A New Mdr2−/− Mouse Model of Sclerosing Cholangitis with Rapid Fibrosis Progression, Early-Onset Portal Hypertension, and Liver Cancer
2014; Elsevier BV; Volume: 185; Issue: 2 Linguagem: Inglês
10.1016/j.ajpath.2014.10.013
ISSN1525-2191
AutoresNaoki Ikenaga, Susan B. Liu, Deanna Sverdlov, Shuhei Yoshida, Imad Nasser, Qingen Ke, Peter M. Kang, Yury Popov,
Tópico(s)Liver physiology and pathology
ResumoWe previously characterized the Mdr2(Abcb4)−/− mouse as a reproducible model of chronic biliary liver disease. However, it demonstrates relatively slow fibrosis progression, possibly due to its fibrosis-resistant genetic background. We aimed to improve the model by moving it onto a fibrosis-susceptible background. We generated novel BALB/c.Mdr2−/− mouse via genetic backcross onto highly fibrosis-susceptible BALB/c substrain, identified in inbred mouse strain screening. Liver fibrosis, portal pressure, and hepatic tumor burden in BALB/c.Mdr2−/− mice were studied up to 1 year of age in direct comparison to parental strain FVB.Mdr2−/−. BALB/c.Mdr2−/− mice developed periductular onion-skin type fibrotic lesions and pronounced ductular reaction starting from 4 weeks of age. Compared to parental strain, BALB/c.Mdr2−/− mice demonstrated dramatically accelerated liver fibrosis, with threefold increase in collagen deposition and bridging fibrosis/early signs of cirrhosis at 12 weeks. This was accompanied by early-onset severe portal hypertension and twofold to fourfold increase in profibrogenic transcripts Col1a1 [procollagen α1(I)], Tgfb1, and Timp1. Primary liver cancers in BALB/c.Mdr2−/− developed earlier, with greater tumor burden compared to FVB.Mdr2−/−. BALB/c.Mdr2−/− mice have unprecedented degree and rapidity of hepatic fibrosis progression and clinically relevant cirrhosis complications, such as early-onset portal hypertension and primary liver cancers. This new model will facilitate development of antifibrotic drugs and studies into mechanisms of biliary fibrosis progression. We previously characterized the Mdr2(Abcb4)−/− mouse as a reproducible model of chronic biliary liver disease. However, it demonstrates relatively slow fibrosis progression, possibly due to its fibrosis-resistant genetic background. We aimed to improve the model by moving it onto a fibrosis-susceptible background. We generated novel BALB/c.Mdr2−/− mouse via genetic backcross onto highly fibrosis-susceptible BALB/c substrain, identified in inbred mouse strain screening. Liver fibrosis, portal pressure, and hepatic tumor burden in BALB/c.Mdr2−/− mice were studied up to 1 year of age in direct comparison to parental strain FVB.Mdr2−/−. BALB/c.Mdr2−/− mice developed periductular onion-skin type fibrotic lesions and pronounced ductular reaction starting from 4 weeks of age. Compared to parental strain, BALB/c.Mdr2−/− mice demonstrated dramatically accelerated liver fibrosis, with threefold increase in collagen deposition and bridging fibrosis/early signs of cirrhosis at 12 weeks. This was accompanied by early-onset severe portal hypertension and twofold to fourfold increase in profibrogenic transcripts Col1a1 [procollagen α1(I)], Tgfb1, and Timp1. Primary liver cancers in BALB/c.Mdr2−/− developed earlier, with greater tumor burden compared to FVB.Mdr2−/−. BALB/c.Mdr2−/− mice have unprecedented degree and rapidity of hepatic fibrosis progression and clinically relevant cirrhosis complications, such as early-onset portal hypertension and primary liver cancers. This new model will facilitate development of antifibrotic drugs and studies into mechanisms of biliary fibrosis progression. Liver fibrosis, characterized by excessive deposition of extracellular matrix, results from chronic liver injury of different etiologies and represents a major worldwide health problem.1Bataller R. Brenner D.A. Liver fibrosis.J Clin Invest. 