1. Volume 137, Issue 1, Pages 297-308.e4 (July 2009)
c-Met Confers Protection Against Chronic Liver Tissue Damage and Fibrosis Progression After Bile Duct Ligation in Mice 
Arne Giebeler, Mark V. Boekschoten, Christian Klein, Malgorzata Borowiak, Carmen Birchmeier, Nikolaus Gassler, Hermann E. Wasmuth, Michael Müller, Christian Trautwein, Konrad L. Streetz 
Gastroenterology 
Volume 137, Issue 1, Pages e4 (July 2009)
DOI: /j.gastro
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2. Figure 1
Generation and characterization of a hepatocyte-specific c-Met knockout mouse. (A) Strategy for the hepatocyte-specific deletion of c-Met. In c-MetloxP/loxP mice the coding region for the transmembrane domain (exon 15) is flanked by 2 loxP sites. c-MetloxP/loxP mice were either crossed with animals expressing the Cre-recombinase under the control of an Alfp-Cre construct (prenatal Cre expression) or under the control of an Alb-Cre construct (postnatal Cre expression). Although Alfp-Cre(+)/c-MetloxP/loxP animals died during embryonal development, Alb-Cre(+)/c-MetloxP/loxP (c-MetΔhepa) mice were viable at normal Mendelian frequency. (B) Deletion of c-Met exon 15 was tested by reverse-transcription polymerase chain reaction. Liver RNA or RNA from isolated hepatocytes derived from either c-MetΔhepa or c-MetloxP/loxP mice were tested via a polymerase chain reaction protocol using primers that are able to detect deletion of exon 15 in the c-Met gene. The position of the deleted (Δc-Met) and the undeleted DNA (wt c-Met) are indicated. Intensity of bands was evaluated densitometrically. (C) HGF induces phosphorylation of c-Met in hepatocytes. Primary hepatocytes were isolated by liver perfusion, plated overnight, and stimulated with 10 ng/μL HGF for 30 minutes. Immunoblot analysis displayed c-Met phosphorylation (pY1230/1234/1235) in c-MetloxP/loxP mice, whereas this regulation was blunted in c-MetΔhepa mice. As a protein loading control, α-tubulin staining was performed.
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3. Figure 2
Changes in serum parameters and liver histology in c-MetΔhepa mice after BDL. (A) Kaplan–Meyer analysis of mouse survival after BDL. All c-MetloxP/loxP mice survived BDL (n = 8), whereas only 88% of c-MetΔhepa mice survived (n = 8). (B) Serum ALT levels were strongly and significantly increased up to 28 days after BDL. No significant differences were found between c-MetΔhepa and c-MetloxP/loxP mice. (C) Total protein in mouse serum 28 days after BDL. c-MetΔhepa mice showed a significantly reduced amount of total serum protein (*P < .05). (D) H&E stainings of paraffin-embedded liver sections of c-MetΔhepa and c-MetloxP/loxP mice before and 7 and 28 days after BDL (magnification, 10×). Necrotic areas that are most apparent in c-MetΔhepa mice are indicated by arrows. (E) Quantification of necrotic areas 3 and 7 days after BDL. At least 10 view-fields/liver in 4 animals of each group were included. Significantly more necrotic areas were found in the livers of c-MetΔhepa mice compared with c-MetloxP/loxP controls (*P < .01).
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4. Figure 3
c-MetΔhepa mice show an enhanced inflammatory response after BDL. (A–C) TNF-α, IL-6, and HGF expression were quantified by real-time polymerase chain reaction at indicated time points before and after BDL. RNA was extracted separately from individual liver tissues (at least 4 samples/time point were included) at the respective time points (*P < .05; **P < .01). (D) Liver-infiltrating neutrophils Ly6G(+) and (E) F4/80(+) macrophages were detected by immunofluorescence staining 3 and 7 days after BDL treatment and quantified (**P < .01; ***P < .001). Photomicrographs are provided as Supplementary Figure 1. (F) KC (Gro-α) chemokine expression was analyzed by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Significant differences in KC liver mRNA and serum levels are indicated between c-MetΔhepa mice and controls (*P < .05; **P < .01).
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5. Figure 4
Higher apoptosis rates and decreased proliferation in livers of c-MetΔhepa mice after BDL. (A) Apoptotic cells were analyzed by terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling staining (green) in livers of c-MetΔhepa and c-MetloxP/loxP controls before and after BDL as indicated. Representative pictures are shown for each time point. (B) Statistical analysis of TUNEL-positive cells in livers of c-MetloxP/loxP controls and c-MetΔhepa mice after BDL (*P < .05). At least 10 view fields of 4 livers per time point were included in this analysis. (C) Hepatocyte proliferation was analyzed by bromodeoxyuridine uptake in livers of c-MetloxP/loxP controls and c-MetΔhepa mice before and after BDL. Anti–bromodeoxyuridine antibodies were labeled with Alexa488 (green). (D) Statistical analysis of bromodeoxyuridine-positive cells in livers of c-MetloxP/loxP controls and c-MetΔhepa mice before and 7 days after BDL (*P < .05). At least 10 view fields of 4 livers per time point were included in this analysis.
