J. A. Zagory1,2, N. Mavila1,2, S. Utley1,2, D. James1,2, K. S. Wang1,2 1Children’s Hospital Los Angeles,Pediatric Surgery,Los Angeles, CA, USA 2Saban Research Institute,Developmental Biology And Regenerative Medicine Program,Los Angeles, CA, USA
Introduction: Biliary atresia (BA) is a congenital progressive fibro-obliterative disease of the extrahepatic biliary tree and is the most common cause of end-stage liver disease in children. BA is characterized by extensive intrahepatic proliferating ductular reaction and biliary fibrosis. The expansion of COLLAGEN-1-expressing cells expressing the stem cell marker PROMININ-1 (PROM1) within the biliary fibrosis is associated with the experimental model of BA induced Rhesus rotavirus (Mavila et al, Hepatology, 2014). However the molecular mechanisms regulating ductular reaction and biliary fibrosis are unclear. Given the role of Notch signaling in biliary development (Kodama et al, Gastroenterology, 2004), we hypothesize that activated Notch signaling is associated with ductular proliferation and biliary fibrosis in cholestatic liver diseases such as BA.
Methods: Statistical analyses were performed on normalized human BA microarray data acquired from http://genet.cchmc.org using Statview software (BA n=66, normal n=7). Liver from mice with experimental cholestasis/biliary fibrosis induced using 3% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet for 2 weeks, human BA collected at the time of Kasai portoenterostomy, and age-matched control liver samples were analyzed histologically using immunofluorescence (IF)/immunohistochemistry (IHC) for the expression of biliary marker Cytokeratin (CK)-19, activated Notch receptors (Notch Intracellular Domain (NICD))-1 & 2, and Notch ligand JAG1. Fibrosis was assessed by Sirius red staining. Gene expression analysis was performed by quantitative Polymerase Chain Reaction (qPCR).
Results: Analysis of human BA microarray data demonstrated significant increases in the expression of JAG1 (2.54 fold, p< 0.001) and its receptor NOTCH2 (1.5-fold, p<0.001). CK19 immunostaining and Sirius red staining confirmed the presence of ductular reactions and fibrosis in both BA and DDC-treated livers compared to normal livers. IHC/IF analysis demonstrated increased expression of activated NICD1 and JAG1 proteins in BA and DDC-treated livers compared to controls. qPCR analysis of DDC-treated livers demonstrated a significant increase in the expression level of Notch-1, Notch-2 and Notch ligands Jag1, Dll1 and Dll4 (p<0.05).
Conclusion: NOTCH signaling is activated in human BA and experimental model of cholestasis and may contribute to the ductular reaction and biliary fibrosis associated with cholestatic liver diseases.