2005; 115: 209-218Crossref PubMed Scopus (4142) Google Scholar, 2Friedman S.L. Mechanisms of hepatic fibrogenesis.Gastroenterology. 2008; 134: 1655-1669Abstract Full Text Full Text PDF PubMed Scopus (2185) Google Scholar Progression of liver fibrosis to cirrhosis leads to complications, including portal hypertension, liver failure, and hepatocellular carcinoma, with high mortality.3Schuppan D. Afdhal N.H. Liver cirrhosis.Lancet. 2008; 371: 838-851Abstract Full Text Full Text PDF PubMed Scopus (1524) Google Scholar Primary sclerosing cholangitis (PSC) is an idiopathic, progressive cholestatic liver disease with a male bias and has characteristics of chronic inflammation and fibrosis of the intrahepatic and extrahepatic bile ducts, leading to biliary cirrhosis and cancer.4Talwalkar J.A. Lindor K.D. Primary sclerosing cholangitis.Inflamm Bowel Dis. 2005; 11: 62-72Crossref PubMed Scopus (123) Google Scholar The etiology of PSC is unknown, and no effective therapy exists to halt or reverse progression to cirrhosis outside of liver transplantation. Thus, safe and effective antifibrotic therapies are urgently needed.5Friedman S.L. Sheppard D. Duffield J.S. Violette S. Therapy for fibrotic diseases: nearing the starting line.Sci Transl Med. 2013; 5: 167sr161Crossref Scopus (487) Google Scholar Robust and reproducible animal models closely resembling the human disease are critical for target discovery and novel drug development. Mouse models are popular because of their small size, short gestation cycle, and straightforward genetic engineering. For antifibrotic drug development, mouse models are advantageous because they allow for cost-effective preclinical studies and permit thorough target validation using genetic manipulation.6Patsenker E. Popov Y. Stickel F. Schneider V. Ledermann M. Sagesser H. Niedobitek G. Goodman S.L. Schuppan D. Pharmacological inhibition of integrin alphavbeta3 aggravates experimental liver fibrosis and suppresses hepatic angiogenesis.Hepatology. 2009; 50: 1501-1511Crossref PubMed Scopus (138) Google Scholar However, mice as a species are relatively resistant to the development of progressive liver fibrosis and cirrhosis.7Bissell D.M. Therapy for hepatic fibrosis: revisiting the preclinical models.Clin Res Hepatol Gastroenterol. 2011; 35: 521-525Crossref PubMed Scopus (12) Google Scholar In addition, commonly used inbred mouse strains demonstrate varying susceptibility to hepatotoxin-induced fibrosis, ranging from the resistant FVB to the susceptible BALB/c strain.8Hillebrandt S. Goos C. Matern S. Lammert F. 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Strain-specific differences in mouse hepatic wound healing are mediated by divergent T helper cytokine responses.Proc Natl Acad Sci U S A. 1997; 94: 10663-10668Crossref PubMed Scopus (284) Google Scholar Mdr2(Abcb4) is a mouse ortholog of human MDR3 (ABCB4) gene encoding for the canalicular phospholipid transporter.11Trauner M. Fickert P. Wagner M. MDR3 (ABCB4) defects: a paradigm for the genetics of adult cholestatic syndromes.Semin Liver Dis. 2007; 27: 77-98Crossref PubMed Scopus (190) Google Scholar Genetic disruption of the Mdr2 gene in mice causes a complete absence of phosphatidylcholine from bile,12Smit J.J.M. Schinkel A.H. Oude Elferink R.P.J. Groen A.K. Wagenaar E. van Deemter L. Mol C.A.A.M. Ottenhoff R. van der Lugt N.M.T. van Roon M.A. van der Valk M.A. Offerhaus G.J.A. Berns A.J.M. Borst P. Homozygous disruption of the murine MDR2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease.Cell. 1993; 75: 451-462Abstract Full Text PDF PubMed Scopus (1332) Google Scholar leading to liver injury, sclerosing cholangitis,13Fickert P. Fuchsbichler A. Wagner M. Zollner G. Kaser A. Tilg H. Krause R. Lammert F. Langner C. Zatloukal K. Marschall H.U. Denk H. Trauner M. Regurgitation of bile acids from leaky bile ducts causes sclerosing cholangitis in Mdr2 (Abcb4) knockout mice.Gastroenterology. 