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Depletion of Apoptosis Signal-Regulating Kinase 1 Prevents Bile Duct Ligation–Induced Necroinflammation and Subsequent Peribiliary Fibrosis

  • Hirotsugu Noguchi
    Affiliations
    Departments of Pathology and Cell Biology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan
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  • Sohsuke Yamada
    Correspondence
    Address correspondence to Sohsuke Yamada, M.D., Ph.D., Department of Pathology and Cell Biology, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan.
    Affiliations
    Departments of Pathology and Cell Biology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan
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  • Atsunori Nabeshima
    Affiliations
    Departments of Pathology and Cell Biology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan
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  • Xin Guo
    Affiliations
    Departments of Pathology and Cell Biology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan
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  • Akihide Tanimoto
    Affiliations
    Department of Molecular and Cellular Pathology, Kagoshima University Graduate School of Medical and Dental Sciences, Kitakyushu, Japan
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  • Ke-Yong Wang
    Affiliations
    Departments of Pathology and Cell Biology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan

    Bio-information Research Center, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan
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  • Shohei Kitada
    Affiliations
    Departments of Pathology and Cell Biology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan

    Department of Urology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan
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  • Takashi Tasaki
    Affiliations
    Departments of Pathology and Cell Biology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan
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  • Tatsuo Takama
    Affiliations
    Departments of Pathology and Cell Biology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan

    Department of Emergency Medicine, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan
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  • Shohei Shimajiri
    Affiliations
    Departments of Pathology and Cell Biology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan
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  • Hasita Horlad
    Affiliations
    Department of Cell Pathology, Faculty of Medical and Pharmaceutical Sciences, Graduate School of Medical Sciences, Kumamoto University, Kitakyushu, Japan
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  • Yoshihiro Komohara
    Affiliations
    Department of Cell Pathology, Faculty of Medical and Pharmaceutical Sciences, Graduate School of Medical Sciences, Kumamoto University, Kitakyushu, Japan
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  • Hiroto Izumi
    Affiliations
    Department of Occupational Pneumology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan
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  • Kimitoshi Kohno
    Affiliations
    Department of Molecular Biology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan
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  • Hidenori Ichijo
    Affiliations
    Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, and Core Research for Evolutional Science and Technology, Tokyo, Japan
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  • Yasuyuki Sasaguri
    Affiliations
    Departments of Pathology and Cell Biology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan
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Open AccessPublished:January 10, 2014DOI:https://doi.org/10.1016/j.ajpath.2013.11.030
      Apoptosis signal-regulating kinase 1 (ASK1), also known as mitogen-activated protein kinase kinase kinase (MAP3K), is ubiquitously expressed and situated in an important upstream position of many signal transduction pathways. ASK1 plays a pivotal role in stressor-induced cell survival and inflammatory reactions. To ascertain the regulatory functions of ASK1 in bile duct ligation (BDL)–induced liver injury, we examined the net effects of ASK1 depletion on hepatic necroinflammation and/or fibrosis. We subjected C57BL/6 wild-type (WT) or ASK1-deficient (ASK1−/−) mice to sham or BDL surgery for 14 days. In day 3 BDL animals, ASK1−/− mice had significantly fewer bile infarcts along with more reduced interlobular or portal inflammatory infiltrate of various immune cells, including neutrophils, compared with WT mice in which ASK1 expression was markedly activated. Morphologically apoptotic hepatocytes or cholangiocytes were negligible in both the sham and BDL animals. In contrast, ASK1−/− mice had significantly less proliferating activity of not only hepatocytes but also large cholangiocytes than WT mice. Day 14 BDL ASK1−/− mice manifested potential antifibrogenic aspects of ASK1 deficiency, characterized by significantly fewer activated peribiliary fibrogenic cells and peribiliary fibrosis. These observations indicate that ASK1-mediated hepatic necroinflammation and proliferation, but not apoptosis, are closely linked to liver fibrosis and fibrogenesis. A specific ASK1 pathway blocker or inhibitor might offer a therapeutic strategy against human cholestatic diseases.
