Marie V. Nguyen

Expansion of prominin‐1‐expressing cells in association with fibrosis of biliary atresia

Biliary atresia (BA), the most common cause of end‐stage liver disease and the leading indication for pediatric liver transplantation, is associated with intrahepatic ductular reactions within regions of rapidly expanding periportal biliary fibrosis. Whereas the extent of such biliary fibrosis is a negative predictor of long‐term transplant‐free survival, the cellular phenotypes involved in the fibrosis are not well established. Using a rhesus rotavirus‐induced mouse model of BA, we demonstrate significant expansion of a cell population expressing the putative stem/progenitor cell marker, PROMININ‐1 (PROM1), adjacent to ductular reactions within regions of periportal fibrosis. PROM1positive (pos) cells express Collagen‐1α1. Subsets of PROM1pos cells coexpress progenitor cell marker CD49f, epithelial marker E‐CADHERIN, biliary marker CYTOKERATIN‐19, and mesenchymal markers VIMENTIN and alpha‐SMOOTH MUSCLE ACTIN (αSMA). Expansion of the PROM1pos cell population is associated with activation of Fibroblast Growth Factor (FGF) and Transforming Growth Factor‐beta (TGFβ) signaling. In vitro cotreatment of PROM1‐expressing Mat1a−/− hepatic progenitor cells with recombinant human FGF10 and TGFβ1 promotes morphologic transformation toward a myofibroblastic cell phenotype with increased expression of myofibroblastic genes Collagen‐1α1, Fibronectin, and α‐Sma. Infants with BA demonstrate similar expansion of periportal PROM1pos cells with activated Mothers Against Decapentaplegic Homolog 3 (SMAD3) signaling in association with increased hepatic expression of FGF10, FGFR1, and FGFR2 as well as mesenchymal genes SLUG and SNAIL. Infants with perinatal subtype of BA have higher tissue levels of PROM1 expression than those with embryonic subtype. Conclusion: Expansion of collagen‐producing PROM1pos cells within regions of periportal fibrosis is associated with activated FGF and TGFβ pathways in both experimental and human BA. PROM1pos cells may therefore play an important role in the biliary fibrosis of BA. (Hepatology 2014;60:941–953)

Recent advances in the pathogenesis and management of biliary atresia.

Purpose of review The purpose of this study is to review advances in both the pathogenesis and clinical management of biliary atresia. Recent findings Immunologic studies have further characterized roles of helper T-cells, B-cells, and natural killer cells in the immune dysregulation following viral replication within and damage of biliary epithelium. Prominin-1-expressing portal fibroblasts may play an integral role in the biliary fibrosis associated with biliary atresia. A number of genetic polymorphisms have been characterized as leading to susceptibility for biliary atresia. Postoperative corticosteroid therapy is not associated with greater transplant-free survival. Newborn screening may improve outcomes of infants with biliary atresia and may also provide a long-term cost benefit. Summary Although recent advances have enhanced our understanding of pathogenesis and clinical management, biliary atresia remains a significant challenge requiring further investigation.

Fibroblast Growth Factor signaling regulates the expansion of A6-expressing hepatocytes in association with AKT-dependent β-catenin activation

BACKGROUND & AIMS Fibroblast Growth Factors (FGFs) promote the proliferation and survival of hepatic progenitor cells (HPCs) via AKT-dependent β-catenin activation. Moreover, the emergence of hepatocytes expressing the HPC marker A6 during 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-induced liver injury is mediated partly by FGF and β-catenin signaling. Herein, we investigate the role of FGF signaling and AKT-mediated β-catenin activation in acute DDC liver injury. METHODS Transgenic mice were fed DDC chow for 14days concurrent with either Fgf10 over-expression or inhibition of FGF signaling via expression of soluble dominant-negative FGF Receptor (R)-2IIIb. RESULTS After 14days of DDC treatment, there was an increase in periportal cells expressing FGFR1, FGFR2, and AKT-activated phospho-Serine 552 (pSer552) β-Catenin in association with up-regulation of genes encoding the FGFR2IIIb ligands, Fgf7, Fgf10, and Fgf22. In response to Fgf10 over-expression, there was an increase in the number of pSer552-β-Catenin((positive)+ive) periportal cells as well as cells co-positive for A6 and hepatocyte marker, Hepatocyte Nuclear Factor-4α (HNF4α). A similar expansion of A6(+ive) cells was observed after Fgf10 over-expression with regular chow and after partial hepatectomy during ethanol toxicity. Inhibition of FGF signaling increased the periportal A6(+ive)HNF4α(+ive) cell population while reducing centrolobular A6(+ive) HNF4α(+ive) cells. AKT inhibition with Wortmannin attenuated FGF10-mediated A6(+ive)HNF4α(+ive) cell expansion. In vitro analyses using FGF10 treated HepG2 cells demonstrated AKT-mediated β-Catenin activation but not enhanced cell migration. CONCLUSIONS During acute DDC treatment, FGF signaling promotes the expansion of A6-expressing liver cells partly via AKT-dependent activation of β-Catenin expansion of A6(+ive) periportal cells and possibly by reprogramming of centrolobular hepatocytes.