2004; 127: 261-274Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar and cholelithiasis.14Lammert F. Wang D.Q. Hillebrandt S. Geier A. Fickert P. Trauner M. Matern S. Paigen B. Carey M.C. Spontaneous cholecysto- and hepatolithiasis in Mdr2-/- mice: a model for low phospholipid-associated cholelithiasis.Hepatology. 2004; 39: 117-128Crossref PubMed Scopus (135) Google Scholar We and others have previously reported the Mdr2(Abcb4)−/− mouse, on an FVB genetic background (FVB.Mdr2−/− mouse), as a reproducible model of spontaneously progressive chronic biliary liver disease associated with fibrosis, with histological lesions closely resembling human PSC.15Mauad T.H. van Nieuwkerk C.M.J. Dingemans K.P. Smit J.J.M. Schinkel A.H. Notenboom R.G.E. van den Bergh Weerman M.A. Verkruisen R.P. Groen A.K. Oude Elferink R.P.J. van der Valk M.A. Borst P. Offerhaus G.J.A. Mice with homozygous disruption of the mdr2 P-glycoprotein gene: a novel animal model for studies of nonsuppurative inflammatory cholangitis and hepatocarcinogenesis.Am J Pathol. 1994; 145: 1237-1245PubMed Google Scholar, 16Fickert P. Zollner G. Fuchsbichler A. Stumptner C. Weiglein A.H. Lammert F. Marschall H.U. Tsybrovskyy O. Zatloukal K. Denk H. Trauner M. Ursodeoxycholic acid aggravates bile infarcts in bile duct-ligated and Mdr2 knockout mice via disruption of cholangioles.Gastroenterology. 2002; 123: 1238-1251Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 17Popov Y. Patsenker E. Fickert P. Trauner M. Schuppan D. Mdr2 (Abcb4)-/- mice spontaneously develop severe biliary fibrosis via massive dysregulation of pro- and antifibrogenic genes.J Hepatol. 2005; 43: 1045-1054Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar More important, mutations in human ortholog gene MDR3 are associated with a spectrum of liver disorders, including progressive familial intrahepatic cholestasis type 3, low phospholipid–associated cholelithiasis, parenteral nutrition-induced cholestasis, sepsis-associated cholestasis, and bile duct injury after liver transplantation.11Trauner M. Fickert P. Wagner M. MDR3 (ABCB4) defects: a paradigm for the genetics of adult cholestatic syndromes.Semin Liver Dis. 2007; 27: 77-98Crossref PubMed Scopus (190) Google Scholar, 18Gonzales E. Davit-Spraul A. Baussan C. Buffet C. Maurice M. Jacquemin E. Liver diseases related to MDR3 (ABCB4) gene deficiency.Front Biosci (Landmark Ed). 2009; 14: 4242-4256Crossref PubMed Scopus (53) Google Scholar Emerging evidence suggests heterozygous MDR3 mutations are frequent in people with cryptogenic biliary cirrhosis.19Ziol M. Barbu V. Rosmorduc O. Frassati-Biaggi A. Barget N. Hermelin B. Scheffer G.L. Bennouna S. Trinchet J.C. Beaugrand M. Ganne-Carrie N. ABCB4 heterozygous gene mutations associated with fibrosing cholestatic liver disease in adults.Gastroenterology. 2008; 135: 131-141Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar However, compared to severe disease caused by homozygous MDR3 mutations in humans,18Gonzales E. Davit-Spraul A. Baussan C. Buffet C. Maurice M. Jacquemin E. Liver diseases related to MDR3 (ABCB4) gene deficiency.Front Biosci (Landmark Ed). 