      It was previously proposed that hepatocytes undergoing apoptosis might provide a critical hit to drive progression from chronic inflammation to cirrhosis, especially in cholestatic injury, such as primary biliary cirrhosis, primary sclerosing cholangitis, biliary atresia, or chronic cholelithiasis, with reference to a single laboratory.
      • Miyoshi H.
      • Rust C.
      • Roberts P.J.
      • Burgart L.J.
      • Gores G.J.
      Hepatocyte apoptosis after bile duct ligation in the mouse involves Fas.
      • Canbay A.
      • Higuchi H.
      • Bronk S.F.
      • Taniai M.
      • Sebo T.J.
      • Gores G.J.
      Fas enhances fibrogenesis in the bile duct ligated mouse: a link between apoptosis and fibrosis.
      • Yoon J.H.
      • Gores G.J.
      Death receptor-mediated apoptosis and the liver.
      • Canbay A.
      • Taimr P.
      • Torok N.
      • Higuchi H.
      • Friedman S.
      • Gores G.J.
      Apoptotic body engulfment by a human stellate cell line is profibrogenic.
      In striking contrast, several other groups
      • Schoemaker M.H.
      • Gommans W.M.
      • Conde de la Rosa L.
      • Homan M.
      • Klok P.
      • Trautwein C.
      • van Goor H.
      • Poelstra K.
      • Haisma H.J.
      • Jansen P.L.
      • Moshage H.
      Resistance of rat hepatocytes against bile acid-induced apoptosis in cholestatic liver injury is due to nuclear factor-kappa B activation.
      • Gujral J.S.
      • Liu J.
      • Farhood A.
      • Jaeschke H.
      Reduced oncotic necrosis in Fas receptor-deficient C57BL/6J-lpr mice after bile duct ligation.
      • Fickert P.
      • Trauner M.
      • Fuchsbichler A.
      • Zollner G.
      • Wagner M.
      • Marschall H.U.
      • Zatloukal K.
      • Denk H.
      Oncosis represents the main type of cell death in mouse models of cholestasis.
      • Nalapareddy P.D.
      • Schüngel S.
      • Hong J.Y.
      • Manns M.P.
      • Jaeschke H.
      • Vogel A.
      The BH3-only protein bid does not mediate death-receptor-induced liver injury in obstructive cholestasis.
      • Mitchell C.
      • Mahrouf-Yorgov M.
      • Mayeuf A.
      • Robin M.A.
      • Mansouri A.
      • Fromenty B.
      • Gilgenkrantz H.
      Overexpression of Bcl-2 in hepatocytes protects against injury but does not attenuate fibrosis in a mouse model of chronic cholestatic liver disease.
      have recently suggested that there is no evidence of hepatocellular apoptosis using morphologic criteria, but not terminal deoxynucleotidyl transferase end-labeling (TUNEL) staining, in or around necrotic foci (ie, bile infarcts) after bile duct ligation (BDL), which is used to induce cholestasis in rodents.
      • Miyoshi H.
      • Rust C.
      • Roberts P.J.
      • Burgart L.J.
      • Gores G.J.
      Hepatocyte apoptosis after bile duct ligation in the mouse involves Fas.
      • Canbay A.
      • Higuchi H.
      • Bronk S.F.
      • Taniai M.
      • Sebo T.J.
      • Gores G.J.
      Fas enhances fibrogenesis in the bile duct ligated mouse: a link between apoptosis and fibrosis.
      • Yoon J.H.
      • Gores G.J.
      Death receptor-mediated apoptosis and the liver.
      • Schoemaker M.H.
      • Gommans W.M.
      • Conde de la Rosa L.
      • Homan M.
      • Klok P.
      • Trautwein C.
      • van Goor H.
      • Poelstra K.
      • Haisma H.J.
      • Jansen P.L.
      • Moshage H.
      Resistance of rat hepatocytes against bile acid-induced apoptosis in cholestatic liver injury is due to nuclear factor-kappa B activation.