Hepatic Prominin-1 expression is associated with biliary fibrosis

Background. Intrahepatic biliary fibrosis, as seen with cholestatic liver injuries such as biliary atresia, is mechanistically distinct from fibrosis caused by hepatocyte toxicity. We previously demonstrated the expansion of cells expressing the stem/progenitor cell marker Prominin‐1, within regions of developing fibrosis in biliary atresia. Thus, we hypothesized that Prominin‐1 expression is biliary fibrosis‐specific. Methods. Gene expression of Prominin‐1 was analyzed in adult mice undergoing either cholestatic bile duct ligation or hepatotoxic carbon tetrachloride administration by quantitative polymerase chair reaction. Lineage tracing of Prominin‐1‐expressing cells and Collagen‐1&agr;‐expressing cells was performed after bile duct ligation in Prominin‐1cre‐ert2−lacz;Gfplsl and Collagen‐1&agr;Gfp transgenic mice, respectively. Results. Prominin‐1 expression increased significantly after bile duct ligation compared with sham (6.6 ± 0.9‐fold change at 2 weeks, P < .05) but not with carbon tetrachloride (−0.7 ± 0.5‐fold change, not significant). Upregulation of Prominin‐1 was observed histologically throughout the liver as early as 5 days after bile duct ligation in Prominin‐1cre‐ert2−lacz mice by LacZ staining in nonhepatocyte cells. Lineage tracing of Prominin‐1‐expressing cells labeled prior to bile duct ligation in Prominin‐1cre‐ert2−lacz;Gfplsl mice, demonstrated increasing colocalization of GREEN FLUORESCENT PROTEIN with biliary marker CYTOKERATIN‐19 within ductular reactions up to 5 weeks after bile duct ligation consistent with biliary transdifferentiation. In contrast, rare colocalization of GREEN FLUORESCENT PROTEIN with mesenchymal marker &agr;‐SMOOTH MUSCLE ACTIN in Prominin‐1cre‐ert2−lacz;Gfplsl mice and some colocalization of GREEN FLUORESCENT PROTEIN with PROMININ‐1 in Collagen‐1&agr;Gfp mice, indicate minimal contribution of Prominin‐1 progenitor cells to the pool of collagen‐producing myofibroblasts. Conclusion. During biliary fibrosis Prominin‐1‐expressing progenitor cells transdifferentiate into cells within ductular reactions. This transdifferentiation may promote fibrosis.

Toll-like receptor 3 mediates PROMININ-1 expressing cell expansion in biliary atresia via Transforming Growth Factor-Beta

BACKGROUND In biliary atresia (BA), epithelial-mesenchymal hepatic progenitor cells (HPC) expressing the stem/progenitor cell marker PROMININ-1 (PROM1) undergo expansion and subsequent transdifferentiation into collagen-producing myofibroblasts within regions of evolving biliary fibrosis under the regulation of Transforming Growth Factor-β (TGFβ) signaling. We hypothesized that pro-inflammatory Toll-like Receptor-3 (TLR3) signal activation promotes the differentiation of PROM1+ HPC via TGFβ pathway activation in vitro. METHODS PROM1+ Mat1a(-/-) HPC were treated with a double-stranded RNA analog, polyionosinic-polycytidylic acid (Poly I:C), ± small molecule inhibitors nafamostat, or SB431542. RESULTS Poly I:C induced myofibroblastic-like morphologic changes, degradation of IκB-α consistent with TLR3-NFκB activation, a 15-fold increase in the expression of Vimentin, a 9-fold increase in Collagen-1a, a 4.6-fold increase in Snail at 24h (p<0.05), and an 8.2-fold increase in Prom1 at 72h (p<0.0001) by qPCR. Immunofluorescence demonstrated nuclear phosphorylated SMAD3, TLR3, and COLLAGEN-1α staining following Poly I:C treatment. Degradation of IκBα was inhibited by nafamostat. Co-treatment with either nafamostat or SB431542 blocked the morphologic change and abrogated the increased expression of Cd133, Collagen, Vimentin, and Snail1. CONCLUSIONS TLR3 activation induces myofibroblastic differentiation of PROM1+ HPC in part via TGFβ pathway activation to promote BA-associated biliary fibrosis.

Congenital Intrahepatic Cholestasis

Progressive familial intrahepatic cholestasis (PFIC) comprises a heterogeneous group of disorders defined by specific mutations in bile salt export proteins and these disorders are further differentiated from Alagille syndrome and other cholestatic disease processes according to their clinical and diagnostic characteristics. Pruritus is a common and debilitating condition associated with cholestasis of any etiology, but pharmacological therapies are limited and often fail. Surgical procedures designed to decrease bile salt load, namely via partial external biliary diversion (PEBD) or ileal exclusion (IE), as well as technical variations and laparoscopic approaches, have been developed as have acceptable therapeutic interventions. Liver transplantation is the only treatment in advanced disease.

Progenitor Cells in Liver Development, Regeneration and Repair

During organogenesis, the liver develops from the foregut endoderm and grows into the adjacent septum transversum resulting in the formation of the liver bud. Growth factors released from the septum transversum and the cardiac mesenchyme induce endodermal differentiation and proliferation, thus, forming the primordial liver and extrahepatic biliary structures. Endodermal precursor cells within the liver bud comprise bi-potential liver progenitor cells called hepatoblasts, which differentiates into hepatocytes and cholangiocytes. While the postnatal liver has remarkable capacity to regenerate via the proliferation of mature hepatocytes, this compensatory mechanism may be overwhelmed during states of chronic injury. Under these conditions, resident stem/progenitor cells proliferate to replace lost liver parenchyma. Significant progress has been made recently in elucidating the role of various signaling pathways and progenitor cells in liver development and regeneration. In this review, we summarize our recent understanding of progenitor cells in liver development, regeneration and repair.