These authors equally contributed to this work.
Autoimmune, Cholestatic and Biliary Disease
Article first published online: 7 APR 2011
Copyright © 2011 American Association for the Study of Liver Diseases
Volume 53, Issue 4, pages 1246–1258, April 2011
How to Cite
Omenetti, A., Bass, L. M., Anders, R. A., Clemente, M. G., Francis, H., D. Guy, C., McCall, S., Choi, S. S., Alpini, G., Schwarz, K. B., Diehl, A. M. and Whitington, P. F. (2011), Hedgehog activity, epithelial-mesenchymal transitions, and biliary dysmorphogenesis in biliary atresia . Hepatology, 53: 1246–1258. doi: 10.1002/hep.24156
Potential conflict of interest: Nothing to report.
Supported by grants from the National Institutes of Health, grant numbers RO1 DK077794 (to A.M.D.), R01DK080736, and R01DK081417 (to R.A.A.). Portions of the study were supported by the Dr. Nicholas C. Hightower Centennial Chair of Gastroenterology to G.A. from Scott & White Hospital and a VA Merit Award, and by funds provided by the Liver Foundation for Kids, Lemont, IL.
- Issue published online: 7 APR 2011
- Article first published online: 7 APR 2011
- Accepted manuscript online: 12 JAN 2011 06:51PM EST
- Manuscript Accepted: 18 DEC 2010
- Manuscript Received: 17 OCT 2010
- National Institutes of Health. Grant Numbers: RO1 DK077794, R01DK080736, R01DK081417
- Scott & White Hospital and a VA Merit Award
- Liver Foundation for Kids, Lemont, IL
Additional Supporting Information may be found in the online version of this article.
|HEP_24156_sm_suppinfofigure1.tif||635K||Supporting Figure 1. Up-regulation of Sonic Hedgehog ligand mRNA in children with biliary atresia and other types of cholestatic liver disease. Patients with BA, children with various other cholestatic liver diseases, and age-matched controls without liver disease (nondiseased, ND), were examined for Shh ligand expression. 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) Shh ligand mRNA expression. Gene expression data in diseased livers are expressed relative to levels in control subjects and graphically depicted using box-and-whisker plots. As evaluated by Wilcoxon rank-sum test, no significant differences in Shh expression were noted among the groups.|
|HEP_24156_sm_suppinfofigure2.tif||4068K||Supporting Figure 2 Strong correlation between expression of Snail and that of other mesenchymal markers in BA patients. (A-C) Covariance between the EMT-driving gene, Snail, and two other typical mesenchymal markers, Vimentin and FSP1, was evaluated in BA patients. Data were plotted to demonstrate the results of linear regression analysis. Significance (P=value) and strength of correlation (coefficient of determination, r2) are shown. (D-E) Pictures demonstrating FSP1 staining in the liver (intrahepatic, D) and extrahepatic biliary remnant (E) of representative BA patients. Magnification 200×.|
|HEP_24156_sm_suppinfofigure3.tif||12458K||Supporting Figure 3 Hh-responsive (Gli2-positive) cells co-express mesenchymal and epithelial markers in BA livers and extrahepatic biliary remnants. Immunohistochemical analysis for Gli2/Vimentin (A-B) and Gli2/KRT7 (C-D) was performed in both livers (A, C) and extrahepatic biliary remnants (B, D) from BA patients. Final magnifications 100×|
|HEP_24156_sm_suppinfofigure4.tif||6565K||Supporting Figure 4 Progenitor marker expression in BA livers and extrahepatic biliary remnants. Immunohistochemical analysis for AE1/AE3 (A-B) and Epcam (C-D) was performed in both livers (A, C) and extrahepatic biliary remnants (B, D) from BA patients. Final magnifications 200×|
|HEP_24156_sm_suppinfofigure5.tif||1655K||Supporting Figure 5. FSP1 expression is Hedgehog-regulated in normal primary rat cholangiocytes. QRT-PCR (A) and immunocytochemistry (B) of pooled small and large primary cholangiocytes from healthy adult rats were used to assess FSP1. (A) FSP1 mRNA expression after treatment with unconditioned medium (white bars) containing control IgG or MF-conditioned with control IgG (black bars) or Hh neutralizing antibody (grey bars). (B) Representative cholangiocyte cytospins. Final Magnification 400×, inserts show magnified field of areas pointed by the arrows. (C) Quantification of FSP1 positive cholangiocytes. FSP1(+) (red bars) and FSP1 (-) (black bars) cholangiocytes were counted under under 200× final magnification. Per each field, FSP1 positive (i.e. red (+) and DAPI(+)) (red bars) and FSP1 negative (i.e. red (-) and DAPI(+)) were counted and the final data were expressed as percentage of total cells/field. (D) QRTPCR analysis of FSP1 expression in small (more immature) and large (mature) subpopulations of primary cholangiocytes isolated from rats with ongoing biliary fibrosis (i.e. BDL). Data are expressed relative to control group (white bars) and displayed as mean ± Standard Error Mean (SEM). Comparisons between groups were performed using the two-tailed Student's t-test. Significance was accepted at the 5% level. (*P<0.05, ** P<0.005).|
|HEP_24156_sm_suppinfotable1.doc||38K||Supporting Table 1. Antibodies and retrieval techniques used for IHC.|
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