      • Gujral J.S.
      • Liu J.
      • Farhood A.
      • Jaeschke H.
      Reduced oncotic necrosis in Fas receptor-deficient C57BL/6J-lpr mice after bile duct ligation.
      • Fickert P.
      • Trauner M.
      • Fuchsbichler A.
      • Zollner G.
      • Wagner M.
      • Marschall H.U.
      • Zatloukal K.
      • Denk H.
      Oncosis represents the main type of cell death in mouse models of cholestasis.
      • Nalapareddy P.D.
      • Schüngel S.
      • Hong J.Y.
      • Manns M.P.
      • Jaeschke H.
      • Vogel A.
      The BH3-only protein bid does not mediate death-receptor-induced liver injury in obstructive cholestasis.
      • Mitchell C.
      • Mahrouf-Yorgov M.
      • Mayeuf A.
      • Robin M.A.
      • Mansouri A.
      • Fromenty B.
      • Gilgenkrantz H.
      Overexpression of Bcl-2 in hepatocytes protects against injury but does not attenuate fibrosis in a mouse model of chronic cholestatic liver disease.
      They also concluded that BDL-induced oncotic necrosis but not apoptosis of hepatocytes is closely correlated with the severity of the inflammatory response, including high chemokine and/or cytokine expression.
      • Gujral J.S.
      • Liu J.
      • Farhood A.
      • Jaeschke H.
      Reduced oncotic necrosis in Fas receptor-deficient C57BL/6J-lpr mice after bile duct ligation.
      • Nalapareddy P.D.
      • Schüngel S.
      • Hong J.Y.
      • Manns M.P.
      • Jaeschke H.
      • Vogel A.
      The BH3-only protein bid does not mediate death-receptor-induced liver injury in obstructive cholestasis.
      • Mitchell C.
      • Mahrouf-Yorgov M.
      • Mayeuf A.
      • Robin M.A.
      • Mansouri A.
      • Fromenty B.
      • Gilgenkrantz H.
      Overexpression of Bcl-2 in hepatocytes protects against injury but does not attenuate fibrosis in a mouse model of chronic cholestatic liver disease.
      Meanwhile, although cholangiocytes are a minor component of liver cells, comprising merely 3% to 4% of the rodent liver, cells lining the large bile ducts (large cholangiocytes) are the main target in animal models of cholestasis and intractable human cholestatic diseases.
      • Lazaridis K.N.
      • Strazzabosco M.
      • Larusso N.F.
      The cholangiopathies: disorders of biliary epithelia.
      • Yahagi K.
      • Ishii M.
      • Kobayashi K.
      • Ueno Y.
      • Mano Y.
      • Niitsuma H.
      • Igarashi T.
      • Toyota T.
      Primary culture of cholangiocytes from normal mouse liver.
      Previous studies revealed that rodent cholangiocytes are mitotically dormant under basal conditions, whereas large but not small cholangiocytes undergo significant proliferation, leading to subsequent liver fibrosis after BDL injury, even though morphologic or functional heterogeneity is noted in large versus small cholangiocytes.
      • Alpini G.
      • Glaser S.
      • Ueno Y.
      • Pham L.
      • Podila P.V.
      • Caligiuri A.
      • LeSage G.
      • LaRusso N.F.
      Heterogeneity of the proliferative capacity of rat cholangiocytes after bile duct ligation.
      • LeSage G.
      • Glaser S.
      • Marucci L.
      • Benedetti A.
      • Phinizy J.L.
      • Rodgers R.
      • Caligiuri A.
      • Papa E.
      • Tretjak Z.
      • Jezequel A.M.
      • Holcomb L.A.
      • Alpini G.
      Acute carbon tetrachloride feeding induces damage of large but not small cholangiocytes from BDL rat liver.
      • Glaser S.
      • Gaudio E.
      • Rao A.
      • Pierce L.M.
      • Onori P.
      • Franchitto A.
      • Francis H.L.
      • Dostal D.E.