2009; 14: 4242-4256Crossref PubMed Scopus (53) Google Scholar liver disease in FVB.Mdr2−/− mice is relatively mild and fibrosis progression is slow.20Van Nieuwkerk C.M. Elferink R.P. Groen A.K. Ottenhoff R. Tytgat G.N. Dingemans K.P. Van Den Bergh Weerman M.A. Offerhaus G.J. Effects of Ursodeoxycholate and cholate feeding on liver disease in FVB mice with a disrupted mdr2 P-glycoprotein gene.Gastroenterology. 1996; 111: 165-171Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar We hypothesized that slow fibrosis progression in FVB.Mdr2−/− mouse is due to fibrosis resistance of its FVB genetic background. In this study, we generated and characterized a congenic Mdr2(Abcb4)−/− mouse on fibrosis-susceptible BALB/c background (BALB/c.Mdr2−/− mouse) via genetic backcross, aiming to improve the FVB.Mdr2−/− mouse model for antifibrotic drug testing. We present detailed phenotypic characterization of disease progression in BALB/c.Mdr2−/− mice in direct comparison with the parental strain for up to 1 year of age. The 5- to 6-week-old wild-type (WT) inbred mouse strains FVB/NJ (number 001800), C57BL/6J (number 000664), and BALB/cJ (number 000651) were purchased from The Jackson Laboratory (Bar Harbor, ME), and BALB/cAnNCrl (number 028) mice were from Charles River Laboratories (Wilmington, MA). All mice were housed with a 12-hour light-dark cycle and permitted ad libitum consumption of water and a standard chow diet. All animal experiments and procedures were reviewed and approved by the Institutional Animal Care and Use Committee at Beth Israel Deaconess Medical Center, Harvard Medical School (Boston, MA) (protocol number 004-2012). Liver fibrosis susceptibility of several inbred mouse strains was analyzed in a repeated liver injury model induced by i.p. thioacetamide (TAA) injections three times per week for 6 weeks, according to the dose-escalating regimen we established previously.21Popov Y. Sverdlov D.Y. Sharma A.K. Bhaskar K.R. Li S. Freitag T.L. Lee J. Dieterich W. Melino G. Schuppan D. Tissue transglutaminase does not affect fibrotic matrix stability or regression of liver fibrosis in mice.Gastroenterology. 2011; 140: 1642-1652Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar BALB/c.Mdr2−/− mice were generated by transferring the Mdr2 gene knockout from FVB.Mdr2−/− mice (FVB.129P2-Abcb4tm1Bor; The Jackson Laboratory). Several commonly used inbred strains were analyzed for susceptibility to TAA-induced liver fibrosis, and the most susceptible substrain, BALB/cAnNCrl, was selected for conventional backcrossing. Briefly, FVB.Mdr2−/− mice were mated to WT BALB/c mice, with heterozygous carriers of Mdr2 mutation in each generation mated to WT BALB/c for 12 generations. The Y chromosome was fixed via breeding female FVB.Mdr2−/− carrier to male WT BALB/c in the first breeding cycle; male Mdr2 mutation carriers were bred to WT BALB/c females for the subsequent 11 generations. Mutation carriers in N12 progeny were intercrossed to obtain mice homozygous for Mdr2 mutation and establish the BALB/c.Mdr2−/− colony. Liver tumors in 7- and 12-month-old mice were evaluated macroscopically and microscopically. Hepatic collagen content was determined as relative hydroxyproline (μg per 100 mg of wet liver) in 300- to 400-mg liver samples from two different lobes (representing >10% of whole liver) after hydrolysis in 6N HCl for 16 hours at 110°C, as described.17Popov Y. Patsenker E. Fickert P. Trauner M. Schuppan D. Mdr2 (Abcb4)-/- mice spontaneously develop severe biliary fibrosis via massive dysregulation of pro- and antifibrogenic genes.J Hepatol. 