Injury, progression, and fibrosis of the extrahepatic bile duct

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PROJECT SUMMARY Biliary atresia (BA) is a fibro-obliterative disease of the bile ducts, especially the extrahepatic ducts (EHBDs), that afflicts neonates around the world. BA is the most common indication for liver transplant in the pediatric population, with 50% of patients requiring transplant by age 2 and most of the rest before adulthood. The etiology and early course of the disease are unknown; however, important recent data suggest that BA results from a prenatal environmental insult (sparing the mother) that is followed by progression of the original injury after birth. This proposal seeks to answer three key questions in BA: 1) Why is toxicity specific to neonates? 2) What determines repair versus progression of cholangiocyte injury? and 3) How and why does fibrosis occur in the EHBD? Our underlying hypothesis is that the answers to these questions are found in the unique features of the neonatal biliary system: that BA results from an injury that occurs in the context of a developmentally immature bile duct with anatomic features that make it susceptible to injury and promote progression of damage and a fibrotic response.Our preliminary work identified key features of the neonatal bile ducts that potentially increase their susceptibility to injury. These include lack of a protective apical glycocalyx on cholangiocytes and immature cholangiocyte cell-cell junctions. We also showed that the submucosa of the neonatal EHBD contains a large population of fibrogenic cells that are “primed' to respond to insults and that the anatomical structure of the neonatal submucosa may propagate injury.We developed two unique tools to study the role of duct immaturity in susceptibility and response to injury. First, we developed a mouse model of prenatal EHBD damage. We identified and synthesized a previously unknown isoflavonoid biliary toxin, biliatresone, that causes EHBD injury in fetal and neonatal mice after treatment of pregnant mothers. Damage is worsened by humanizing the bile acid profile. This model will enable us to use genetically-modified and specially-treated mice to investigate the importance of neonatal duct susceptibility factors in injury. Second, we developed a microfluidic bile duct-on-a-chip device that allows us to culture neonatal, adult, and genetically- modified cholangiocytes in a confluent, impermeable monolayer, to apply various treatments selectively to the apical or basal surface, and to determine their impact on key cholangiocyte functions, including the permeability barrier.Our three specific aims use these and other tools we have developed to study: 1) neonatal susceptibility to injury, including the role of the glycocalyx; 2) the determinants of injury progression, including the role of bile acids; and 3) the identity of the fibrogenic cells of the EHBD and the role of submucosal architecture in the spread of injury. This work has the potential to both significantly shift our understanding of BA and lead to new therapeutic options.