      • Venter J.K.
      • DeMorrow S.
      • Mancinelli R.
      • Carpino G.
      • Alvaro D.
      • Kopriva S.E.
      • Savage J.M.
      • Alpini G.
      Morphological and functional heterogeneity of the mouse intrahepatic biliary epithelium.
      Nevertheless, the regulation of biliary injury and proliferation during cholestasis is multifactorial and complex, and the various roles of large cholangiocytes in the initiation and progression of cholestatic liver diseases are still debatable.
      • Alpini G.
      • Glaser S.
      • Ueno Y.
      • Pham L.
      • Podila P.V.
      • Caligiuri A.
      • LeSage G.
      • LaRusso N.F.
      Heterogeneity of the proliferative capacity of rat cholangiocytes after bile duct ligation.
      • Glaser S.
      • Gaudio E.
      • Rao A.
      • Pierce L.M.
      • Onori P.
      • Franchitto A.
      • Francis H.L.
      • Dostal D.E.
      • Venter J.K.
      • DeMorrow S.
      • Mancinelli R.
      • Carpino G.
      • Alvaro D.
      • Kopriva S.E.
      • Savage J.M.
      • Alpini G.
      Morphological and functional heterogeneity of the mouse intrahepatic biliary epithelium.
      Hence, key factors and signaling pathways that control the responses to injury in not only hepatocytes but also cholangiocytes need to be identified.
      Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase (MAP3K) family member that is activated through distinct mechanisms in response to various cytotoxic stressors, including oxidative stress mediated by hydrogen peroxide, endoplasmic reticulum (ER) stress, and immune system mediators, such as tumor necrosis factor (TNF)-α, IL-1β, or Fas ligands.
      • Ichijo H.
      • Nishida E.
      • Irie K.
      • Digike P.T.
      • Saitoh M.
      • Moriguchi T.
      • Takagi M.
      • Matsumoto K.
      • Miyazono K.
      • Gotoh Y.
      Induction of apoptosis by ASK1, mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways.
      • Tobiume K.
      • Matsuzawa A.
      • Takahashi T.
      • Nishitoh H.
      • Morita K.
      • Takeda K.
      • Minowa O.
      • Miyazono K.
      • Noda T.
      • Ichijo H.
      ASK1 is required for sustained activations of JNK/p38 MAP kinases and apoptosis.
      ASK1 is situated in an important upstream position for many signal transduction pathways, such as the c-Jun N-terminal kinase (JNK) and p38 MAP kinase (MAPK), which subsequently induce inflammation and intrinsic apoptotic signaling through mitochondria-dependent caspase activation.
      • Ichijo H.
      • Nishida E.
      • Irie K.
      • Digike P.T.
      • Saitoh M.
      • Moriguchi T.
      • Takagi M.
      • Matsumoto K.
      • Miyazono K.
      • Gotoh Y.
      Induction of apoptosis by ASK1, mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways.
      • Tobiume K.
      • Matsuzawa A.
      • Takahashi T.
      • Nishitoh H.
      • Morita K.
      • Takeda K.
      • Minowa O.
      • Miyazono K.
      • Noda T.
      • Ichijo H.
      ASK1 is required for sustained activations of JNK/p38 MAP kinases and apoptosis.
      • Yamada S.
      • Ding Y.
      • Tanimoto A.
      • Wang K.Y.
      • Guo X.
      • Li Z.
      • Tasaki T.
      • Nabesima A.
      • Murata Y.
      • Shimajiri S.
      • Kohno K.
      • Ichijo H.
      • Sasaguri Y.
      Apoptosis signal-regulating kinase 1 deficiency accelerates hyperlipidemia-induced atheromatous plaques via suppression of macrophage apoptosis.
      Previous reports using ASK1−/− mice or ASK1 overexpressing transgenic mice confirmed that ASK1 plays a pivotal role in the regulation of cardiomyocyte apoptosis in ischemia-reperfusion injury models.
      • Watanabe T.
      • Otsu K.
      • Takeda T.