2005; 43: 1045-1054Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar Total hydroxyproline (mg/whole liver) was calculated on the basis of individual liver weights and the corresponding relative hydroxyproline content.22Popov Y. Sverdlov D.Y. Bhaskar K.R. Sharma A.K. Millonig G. Patsenker E. Krahenbuhl S. Krahenbuhl L. Schuppan D. Macrophage-mediated phagocytosis of apoptotic cholangiocytes contributes to reversal of experimental biliary fibrosis.Am J Physiol Gastrointest Liver Physiol. 2010; 298: G323-G334Crossref PubMed Scopus (107) Google Scholar Extracellular membrane stability was assessed biochemically ex vivo by complete fibrotic matrix fractionation via serial extractions, and solubilized collagen quantification in each fraction was performed as previously described,21Popov Y. Sverdlov D.Y. Sharma A.K. Bhaskar K.R. Li S. Freitag T.L. Lee J. Dieterich W. Melino G. Schuppan D. Tissue transglutaminase does not affect fibrotic matrix stability or regression of liver fibrosis in mice.Gastroenterology. 2011; 140: 1642-1652Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar with modifications. Briefly, 500 mg of snap-frozen tissue from two liver lobes was homogenized, and a series of overnight extractions (dilution 1:20, w/v) in increasingly harsh conditions was performed to obtain the following collagen fractions: acetic acid–soluble fraction (soluble, freshly secreted collagens and mature collagens), pepsin-soluble fraction (fibrillar and moderately cross-linked collagens), and insoluble, highly cross-linked collagens. Collagen content solubilized in each step was quantified biochemically via hydroxyproline after complete acidic hydrolysis and expressed as percentage of hydroxyproline recovered in all fractions. Immunohistochemical (IHC), hematoxylin and eosin, connective tissue (Sirius Red), and reticulin stains were performed in formalin-fixed, paraffin-embedded liver sections of snap-frozen liver pieces, as described previously.22Popov Y. Sverdlov D.Y. Bhaskar K.R. Sharma A.K. Millonig G. Patsenker E. Krahenbuhl S. Krahenbuhl L. Schuppan D. Macrophage-mediated phagocytosis of apoptotic cholangiocytes contributes to reversal of experimental biliary fibrosis.Am J Physiol Gastrointest Liver Physiol. 2010; 298: G323-G334Crossref PubMed Scopus (107) Google Scholar, 23Popov Y. Patsenker E. Stickel F. Zaks J. Bhaskar K.R. Niedobitek G. Kolb A. Friess H. Schuppan D. Integrin alphavbeta6 is a marker of the progression of biliary and portal liver fibrosis and a novel target for antifibrotic therapies.J Hepatol. 2008; 48: 453-464Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 24Ferrell L.D. Crawford J.M. Dhillon A.P. Scheuer P.J. Nakanuma Y. Proposal for standardized criteria for the diagnosis of benign, borderline, and malignant hepatocellular lesions arising in chronic advanced liver disease.Am J Surg Pathol. 1993; 17: 1113-1123Crossref PubMed Scopus (153) Google Scholar Anti–α-smooth muscle actin (α-SMA; ab5694; dilution 1:400; Abcam, Cambridge, MA) and anti–pan-cytokeratin (Z0622; dilution 1:50; Dako, Glostrup, Denmark) antibodies were used as primary antibodies for IHC. Images were captured using Zeiss Axioimager M1 (Carl Zeiss, Oberkochen, Germany). For Western blot analysis, 15 μg of tissue lysate per lane was probed with anti–α-SMA antibody (M0851; dilution 1:1000; Dako) or anti-β actin (number 4970; dilution 1:2000; Cell Signaling Technology, Danvers, MA), as described.22Popov Y. Sverdlov D.Y. Bhaskar K.R. Sharma A.K. Millonig G. Patsenker E. Krahenbuhl S. Krahenbuhl L. Schuppan D. Macrophage-mediated phagocytosis of apoptotic cholangiocytes contributes to reversal of experimental biliary fibrosis.Am J Physiol Gastrointest Liver Physiol. 