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, 53 (4), 1246-58

Hedgehog Activity, Epithelial-Mesenchymal Transitions, and Biliary Dysmorphogenesis in Biliary Atresia

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Hedgehog Activity, Epithelial-Mesenchymal Transitions, and Biliary Dysmorphogenesis in Biliary Atresia

Alessia Omenetti et al. Hepatology.

Abstract

Biliary atresia (BA) is notable for marked ductular reaction and rapid development of fibrosis. Activation of the Hedgehog (Hh) pathway promotes the expansion of populations of immature epithelial cells that coexpress mesenchymal markers and may be profibrogenic. We examined the hypothesis that in BA excessive Hh activation impedes ductular morphogenesis and enhances fibrogenesis by promoting accumulation of immature ductular cells with a mesenchymal phenotype. Livers and remnant extrahepatic ducts from BA patients were evaluated by quantitative reverse-transcription polymerase chain reaction (QRT-PCR) and immunostaining for Hh ligands, target genes, and markers of mesenchymal cells or ductular progenitors. Findings were compared to children with genetic cholestatic disease, age-matched deceased donor controls, and adult controls. Ductular cells isolated from adult rats with and without bile duct ligation were incubated with Hh ligand-enriched medium ± Hh-neutralizing antibody to determine direct effects of Hh ligands on epithelial to mesenchymal transition (EMT) marker expression. Livers from pediatric controls showed greater innate Hh activation than adult controls. In children with BA, both intra- and extrahepatic ductular cells demonstrated striking up-regulation of Hh ligand production and increased expression of Hh target genes. Excessive accumulation of Hh-producing cells and Hh-responsive cells also occurred in other infantile cholestatic diseases. Further analysis of the BA samples demonstrated that immature ductular cells with a mesenchymal phenotype were Hh-responsive. Treating immature ductular cells with Hh ligand-enriched medium induced mesenchymal genes; neutralizing Hh ligands inhibited this.

Conclusion: BA is characterized by excessive Hh pathway activity, which stimulates biliary EMT and may contribute to biliary dysmorphogenesis. Other cholestatic diseases show similar activation, suggesting that this is a common response to cholestatic injury in infancy.

