Coordinated development of the mouse extrahepatic bile duct: implications for neonatal susceptibility to biliary injury

Abstract

Background & Aims: The extrahepatic bile duct is the primary tissue initially affected by the cholangiopathy biliary atresia. Biliary atresia affects neonates exclusively and current animal models suggest that the developing bile duct is uniquely susceptible to damage. In this study, we aimed to define the anatomical and functional differences between the neonatal and adult mouse extrahepatic bile ducts.Methods: We studied mouse passaged cholangiocytes, mouse BALB/c neonatal and adult primary cholangiocytes and isolated extrahepatic bile ducts, and a collagen reporter mouse. Methods included transmission electron microscopy, lectin staining, immunostaining, rhodamine uptake assays, bile acid toxicity assays, and in vitro modeling of the matrix.Results: The cholangiocyte monolayer of the neonatal extrahepatic bile duct was immature, lacking the uniform apical glycocalyx and mature cell-cell junctions typical of adult cholangiocytes. Functional studies showed that the glycocalyx protected against bile acid injury and that neonatal cholangiocyte monolayers were more permeable than adult monolayers. In adult ducts, the submucosal space was filled with collagen I, elastin, hyaluronic acid, and proteoglycans. In contrast, the neonatal submucosa had little collagen I and elastin, although both increased rapidly after birth.In vitro modeling suggested that the composition of the neonatal submucosa relative to the adult submucosa led to increased diffusion of bile. A Col-GFP reporter mouse showed that cells in the neonatal but not adult submucosa were actively producing collagen. Conclusion:We identified four key differences between the neonatal and adult extrahepatic bile duct. We showed that these features may have functional implications, suggesting the neonatal extrahepatic bile ducts are particularly susceptible to injury and fibrosis.Lay Summary Biliary atresia is a disease that affects newborns and is characterized by extrahepatic bile duct injury and obstruction with resulting liver injury. We identify four key differences between the epithelial and submucosal layers of the neonatal and adult extrahepatic bile duct and show that these may render the neonatal duct particularly susceptible to injury. Highlights 1) The apical glycocalyx is thin and patchy in neonatal compared to adult cholangiocytes 2) Neonatal cholangiocytes have immature cell-cell junctions and increased permeability 3) The neonatal submucosal space has minimal collagen I or elastin 4) The neonatal submucosal space contains many actively collagen-secreting cells Graphical abstract

Photochemically Activated Notch Signaling Hydrogel Preferentially Differentiates Human Derived Hepatoblasts to Cholangiocytes

“…[15][16][17][18] HA is found in the submucosal space of neonatal and adult extrahepatic bile ducts, and both hepatoblasts and cholangiocytes express CD44 receptors. [19,20] HA localization in the vicinity of liver bile ducts coupled with the presence of the CD44 receptor in cholangiocytes, and the lack of CD44 in hepatocytes, [21] suggests that HA regulates cholangiocyte function through HA-CD44 interactions. [22] HA also binds to toll like receptor 4 (TLR4), which is expressed by cholangiocytes.…”

Interstitial spaces are continuous across tissue and organ boundaries in humans

“…between cells and around capillaries) as well as the larger fibroconnective tissue spaces we recently identified. 10,[14][15][16] We demonstrate continuity across organ boundaries and between spaces in all fibrous tissues studied, including the perineurium and vascular adventitia within them. We suggest that there is a broad and interconnected network of interstitial fluid-filled channels throughout the body, including the structural coverings of nerves and vessels, and that this has significant implications for molecular signaling, cell trafficking, and the spread of malignant and infectious disease.…”

“…HA is found in interstitial spaces throughout the body at all stages of development . 10,[14][15][16] The physical properties of HA suggest that it regulates flow of fluid and other solutes and small molecules within interstitial fluid; 17 it did not prevent filling by fluorescein in vivo. 11 We confirmed by staining with HA binding protein (HABP) that it is found broadly in intercellular, pericapillary and perineural, and submucosal and dermal interstitial spaces.…”

Tissue engineering of the biliary tract and modelling of cholestatic disorders

“…93 Conversely, biological hydrogels encompassing bioactive components of the native bile duct ECM, such as collagen I, III and IV, fibronectin, laminin and elastin provide a more physiological environment compared to their synthetic counterparts. [94][95][96] Furthermore, hydrogels combining multiple components recapitulate the natural ECM more closely than single component matrices. Accordingly, cholangiocytes in hydrogels generated from digested whole liver ECM enable studies on branching morphogenesis, 97 which are not possible with single component hydrogels, such as collagen I gels, because of the lack of biochemical cues provided by other ECM components.…”