2010; 298: G323-G334Crossref PubMed Scopus (107) Google Scholar Relative mRNA levels were quantified in total liver RNA by real-time quantitative RT-PCR on a LightCycler 1.5 instrument (Roche, Mannheim, Germany) using the TaqMan method, as described in detail.17Popov Y. Patsenker E. Fickert P. Trauner M. Schuppan D. Mdr2 (Abcb4)-/- mice spontaneously develop severe biliary fibrosis via massive dysregulation of pro- and antifibrogenic genes.J Hepatol. 2005; 43: 1045-1054Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 21Popov Y. Sverdlov D.Y. Sharma A.K. Bhaskar K.R. Li S. Freitag T.L. Lee J. Dieterich W. Melino G. Schuppan D. Tissue transglutaminase does not affect fibrotic matrix stability or regression of liver fibrosis in mice.Gastroenterology. 2011; 140: 1642-1652Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 23Popov Y. Patsenker E. Stickel F. Zaks J. Bhaskar K.R. Niedobitek G. Kolb A. Friess H. Schuppan D. Integrin alphavbeta6 is a marker of the progression of biliary and portal liver fibrosis and a novel target for antifibrotic therapies.J Hepatol. 2008; 48: 453-464Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar Sequences of primers and probes used in this study are summarized in Table 1.Table 1Primers and Probes Used in Real-Time Quantitative RT-PCRTarget description (gene)PrimerSequenceβ2MG (B2m)Forward5′-CTGATACATACGCCTGCAGAGTTAA-3′Probe5′-GACCGTCTACTGGGATCGAGACATGTG-3′Reverse5′-ATGAATCTTCAGAGCATCATGAT-3′Procollagen α1(I) (Col1a1)Forward5′-TCCGGCTCCTGCTCCTCTTA-3′Probe5′-TTCTTGGCCATGCGTCAGGAGGG-3′Reverse5′-GTATGCAGCTGACTTCAGGGATGT-3′TGF-β1 (Tgfb1)Forward5′-AGAGGTCACCCGCGTGCTAA-3′Probe5′-ACCGCAACAACGCCATCTATGAGAAAACCA-3′Reverse5′-TCCCGAATGTCTGACGTATTGA-3′TGF-β2 (Tgfb2)Forward5′-GTCCAGCCGGCGGAA-3′Probe5′-CGCTTTGGATGCTGCCTACTGCTTTAGAAAT-3′Reverse5′-GCGAAGGCAGCAATTATCCT-3′Integrin β6 (Itgb6)Forward5′-GCAGAACGCTCTAAGGCCAA-3′Probe5′-TGGCAAACGGGAACCAATCCTCTGT-3′Reverse5′-AAAGTGCTGGTGGAACCTCG-3′Mmp-2 (Mmp2)Forward5′-CCGAGGACTATGACCGGGATAA-3′Probe5′-TCTGCCCCGAGACCGCTATGTCCA-3′Reverse5′-CTTGTTGCCCAGGAAAGTGAAG-3′Mmp-3 (Mmp3)Forward5′-GATGAACGATGGACAGAGGATG-3′Probe5′-TGGTACCAACCTATTCCTGGTTGCTGC-3′Reverse5′-AGGGAGTGGCCAAGTTCATG-3′Mmp-13 (Mmp13)Forward5′-GGAAGACCCTCTTCTTCTCT-3′Probe5′-TCTGGTTAACATCATCATAACTCCACACGT-3′Reverse5′-TCATAGACAGCATCTACTTTGTT-3′Timp-1 (Timp1)Forward5′-TCCTCTTGTTGCTATCACTGATAGCTT-3′Probe5′-TTCTGCAACTCGGACCTGGTCATAAGG-3′Reverse5′-CGCTGGTATAAGGTGGTCTCGTT-3′MG, microglobulin; MMP, matrix metalloproteinase; TGF, transforming growth factor; TIMP, tissue inhibitor of metalloproteinases. Open table in a new tab MG, microglobulin; MMP, matrix metalloproteinase; TGF, transforming growth factor; TIMP, tissue inhibitor of metalloproteinases. Mice were anesthetized with isoflurane (1.5% v/v) via precise vaporizer. After laparotomy, the portal vein was cannulated through an iliocolic vein, and portal pressure was measured directly by inserting a 1.2-Fr high-fidelity pressure catheter (Scisense, London, ON, Canada). Pressure signals were recorded at 2 kHz for 5 minutes, and analyzed using PowerLab software chart 5.5.6 (ADInstruments, Colorado Springs, CO). Serum levels of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase (ALP), and total bilirubin were measured using an automated Catalyst Dx Chemistry Analyzer (IDEXX Laboratories, Inc., Westbrook, ME), according to the manufacturer's recommendations. Data are expressed as means ± SEM, and statistical analyses were performed using Microsoft Excel (Microsoft, Redmond, WA) and Graph-Pad Prism 5 (GraphPad Software, San Diego, CA). Multiple comparisons were performed by two-way analysis of variance, followed by Bonferroni post test, and comparisons between two groups were performed by the Student's t-test. Differences between experimental groups with P < 0.05 were considered