial AW/ID ratio in mice hilar was similar to the increase of arterial AW/ID ratio in human portal area.2.Abnormal HSVP before obvious fibrosis in BA Typical HSST sign was observed under laparoscopy in 12 BA infants younger than 30 days with pathology staging of liver fibrosis at stages 1 and 2.Besides,9/12 mice at14-17 days after RRV injection appeared abnormal vascular plexus on both visceral and diaphragmatic facies of the liver;however,the liver capsule of mice in the NC group was clean without vascular plexus.3.Overexpression of Notch3 and phenotype changes of arterial smooth muscle cells in BA The m RNA expression of Notch3 in liver tissue from BA infants was significantly higher than that of non-CC or non-BA groups.The main downstream target gene was Hey1.It was verified by the WB test that Notch3 and Hey1 proteins were at higher levels in liver tissue from BA infants.Results of OPAL analysis showed that Notch3 expression was located in the hepatic artery system;Hypoxia in the portal area was significantly severer in BA infant;The number of bile duct epithelial cells expressing HIF-1? and SMCs with synthetic phenotype was more in BA infants.Both Notch2 and Notch3 genes in the RRV mice models were significantly higher than control.The expression of Notch3 m RNA peaked at Day7 and remained high until Day10.The dynamic expression of Notch3 protein was consistent with PCR results,while the increase of VEGF was relatively delayed.HIF-1? seldom expressed at an early stage of BA but increased in the middle and advanced stages.After isolated from the mice,SMCs from hilar vessels in the RRV group were more immature than NC group by IF analysis,suggesting a phenotypic disturbance of SMCs in the hepatic arterial system in experimental BA.4.Overactivation of Notch3 pathway as an accelerating factor in experimental BA Early overexpression of Notch3 in normal neonatal mice unexpectedly replicated the HSVP similar to the BA model.Mice in the Ade-NICD3 group showed stagnation in long-term weight gain rather than lethal toxicity.HIF-1? m RNA exceeded the expression level of controls at Day14 and Day21.Expressions of inflammatory cytokines in the Ade-NICD3 group were significantly higher than Ade-null controls at Day14.The early activation of Notch3 in the RRV model resulted in an accelerated progression of BA.Mice were poorly coated and smaller in size.All animals died rapidly within Day12.Extensive hemorrhagic spots spread across the liver capsule,meanwhile,a small number of vascular clusters growing to the surface.Disordered VSMCs proliferated as a mass network.Small blood vessels and bile ducts in the portal area quickly lost their normal structure.The m RNA levels of IL-6,Cxcl1,Cxcl2 and TNF-? in the RRV+ Ade-NICD3 group were higher at Day7 compared to the RRV+PBS controls.5.Therapeutic inhibition of Notch3 pathway in WT and Notch3-/-neonatal mice after RRV challenge Early intervention with Notch3-Ab exerted a protective influence on neonatal WT mice after the RRV challenge.Anatomical findings at Day14 showed fibrosis of the gallbladder and atresia of the extrahepatic bile duct in RRV control.With early antibody blocking,the formation of SVP on the liver capsule was prevented.The expression of HIF-1? in the portal area decreased suggestive of better oxygen supply.Expression of inflammatory cytokinesincreased from Day3 to Day7 but remained stable or decreased afterward.Also,the number of DC and NK cells in liver tissue decreased,indicating an inhibition of the inflammatory response in liver.The intervention with Ig G had insignificant influence on survival or weight.DAPT at the dose of 10 ug had limited therapeutic effects on WT mice.The typical symptoms of BA including clay stool,ascites,jaundice,abnormal hair,and growth retardation still appeared,indicating that DAPT may help slow down the process rather than preventing the final formation of BA phenotype.When BALB/c Notch3-/-mice were challenged with RRV by intraperitoneal injection,animals obtained improved symptoms and increased survival rate.There was no presence of typical HSVP.Conclusion:The structural disorders of peribiliary arterioles play a role in the pathogenesis of BA.The overactivated Notch3 signaling mediates both the formation of HSVP and remodeling of the hepatic arterial system.A persistent remodeling of arterioles aggravates the ischemia injury in the portal area and the extrahepatic peribiliary region in both clinical and experimental BA.