Volume 123, Issue 4, Pages 1323-1330 (October 2002) Fas enhances fibrogenesis in the bile duct ligated mouse: A link between apoptosis and fibrosis  Ali Canbay, Hajime Higuchi, Steven F. Bronk, Makiko Taniai, Tom J. Sebo, Gregory J. Gores  Gastroenterology  Volume 123, Issue 4, Pages 1323-1330 (October 2002) DOI: 10.1053/gast.2002.35953 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 Hepatocyte apoptosis and ALT values in 3-day BDL mice are significantly increased in WT compared with lpr mice. WT and Fas-deficient lpr mice underwent a sham operation or ligation of their common bile duct. Three days after the surgical procedure, the mice were anesthetized, liver tissue procured, and serum obtained. (A) Fixed liver specimens from WT and lpr bile duct ligated mice were prepared for H&E and TUNEL assays. Bile duct proliferation, portal edema, and mild portal infiltrates were present in both BDL WT and lpr mice. However, clusters of apoptotic cells only occurred in WT BDL mice. (B) Fixed liver specimens were stained with the TUNEL assay to identify apoptotic hepatocytes. The magnitude of apoptosis occurring in WT mice was greater than in sham-operated control or BDL lpr mice (P <.001 by ANOVA, n = 3 for each experimental group). (C) Serum ALT values are greater in WT than in lpr mice 3 days after BDL (P <.001 by ANOVA, n = 3 for each experimental group). Gastroenterology 2002 123, 1323-1330DOI: (10.1053/gast.2002.35953) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 α-SMA and PDGF receptor β mRNA expression, markers for stellate cell activation, are increased in WT compared with lpr BDL mice. Mice underwent BDL or a sham operation. Three days after the surgical procedure, liver tissue was procured under deep anesthesia and total hepatic RNA isolated using the Trizol Reagent (Invitrogen, Carlsbad, CA). α-SMA and PDGF receptor β mRNA was quantitated by real-time PCR as extensively described in the Experimental Procedures section. The expression was normalized as a ratio using 18s mRNA as a housekeeping gene. A value of one for this ratio was arbitrarily assigned to the data obtained from sham operated WT mice. (A) The magnitude of α-SMA mRNA expression in WT BDL was greater than in lpr BDL mice (P <.03 by ANOVA, n = 3 for each group). (B) The expression of PDGF receptor β mRNA was also greater in WT versus lpr BDL mice (P <.001, n = 3 for each group). Gastroenterology 2002 123, 1323-1330DOI: (10.1053/gast.2002.35953) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 TGF-β, collagen 1α(I) (COL1A1), and TIMP mRNA expression, markers for stellate cell fibrogenic activity, are increased in WT compared with lpr BDL mice. Mice underwent BDL or a sham operation. Three days after the surgical procedure, liver tissue was procured under deep anesthesia and total RNA isolated using the Trizol Reagent (Invitrogen, Carlsbad, CA). TGF-β, collagen 1α(I), and TIMP mRNA was quantitated by real-time PCR as extensively described in the Experimental Procedures section. The expression was normalized as a ratio using 18s ribosomal mRNA as a housekeeping gene. A value of 1 for this ratio was arbitrarily assigned to the data obtained from sham-operated WT mice. (A) The magnitude of TGF-β mRNA expression was greater in WT mice than lpr BDL mice (P <.001 by ANOVA, n = 3 for each group). (B) Collagen α1(I) expression was significantly greater in WT than in lpr BDL mice (P <.001 by ANOVA, n = 3 for each group). (C) TIMP mRNA expression, a marker for progression of fibrosis, was also increased in WT compared with lpr BDL mice (P <.03 by ANOVA, n = 3 for each experimental group). Gastroenterology 2002 123, 1323-1330DOI: (10.1053/gast.2002.35953) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 α-SMA liver immunostaining 3 weeks after bile duct ligation. Mice underwent bile duct ligation or a sham operation. Three weeks after the surgical procedure, liver tissue was obtained under deep anesthesia and fixed in 4% paraformaldehyde. Immunostaining for α-SMA was performed as described under the Experimental Procedures. Immunoreactivity for α-SMA was greater in WT BDL compared with lpr BDL mice. Liver sections from WT sham and lpr sham-operated mice displayed little to no α-SMA immunoreactivity. (Original magnification 100×.) Gastroenterology 2002 123, 1323-1330DOI: (10.1053/gast.2002.35953) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 Sirius red staining is increased in WT BDL compared with lpr BDL mice. Mice underwent BDL or a sham operation. Three weeks after the surgical procedure, liver tissue was obtained under deep anesthesia and fixed in 4% paraformaldehyde. Collagen fibers were stained with Sirius red as described in the Experimental Procedures section (A) and the surface area stained with Sirius red quantitated using digital image analysis. (B) Sirius red staining was quantitatively greater in WT BDL than in lpr BDL mice (P <.001 by ANOVA, n = 3 for each group). In contrast, there was minimal Sirius red staining and no difference between sham-operated mice from the 2 groups of animals. (Original magnification 100×.) Gastroenterology 2002 123, 1323-1330DOI: (10.1053/gast.2002.35953) Copyright © 2002 American Gastroenterological Association Terms and Conditions