      • Yamaguchi O.
      • Hikoso S.
      • Kashiwase K.
      • Higuchi Y.
      • Taniike M.
      • Nakai A.
      • Matsumura Y.
      • Nishida K.
      • Ichijo H.
      • Hori M.
      Apoptosis signal-regulating kinase 1 is involved not only in apoptosis but also in non-apoptotic cardiomyocyte death.
      • Liu Q.
      • Sargent M.A.
      • York A.J.
      • Molkentin J.D.
      ASK1 regulates cardiomyocyte death but not hypertrophy in transgenic mice.
      Moreover, we recently found that activation of ASK1 signaling enhances hyperlipidemia-induced necrotic lipid core formation by inducing macrophage apoptosis and accelerates mechanical injury–induced vascular remodeling via increased neovascularization and proinflammatory reaction and by stimulating apoptosis of smooth muscle cells and/or endothelial cells.
      • Yamada S.
      • Ding Y.
      • Tanimoto A.
      • Wang K.Y.
      • Guo X.
      • Li Z.
      • Tasaki T.
      • Nabesima A.
      • Murata Y.
      • Shimajiri S.
      • Kohno K.
      • Ichijo H.
      • Sasaguri Y.
      Apoptosis signal-regulating kinase 1 deficiency accelerates hyperlipidemia-induced atheromatous plaques via suppression of macrophage apoptosis.
      • Tasaki T.
      • Yamada S.
      • Guo X.
      • Tanimoto A.
      • Wang K.Y.
      • Nabeshima A.
      • Kitada S.
      • Noguchi H.
      • Kimura S.
      • Shimajiri S.
      • Kohno K.
      • Ichijo H.
      • Sasaguri Y.
      Apoptosis signal-regulating kinase 1 deficiency attenuates vascular injury-induced neointimal hyperplasia by suppressing apoptosis in smooth muscle cells.
      ASK1 plays a significant role in the regulation of vascular cell apoptosis and inflammatory signaling in vivo and is correlated with plaque vulnerability in atherosclerosis.
      • Tasaki T.
      • Yamada S.
      • Guo X.
      • Tanimoto A.
      • Wang K.Y.
      • Nabeshima A.
      • Kitada S.
      • Noguchi H.
      • Kimura S.
      • Shimajiri S.
      • Kohno K.
      • Ichijo H.
      • Sasaguri Y.
      Apoptosis signal-regulating kinase 1 deficiency attenuates vascular injury-induced neointimal hyperplasia by suppressing apoptosis in smooth muscle cells.
      Other studies report that ASK1 expression plays a critical role in vivo in the regulation of apoptosis in type 2 pneumocytes in lung injury, with both antiapoptotic and anti-inflammatory properties.
      • Makena P.S.
      • Gorantla V.K.
      • Ghosh M.C.
      • Bezawada L.
      • Kandasamy K.
      • Balazs L.
      • Luellen C.L.
      • Thompson K.E.
      • Parthasarathi K.
      • Ichijo H.
      • Waters C.M.
      • Sinclair S.E.
      Deletion of apoptosis signal-regulating kinase-1 prevents ventilator-induced lung injury in mice.
      However, few studies have investigated the relationship between ASK1 signaling pathway and cholestasis-induced injury, even though ASK1 is ubiquitously expressed.
      In the current study, we examined the roles of ASK1 in BDL-induced cholestatic liver injury using mice genetically deficient for ASK1 (ASK1−/−). Furthermore, we aimed to determine the net effects and key factors of ASK1 in liver necroinflammation or subsequent fibrosis.

      Materials and Methods

       Animals and BDL Model

      Experiments were performed using 6- to 8-week-old, male, C57BL/6 wild-type (WT) and ASK1−/− mice,
      • Yamada S.
      • Ding Y.
      • Tanimoto A.
      • Wang K.Y.
      • Guo X.
      • Li Z.
      • Tasaki T.
      • Nabesima A.
      • Murata Y.
      • Shimajiri S.
