Viral factors induce Hedgehog pathway activation in humans with viral hepatitis, cirrhosis, and hepatocellular carcinoma - PubMed (original) (raw)

. 2010 Dec;90(12):1690-703.

doi: 10.1038/labinvest.2010.147. Epub 2010 Aug 9.

Rafal P Witek, Wing-Kin Syn, Steve S Choi, Shelton Bradrick, Gamze F Karaca, Kolade M Agboola, Youngmi Jung, Alessia Omenetti, Cynthia A Moylan, Liu Yang, Martin E Fernandez-Zapico, Ravi Jhaveri, Vijay H Shah, Fausto E Pereira, Anna M Diehl

Affiliations

Viral factors induce Hedgehog pathway activation in humans with viral hepatitis, cirrhosis, and hepatocellular carcinoma

Thiago de Almeida Pereira et al. Lab Invest. 2010 Dec.

Abstract

Hedgehog (Hh) pathway activation promotes many processes that occur during fibrogenic liver repair. Whether the Hh pathway modulates the outcomes of virally mediated liver injury has never been examined. Gene-profiling studies of human hepatocellular carcinomas (HCCs) demonstrate Hh pathway activation in HCCs related to chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV). Because most HCCs develop in cirrhotic livers, we hypothesized that Hh pathway activation occurs during fibrogenic repair of liver damage due to chronic viral hepatitis, and that Hh-responsive cells mediate disease progression and hepatocarciongenesis in chronic viral hepatitis. Immunohistochemistry and qRT-PCR analysis were used to analyze Hh pathway activation and identify Hh-responsive cell types in liver biopsies from 45 patients with chronic HBV or HCV. Hh signaling was then manipulated in cultured liver cells to directly assess the impact of Hh activity in relevant cell types. We found increased hepatic expression of Hh ligands in all patients with chronic viral hepatitis, and demonstrated that infection with HCV stimulated cultured hepatocytes to produce Hh ligands. The major cell populations that expanded during cirrhosis and HCC (ie, liver myofibroblasts, activated endothelial cells, and progenitors expressing markers of tumor stem/initiating cells) were Hh responsive, and higher levels of Hh pathway activity associated with cirrhosis and HCC. Inhibiting pathway activity in Hh-responsive target cells reduced fibrogenesis, angiogenesis, and growth. In conclusion, HBV/HCV infection increases hepatocyte production of Hh ligands and expands the types of Hh-responsive cells that promote liver fibrosis and cancer.

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Figures

Figure 1

Figure 1. Liver cell production of Sonic Hh ligands (Shh) increases during chronic viral hepatitis

A) Liver section from a representative control liver without viral hepatitis (NRL). Final magnification ×200. B) qRT-PCR analysis of RNA isolated from normal liver, and HBV- or HCV-infected livers with F0/1, F2, or F3/4 fibrosis (n = 3/group). Results were normalized to Shh expression in normal control livers; means ± SEM are displayed. **P < 0.005 vs. controls. C) Immunohistochemical staining for Shh in representative sections from patients with HBV (top) and HCV (bottom). Left panels display findings in livers with F0 fibrosis; right panels display findings in livers with F3-4 fibrosis. Final magnification ×200. D-E) qRT-PCR analysis of Shh expression in Huh-7 cells infected with HCV JFH-1 (D). Results were normalized to the respective controls and mean ± SEM data are shown. **P < 0.005 vs. controls.

Figure 2

Figure 2. Liver cells that express the Hh-regulated transcription factor, Gli2, accumulate during chronic viral hepatitis

A) Liver section from a representative control liver without viral hepatitis (NRL). Final magnification ×200. B) qRT-PCR analysis of RNA isolated from normal liver, and HBV- or HCV-infected livers with F0/1, F2, or F3/4 fibrosis (n = 3/group). Results were normalized to Gli2 expression in normal control livers; mean ± SEM data are displayed. **P < 0.005 vs. controls. C) Immunohistochemical staining for Gli2 in representative sections from patients with HBV (top) and HCV (bottom). Left panels display findings in livers with F0 fibrosis; right panels display findings in livers with F3-4 fibrosis. Final magnification ×200.