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6. Figure 5
Microarray analysis revealed dysregulation of anti-apoptotic genes and higher sensitivity to Fas-induced apoptosis in livers of c-MetΔhepa mice after BDL. (A) C-MetloxP/loxP and c-MetΔhepa mice were stimulated with 2 μg recombinant murine HGF. Total protein was isolated and Western blot analysis for the detection of phosphorylated STAT3 was performed. The position of phospho-STAT3 (pSTAT3) is indicated. (B) For gene array analysis c-MetloxP/loxP controls were stimulated for 2 hours with 2 μg recombinant murine HGF. Liver RNA was isolated from animals before and after HGF stimulation. Detailed analysis of gene expression unraveled a set of 463 genes in total being up-regulated or down-regulated more then 2-fold by HGF/c-Met (limma P value < .01). (C) Affymetrix GeneChip analysis of liver RNA was used to detect gene expression of HGF-stimulated c-MetΔhepa and c-MetloxP/loxP mice. Three animals per group were treated in parallel, before and after intraperitoneal injection of recombinant HGF or NaCl. Examples of genes that are regulated HGF/c-Met–dependent and cell damage and apoptosis related are depicted. gcRMA normalized intensity values are provided before and after treatment with corresponding fold changes and limma P values. Microarray data can be found under GEO series accession number GSE (D) TNFAIP2 expression was quantified by real-time polymerase chain reaction before and 3 days after BDL as indicated. RNA was extracted separately from individual liver tissues (at least 4 samples/time point were included) at the respective time points (*P < .05). (E) Bile duct–ligated mice were stimulated 3 days after BDL with Jo-2 (6 h) antibody (400 ng/g body weight). (F) c-MetΔhepa mice displayed significantly higher counts of terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling–positive cells. Statistical analysis of terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling–positive cells in c-MetloxP/loxP controls and c-MetΔhepa in 3-day bile duct–ligated mice that were stimulated for 6 hours with Jo-2 antibody (*P < .05). At least 10 view fields of 4 livers per time point were included in this analysis.
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7. Figure 6
c-Met deletion in hepatocytes induces enhanced stellate cell activation after BDL. (A, B, and D) Transforming growth factor-β1 (tgf-β1), TLR4, and α-smooth muscle actin (α-sma) expression were quantified by real-time polymerase chain reaction at time points before and after BDL as indicated. RNA was extracted separately from individual liver tissues (at least 4 samples/time point were included) at the respective time points (*P < .05; **P < .01). Analysis for c-MetΔhepa and c-MetloxP/loxP controls are shown. (C) Immunofluorescence analysis of α-SMA expression (Alexa488 green) in livers of bile duct–ligated c-MetΔhepa and control mice before and 3 days after BDL, indicating a stronger activation of HSCs in c-MetΔhepa mice that underwent BDL are displayed. Representative pictures are shown for each time point.
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8. Figure 7
Enhanced progression of liver fibrosis in c-MetΔhepa mice after BDL. (A) Sirius red staining of paraffin-embedded liver tissue derived from livers of c-MetΔhepa and c-MetloxP/loxP controls 28 days after BDL. Representative photomicrographs taken under polarized light are shown for each time point (magnification, 20×). (B) Graphic analysis of liver Sirius red staining in c-MetΔhepa mice and c-MetloxP/loxP controls 28 days after BDL. To graphically visualize the difference in collagen fiber accumulation, the intensity of Sirius red staining in bile duct–ligated mice (as shown in Figure 6A) was determined. Photomicrographs of Sirius red–stained slides were analyzed using the open source ImageJ software (livers from non–bile duct–ligated mice were used as controls and set as zero, **P < .01). (C) Collagen-1α, (D) MMP-13, (E) TIMP-1, and (F) PDGF-D expression was quantified by real-time polymerase chain reaction before and 28 days after BDL as indicated. RNA was extracted separately from individual liver tissues (at least 4 samples/time point were included) at the respective time points (*P < .05).
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9. Supplementary Figure 1
Cryosections were immunostained to display the infiltration of (A) Ly6G(+) neutrophils and (B) F4/80(+) macrophages. c-MetΔhepa mice show a significantly higher number of either cell population compared with c-MetloxP/loxP animals (a quantitative analysis is provided under Figure 3D and E).
Gastroenterology  , e4DOI: ( /j.gastro )
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10. Supplementary Figure 2
(A) Cytokeratin-19 immunofluorescence staining (Alexa488 green) in livers of bile duct–ligated c-MetΔhepa and control mice before and 3 days after BDL were performed. Representative pictures are shown for each time point. Analyses for c-MetΔhepa and c-MetloxP/loxP controls are shown. (B) Statistical analysis of cytokeratin-19 (ck-19)–positive cells in 28-day bile duct–ligated c-MetloxP/loxP controls and c-MetΔhepa mice are shown (*P < .05).
Gastroenterology  , e4DOI: ( /j.gastro )
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