Figures

Figure 1
Figure 1. Liver from child-aged controls displays higher Hedgehog pathway activation and FSP1 expression compared to adult controls
Non-disease liver (CTL) from adult and pediatric controls were analyzed at protein and gene levels for Gli2 and FSP1 expression. (A-D) IHC was performed in representative patients (N=2 per group) to localize the cell types expressing the Hedgehog transcription factor Gli2 (A, C) and the EMT marker FSP1 (B, D) at baseline. Final magnification 630X. (E-F) QRT-PCR was performed to quantitative differences in gene expression of Gli2 and FSP1 between child-aged (N=6) and adult (N=5) CTL livers. Data are displayed by box-and-whisker plots. Significance of difference in gene expression was evaluated by Wilcoxon rank-sum test. ** Significant Wilcoxon rank-sum test vs NL control.
Figure 2
Figure 2. Up-regulation of Sonic Hedgehog ligand production in children with biliary atresia and other types of cholestatic liver disease
Livers from patients with BA (N=12), children with various other types of cholestatic liver disease (AGS, FIC1 and BSEP, N=18), and age-matched controls without liver disease (nondiseased, ND, N=6), and all available extrahepatic biliary remnants from BA patients (N=5) were examined for Shh ligand production by IHC. Representative pictures at 400X of magnification, with inserts showing magnified views to facilitate visualization of Shh-producing cells.
Figure 3
Figure 3. Up-regulation of Hedgehog target gene expression in children with biliary atresia and other types of cholestatic liver disease
Patients with BA, children with various other types of cholestatic liver disease, and age-matched controls without liver disease (non diseased, ND), were examined for expression of the Hh-target genes. QRT-PCR analysis was performed in liver tissue from patients with BA (N=9), Alagille's syndrome (AGS, N=5), progressive familial intrahepatic cholestasis type 1 (FIC1, N=7), progressive intrahepatic cholestasis type 2 (BSEP, N=6), and age-matched CTL (N=6). (A-E) mRNA expression of Gli1, Gli2, Gli3, Ptc, and Hhip. Gene expression data in diseased livers are expressed relative to expression levels in control subjects and graphically depicted as box-and-whisker plots. Significance was evaluated by Wilcoxon rank-sum test. ** Significant Wilcoxon rank-sum test vs NL control.
Figure 4
Figure 4. Nuclear accumulation of Gli2 protein in children with biliary atresia and other types of cholestatic liver disease
(A-D) Gli2 protein expression was confirmed by IHC analysis in all ND livers (Figure 1) and all available extrahepatic biliary remnants from BA patients (A), BA livers (B), and all livers from patients with AGS (C), FIC1 (D) and BSEP (E). Representative pictures at 400X of magnification, with inserts showing magnified views to facilitate visualization of cells with nuclear accumulation of Gli2 protein.
Figure 5
Figure 5. Hedgehog activation in biliary atresia was accompanied by EMT in ductular cells
(A-D) QRT-PCR analysis of EMT markers (i.e. N-Cadherin, Vimentin,, Snail, FSP1) in livers from pediatric patients with BA (N=9) and age-matched controls (CTL) (N=6). Gene expression data in disease livers are expressed relative to levels in control subjects and presented as box-and-whisker plots. Significance was evaluated by Wilcoxon rank-sum test. ** Significant Wilcoxon rank-sum test vs NL control. (E-F) Immunohistochemical analysis for FSP1 was performed in both livers (E) and extrahepatic biliary remnants (F) from BA patients. Final magnifications 630X (E-F).
Figure 6
Figure 6. Expressions of mesenchymal marker (FSP1) and marker of immature liver epithelial cells (KRT7) co-localize in ductular epithelial of BA patients
To further characterize the FSP1 expressing cells, additional double immunocytochemistry (A-D) for FSP1 (stained brown) and KRT7 (stained pink) was performed in the same sections. (A, C) FSP1(+)/KRT7(+) intrahepatic bile ducts of representative patients with BA. (B, D) Similar staining in the extrahepatic biliary remnants from representative BA patients. Final magnification 200X (A-B) and 630X (C-D). Inserts show magnified view of double positive cells in intrahepatic ducts (C) and extrahepatic biliary epithelium (D).
Figure 7
Figure 7. Cells undergoing EMT in liver samples from BA patients are Hedgehog responsive
(A-B) Covariance between the Hh target gene Gli2 and the EMT markers FSP1 and Vimentin was analyzed in patients with BA; data were plotted to demonstrate results of the linear regression analysis. Significance (P=value) and strength of correlation (coefficient of determination, r2) are indicated. (C-D) Double immunohistochemistry for Gli2 (brown) and Vimentin (blue) was performed to confirm that ductular cells acquiring mesenchymal markers were Hh responsive. Representative pictures of BA liver are presented at 630X (C), with magnified view in inserts. Similar staining results were observed in the extrahepatic biliary remnants of representative BA patients (D). Double immunohistochemistry for Gli2 (brown) and the progenitor marker, KRT7 (pink), were done in serial sections from the same cases. Gli2/KRT7 staining in intrahepatic bile ducts (E) and extrahepatic biliary remnants (F). Final magnifications 630X (E-F).
Figure 8
Figure 8. EMT localizes to the progenitor cell compartment in human BA samples
(A-D) Immunostaining for additional progenitor markers was performed to further assess the accumulation of immature ductular cells in BA livers (A, C) and extrahepatic biliary remnants (B, D). (A-B) AE1/AE3, (C-D) Epcam. Representative pictures are displayed at 630X. Higher magnification views are also provided in inserts to facilitate visualization of marker-positive cells.

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