      • Kohno K.
      • Ichijo H.
      • Sasaguri Y.
      Apoptosis signal-regulating kinase 1 deficiency accelerates hyperlipidemia-induced atheromatous plaques via suppression of macrophage apoptosis.
      • Tasaki T.
      • Yamada S.
      • Guo X.
      • Tanimoto A.
      • Wang K.Y.
      • Nabeshima A.
      • Kitada S.
      • Noguchi H.
      • Kimura S.
      • Shimajiri S.
      • Kohno K.
      • Ichijo H.
      • Sasaguri Y.
      Apoptosis signal-regulating kinase 1 deficiency attenuates vascular injury-induced neointimal hyperplasia by suppressing apoptosis in smooth muscle cells.
      weighing 18 to 22 g, maintained in a temperature and light-controlled facility with free access to standard rodent chow diet and water. ASK1−/− mice were developed on a C57BL/6 background.
      • Yamada S.
      • Ding Y.
      • Tanimoto A.
      • Wang K.Y.
      • Guo X.
      • Li Z.
      • Tasaki T.
      • Nabesima A.
      • Murata Y.
      • Shimajiri S.
      • Kohno K.
      • Ichijo H.
      • Sasaguri Y.
      Apoptosis signal-regulating kinase 1 deficiency accelerates hyperlipidemia-induced atheromatous plaques via suppression of macrophage apoptosis.
      • Tasaki T.
      • Yamada S.
      • Guo X.
      • Tanimoto A.
      • Wang K.Y.
      • Nabeshima A.
      • Kitada S.
      • Noguchi H.
      • Kimura S.
      • Shimajiri S.
      • Kohno K.
      • Ichijo H.
      • Sasaguri Y.
      Apoptosis signal-regulating kinase 1 deficiency attenuates vascular injury-induced neointimal hyperplasia by suppressing apoptosis in smooth muscle cells.
      To produce a ligation-induced cholestatic liver injury (BDL) model, the peritoneal cavity was opened after a midline upper-abdominal incision, and the common bile duct was double-ligated with sterile surgical 7-0 silk sutures (Alfresa Pharma Corp., Tokyo, Japan) and cut between the ligatures in two groups of mice at 6 to 8 weeks of age under anesthesia [intraperitoneal injection of ketamine (100 mg/kg) (Daiichi Sankyo Co., Tokyo, Japan) and medetamidine (2 mg/kg) (Meiji Yakuhin Co., Tokyo, Japan)], as previously described.
      • Tasaki T.
      • Yamada S.
      • Guo X.
      • Tanimoto A.
      • Wang K.Y.
      • Nabeshima A.
      • Kitada S.
      • Noguchi H.
      • Kimura S.
      • Shimajiri S.
      • Kohno K.
      • Ichijo H.
      • Sasaguri Y.
      Apoptosis signal-regulating kinase 1 deficiency attenuates vascular injury-induced neointimal hyperplasia by suppressing apoptosis in smooth muscle cells.
      Sham-operated mice, as controls, underwent laparotomy with exposure but no ligation of the common bile duct. The fascia and skin of the midline abdominal incision were closed with sterile surgical 6-0 silk sutures (Alfresa Pharma Corp.). After a defined period of BDL or sham operation at 3 or 14 days, animals were euthanized by exsanguination under reanesthetization with an i.p. injection of ketamine-medetamidine, as follows: the peritoneal cavity was reopened, and blood samples were taken from the inferior vena cava, followed by immediate cannulation of the suprahepatic vena cava. In all animals, after blood was flushed out of the liver via the suprahepatic vena cava catheter, livers were excised and cut into small pieces and used for various experiments as described below. The 24-hour urine of day 3 BDL mice was collected using mouse metabolic cages (Sugiyama-Gen Co., Ltd., Tokyo, Japan).
      The Ethics Committee of Animal Care and Experimentation, University of Occupational and Environmental Health (Japan), approved the protocols. They were performed according to the Institutional Guidelines for Animal Experiments and the Law (no. 105) and Notification (no. 6) of the Japanese government. The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.