Figure 3

Figure 3. Myofibroblastic cells that accumulate during chronic viral hepatitis are Hh-responsive and Hh signaling is necessary for fibrogenic gene expression

A) RNA was isolated from normal liver, and HBV- or HCV-infected livers with F0/1, F2, or F3/4 fibrosis (n = 3/group); qRT-PCR was done to assess expression of α-sma (left panel); accumulation of αSMA-expressing cells (middle panel) and Sirius red stained fibrils (right panel) were quantified in serial liver sections using morphometric analysis. Means ± SEM results are graphed. *P < 0.05, **P < 0.005 vs controls. B) Immunohistochemistry demonstrating sinusoidal cell expression of αSMA (left panel), Ihh (middle panel), and Patched (Ptc) (right panel) in representative liver sections with F0-1 fibrosis. Final magnification ×600. C-D) Hepatic stellate cells were isolated from non-diseased human liver and cultured to induce transition to myofibroblastic cells (MF); then treated with tomatidine (T) or cyclopamine (Cy) for an additional 48h. RNA and protein were isolated from triplicate plates and qRT-PCR (C) and Western blot (D) were used to assess effects on gene expression. Experiments were replicated a total of three times; data were normalized to tomatidine-treated cultures. Means ± SEM are displayed. * P< 0.05, **P < 0.005 vs tomatidine-treated cultures.

Figure 4

Figure 4. Activated endothelial cells are Hh responsive during chronic viral hepatitis and Hh pathway activation promotes vascular remodeling responses

A) Immunohistochemistry was used to characterize the types of cells that accumulated in fibrotic septae. Representative photomicrograph from patient with stage F3-4 chronic hepatitis C is shown. Double immunohistochemistry for Gli2 (brown) and CD31 (blue). Final magnification ×600. B) Primary liver endothelial cells were isolated from a non-diseased liver; RNA was harvested from freshly-isolated cells and from triplicate plates of cells that had been cultured to induce transition to an activated phenotype and then treated with either tomatidine (T) or cyclopamine (Cy) for an additional 48h. qRTPCR analysis was done to determine effects on expression of CD31 (left) and Gli2 (right). Data were normalized to gene expression in the freshly-isolated cells. Means ± SEM results are displayed. * P < 0.05, ** P < 0.005, + P < 0.01 vs fresh cells. C-D) SECs were seeded into matrigel and treated with vehicle or Shh ligand (5μg/ml) (C) and HSC condition media (CM) with or without Cy (D). E) SECs were pre-treated with lentivirus containing Smo scramble (scr) shRNA or Smo shRNA. Effects on vascular tube formation were assessed in triplicate experiments. Representative results are displayed as arbitrary units. Means ± SEM are graphed. *P < 0.05 vs control; # P < 0.05 vs condition medium alone.

Figure 5

Figure 5. The ductular reaction that occurs during chronic viral hepatitis is enriched with Hh-responsive ductular cells that co-express epithelial progenitor cell and mesenchymal cell markers

Immunohistochemistry was used to characterize the types of cells that accumulated in fibrotic septae. Representative photomicrographs from patients with stage F3-4 chronic hepatitis C are shown. Mono-staining for A) Shh, B) Gli2, C) Krt 7, D) CD133, E) αSMA, F) S100A4. Final magnifications ×600.

Figure 6

Figure 6. Peri-tumoral stromal tissue adjacent to HCC is enriched with Hh-responsive stromal cells and ductular cells that express markers of tumor stem/progenitor cells

A) Nodules of HCC (N) and adjacent peri-tumoral tissue in representative patient with chronic hepatitis C and HCC. Final magnification ×100. B-E) Immunohistochemistry for Ptc (B), S100A4 (C-D) and Survivin (E) in serial sections from this patient. Dotted lines delineate in (E) margins of samples that were procured using laser capture dissection microscopy (LCM). Final magnifications ×100 (B-C), ×400 (D) and ×200 (E). F) qRT-PCR analysis of peritumoral fibrotic septae and nodules of malignant hepatocytes from 3 patients with chronic viral hepatitis and HCC. Data were normalized to Ptc gene expression in the tumor nodules. Means ± SEM are displayed. ** P < 0.005 vs tumor nodules.

Figure 7

Figure 7. Peri-tumoral tissue adjacent to HCC is enriched with Hh-responsive cells

Immunohistochemistry was used to characterize the types of cells that accumulated in peri-tumoral fibrotic tissue. Representative photomicrographs are shown. Mono-staining (arrowheads) for A) Shh, B) Ihh, C) Gli2, D) Krt7, E) CD133, F) EpCam. Final magnifications ×600 (A, B, D, E and F) or ×400 (C).

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