significant. We evaluated susceptibility to liver fibrosis induced by repeated TAA injections for 6 weeks in four commonly used strains of inbred mice [FVB/NJ, C57BL/6J, and two BALB/c substrains (BALB/cJ and BALB/cAnNCrl)]. Connective tissue staining revealed significant differences in fibrosis degree (FVB/NJ < C57BL/6J < BALB/cJ < BALB/cAnNCrl), ranging from absence of significant fibrosis in FVB/NJ mice to advanced, bridging fibrosis in BALB/cAnNCrl (Figure 1A). Liver collagen content, as determined biochemically via hydroxyproline, increased significantly in all strains, but to a dramatically different extent: relative hydroxyproline (per 100 mg liver) increased by 24% (FVB/NJ), 85% (C57BL/6J), 143% (BALB/cJ), and 205% (BALB/cAnNCrl) compared to their healthy strain-matched controls. Differences were even more significant for total collagen deposition (per whole liver), which was 1.4-fold in FVB/NJ, 1.95-fold in C57BL/6J, 2.52-fold in BALB/cJ, and 3.67-fold in BALB/cAnNCrl (Figure 1B and Supplemental Table S1). Interestingly, BALB/c substrains demonstrated considerable differences in fibrosis, with almost twofold greater absolute amount of collagen deposited in BALB/cAnNCrl compared to BALB/cJ. Splenomegaly developed only in BALB/cAnNCrl mice among all strains tested (Supplemental Table S1). On the basis of these results, BALB/cAnNCrl substrain was selected for Mdr2−/− mutation transfer from the fibrosis-resistant FVB background. Congenic Mdr2−/− mouse on BALB/cAnNCrl genetic background (BALB/c.Mdr2−/− from here forward) was generated using conventional backcross onto BALB/cAnNCrl from parental FVB.129P2-Abcb4tm1Bor (FVB.Mdr2−/− from here forward) (The Jackson Laboratory) for 12 generations, as described in detail in Materials and Methods. Homozygous BALB/c.Mdr2−/− mice were viable and fertile, with no gross abnormalities or significant mortality compared to parental FVB.Mdr2−/− strain when observed up to 1 year of age. Homozygous mating of both FVB.Mdr2−/− and BALB/c.Mdr2−/− was used to generate mice for phenotype analysis. We performed direct phenotypic comparison of liver fibrosis development in novel BALB/c.Mdr2−/− mice and parental FVB.Mdr2−/− mice at early time points of 4, 8, and 12 weeks of age. Both strains spontaneously developed periductal onion-skin type fibrotic lesions starting from 4 weeks of age (Figure 2, A and B). When compared to FVB.Mdr2−/− mice, fibrosis in BALB/c.Mdr2−/− mice was dramatically accelerated, with histological signs of bridging fibrosis already at 8 weeks and thick septae formation by 12 weeks (Figure 2A). Quantitatively, collagen content in the liver of male BALB/c.Mdr2−/− mice was significantly higher at any time point than that of FVB.Mdr2−/− mice: by 2.2-fold at week 4, 3.1-fold at week 8, and 3.3-fold at week 12 (Figure 2C and Supplemental Table S2). Net collagen deposition from week 4 through 12, calculated from an absolute increase in total hydroxyproline content (per whole liver), was also approximately threefold greater in BALB/c.Mdr2−/− mice, 1.435 mg compared to 0.535 mg of hydroxyproline in FVB.Mdr2−/− mice (Figure 2C and Supplemental Table S3). We evaluated fibrotic matrix stability in both strains at 8 weeks of age to assess the extent of collagen cross-linking using stepwise collagen extractions.21Popov Y. Sverdlov D.Y. Sharma A.K. Bhaskar K.R. Li S. Freitag T.L. Lee J. Dieterich W. Melino G. Schuppan D. Tissue transglutaminase does not affect fibrotic matrix stability or regression of liver fibrosis in mice.Gastroenterology. 