       Histopathology

      Liver specimens were stained with H&E, Masson's trichrome, or picrosirius red stain, or immunohistochemistry (IHC) preparations in sequential sections, after fixation in 10% neutral buffered formalin for 24 hours.
      • Yamada S.
      • Ding Y.
      • Tanimoto A.
      • Wang K.Y.
      • Guo X.
      • Li Z.
      • Tasaki T.
      • Nabesima A.
      • Murata Y.
      • Shimajiri S.
      • Kohno K.
      • Ichijo H.
      • Sasaguri Y.
      Apoptosis signal-regulating kinase 1 deficiency accelerates hyperlipidemia-induced atheromatous plaques via suppression of macrophage apoptosis.
      • Tasaki T.
      • Yamada S.
      • Guo X.
      • Tanimoto A.
      • Wang K.Y.
      • Nabeshima A.
      • Kitada S.
      • Noguchi H.
      • Kimura S.
      • Shimajiri S.
      • Kohno K.
      • Ichijo H.
      • Sasaguri Y.
      Apoptosis signal-regulating kinase 1 deficiency attenuates vascular injury-induced neointimal hyperplasia by suppressing apoptosis in smooth muscle cells.
      • Yamada S.
      • Wang K.Y.
      • Tanimoto A.
      • Fan J.
      • Shimajiri S.
      • Kitajima S.
      • Morimoto M.
      • Tsutui M.
      • Watanabe T.
      • Yasumoto K.
      • Sasaguri Y.
      Matrix metalloproteinase 12 accelerates the initiation of atherosclerosis and stimulates the progression of fatty streaks to fibrous plaques in transgenic rabbits.
      • Ding Y.
      • Yamada S.
      • Wang K.Y.
      • Shimajiri S.
      • Guo X.
      • Tanimoto A.
      • Murata Y.
      • Kitajima S.
      • Watanabe T.
      • Izumi H.
      • Kohno K.
      • Sasaguri Y.
      Overexpression of peroxiredoxin 4 protects against high-dose streptozotocin-induced diabetes by suppressing oxidative stress and cytokines in transgenic mice.
      • Guo X.
      • Yamada S.
      • Tanimoto A.
      • Ding Y.
      • Wang K.Y.
      • Shimajiri S.
      • Murata Y.
      • Kimura S.
      • Tasaki T.
      • Nabeshima A.
      • Watanabe T.
      • Kohno K.
      • Sasaguri Y.
      Overexpression of peroxiredoxin 4 attenuates atherosclerosis in apolipoprotein E knockout mice.
      • Nabeshima A.
      • Yamada S.
      • Guo X.
      • Tanimoto A.
      • Wang K.Y.
      • Shimajiri S.
      • Kimura S.
      • Tasaki T.
      • Noguchi H.
      • Kitada S.
      • Watanabe T.
      • Fujii J.
      • Kohno K.
      • Sasaguri Y.
      Peroxiredoxin 4 protects against nonalcoholic steatohepatitis and type 2 diabetes in a nongenetic mouse model.
      Analyses were performed in BDL-induced cholestatic livers in all experiments, whereas the sham-operated livers served as controls.
      Livers embedded in paraffin for histologic examination were cut systematically in sequential sections of 4-μm thickness using a sliding microtome (Leica SM2010R; Leica Microsystems, Wetzler, Germany). For histologic analyses of the liver, images of H&E and special stained sections or IHC sections were captured and quantified using NanoZoomer Digital Pathology Virtual Slide Viewer software version 2.0 (Hamamatsu Photonics Corp., Hamamatsu, Japan). H&E-stained liver sections were used to measure the areas of bile infarcts (hepatic necrosis) and calculate the percentage of necrotic area divided by total area. In addition, apoptotic cells were identified in 10 randomly selected fields of interlobular and portal tracts per these H&E sections (original magnification, ×400), using morphologic criteria (cell shrinkage, chromatin condensation and margination, and apoptotic bodies).