2011; 140: 1642-1652Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar In FVB.Mdr2−/− mice, highly cross-linked (insoluble) collagen fraction represented 19.9% ± 1.4% of total, a 73% increase compared to age-matched WT FVB mice (11.5% ± 0.6%) (P < 0.01). In BALB/c.Mdr2−/− mice, insoluble collagens represented 42.2% ± 1.8% fraction in BALB/c.Mdr2−/− mice, a 210% increase compared to healthy WT controls (13.6% ± 0.8%) (P < 0.001), which was significantly greater than in parental strain (P < 0.001) (Figure 2D). This suggests that fibrotic matrix in BALB/c.Mdr2−/− mice is cross-linked to a greater degree than in FVB.Mdr2−/− mice. Rapid fibrosis progression in BALB/c.Mdr2−/− mice cannot be explained by greater inflammation or liver injury, because both strains demonstrated comparable increases in serum alanine aminotransferase and aspartate aminotransferase (Supplemental Figure S1). Interestingly, although FVB.Mdr2−/− mice demonstrated more severe fibrosis in females, as expected,25van Nieuwerk C.M. Groen A.K. Ottenhoff R. van Wijland M. van den Bergh Weerman M.A. Tytgat G.N. Offerhaus J.J. Oude Elferink R.P. The role of bile salt composition in liver pathology of mdr2 (-/-) mice: differences between males and females.J Hepatol. 1997; 26: 138-145Abstract Full Text PDF PubMed Scopus (68) Google Scholar significant male bias was found in BALB/c.Mdr2−/− mice, with significantly higher hepatic collagen content in males starting at 8 weeks of age (P < 0.01) (Supplemental Table S2). To investigate the relative contribution of increased fibrogenesis versus inhibited fibrolysis in the rapid fibrosis progression of BALB/c.Mdr2−/− mice, the hepatic expression levels of several profibrogenic genes and putatively profibrolytic matrix metalloproteinases (MMP) in males of both strains were analyzed by real-time quantitative RT-PCR. Aggressive fibrosis in BALB/c.Mdr2−/− mice was associated with robust increases in profibrogenic transcripts, such as procollagen α1(I) and (cholangiocyte-derived26Milani S. Herbst H. Schuppan D. Stein H. Surrenti C. Transforming growth factors beta 1 and beta 2 are differentially expressed in fibrotic liver disease.Am J Pathol. 1991; 139: 1221-1229PubMed Google Scholar) transforming growth factor (Tgf)-β2, twofold to fivefold above parental strain levels17Popov Y. Patsenker E. Fickert P. Trauner M. Schuppan D. Mdr2 (Abcb4)-/- mice spontaneously develop severe biliary fibrosis via massive dysregulation of pro- and antifibrogenic genes.J Hepatol. 2005; 43: 1045-1054Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 23Popov Y. Patsenker E. Stickel F. Zaks J. Bhaskar K.R. Niedobitek G. Kolb A. Friess H. Schuppan D. Integrin alphavbeta6 is a marker of the progression of biliary and portal liver fibrosis and a novel target for antifibrotic therapies.J Hepatol. 2008; 48: 453-464Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar at ages of 8 and 12 weeks (Figure 3A). This did not occur with other profibrogenic genes, such as TGF-β1, which was only transiently elevated at 4 weeks, and integrin β6, which was overexpressed at similar levels in both strains (Figure 3A). Interestingly, profibrolytic Mmp-13 expression was suppressed in BALB/c.Mdr2−/− mice at 8 and 12 weeks compared to parental strain, whereas Mmp-3 levels showed no significant differences (Figure 3B). On the other hand, endogenous MMP inhibitor, tissue inhibitor of metalloproteinases 1, was up-regulated up to fivefold at every time point in BALB/c.Mdr2−/− mice compared to FVB.Mdr2−/− mice (Figure 3A). Biliary liver fibrosis is associated with proliferation of cholangiocytes and adult hepatic progenitors (ductular reaction) and activation of hepatic stellate cells (HSCs)/portal myofibroblasts (MFs). We assessed ductular reactio
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