      • Schoemaker M.H.
      • Gommans W.M.
      • Conde de la Rosa L.
      • Homan M.
      • Klok P.
      • Trautwein C.
      • van Goor H.
      • Poelstra K.
      • Haisma H.J.
      • Jansen P.L.
      • Moshage H.
      Resistance of rat hepatocytes against bile acid-induced apoptosis in cholestatic liver injury is due to nuclear factor-kappa B activation.
      • Gujral J.S.
      • Liu J.
      • Farhood A.
      • Jaeschke H.
      Reduced oncotic necrosis in Fas receptor-deficient C57BL/6J-lpr mice after bile duct ligation.
      • Fickert P.
      • Trauner M.
      • Fuchsbichler A.
      • Zollner G.
      • Wagner M.
      • Marschall H.U.
      • Zatloukal K.
      • Denk H.
      Oncosis represents the main type of cell death in mouse models of cholestasis.
      • Nalapareddy P.D.
      • Schüngel S.
      • Hong J.Y.
      • Manns M.P.
      • Jaeschke H.
      • Vogel A.
      The BH3-only protein bid does not mediate death-receptor-induced liver injury in obstructive cholestasis.
      • Mitchell C.
      • Mahrouf-Yorgov M.
      • Mayeuf A.
      • Robin M.A.
      • Mansouri A.
      • Fromenty B.
      • Gilgenkrantz H.
      Overexpression of Bcl-2 in hepatocytes protects against injury but does not attenuate fibrosis in a mouse model of chronic cholestatic liver disease.
      Liver fibrosis was quantified by Masson's trichrome and picrosirius red staining (Picrosirius Red Stain Kit; Polysciences, Inc., Warrington, PA) in 10 randomly selected fields of portal and periportal tracts per section (original magnification, ×400). In periportal tracts, fibers stained red with yellow birefringence under polarized light were defined as expressing collagen type I.
      • Nabeshima A.
      • Yamada S.
      • Guo X.
      • Tanimoto A.
      • Wang K.Y.
      • Shimajiri S.
      • Kimura S.
      • Tasaki T.
      • Noguchi H.
      • Kitada S.
      • Watanabe T.
      • Fujii J.
      • Kohno K.
      • Sasaguri Y.
      Peroxiredoxin 4 protects against nonalcoholic steatohepatitis and type 2 diabetes in a nongenetic mouse model.
      The proportions of collagen content were calculated for individual images in cholestatic BDL livers, excluding large bile ducts (>15-μm diameter) and vessels.

       Analyses of Hepatic Cholestasis (BDL)–Induced Injury

      Serum levels of total bilirubin and hepatic injury–related enzymes, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase, and total bile acids, were measured using commercial assay kits (Wako Pure Chemical Co., Osaka, Japan). To examine the hepatic bile acids, snap frozen liver tissue (30 mg) was homogenized and extracted with chloroform-methanol (2/1 v/v) solution. The organic phase was dried and resolubilized in 2-propanol. Then the hepatic contents of bile acids were determined using commercial assay kits (Wako Pure Chemical Co.). Urinary bilirubin concentrations were normalized with creatinine levels.

       IHC and Double-Immunofluorescence Staining

      One representative sequential section per mouse was prepared for IHC staining and was captured and evaluated by a NanoZoomer Digital Pathology Virtual Slide Viewer (Hamamatsu Photonics Corp.) to avoid potential bias.
      • Yamada S.
      • Ding Y.
      • Tanimoto A.
      • Wang K.Y.
      • Guo X.
      • Li Z.
      • Tasaki T.
      • Nabesima A.
      • Murata Y.
      • Shimajiri S.
      • Kohno K.
      • Ichijo H.
      • Sasaguri Y.
      Apoptosis signal-regulating kinase 1 deficiency accelerates hyperlipidemia-induced atheromatous plaques via suppression of macrophage apoptosis.
      • Tasaki T.
      • Yamada S.
      • Guo X.
      • Tanimoto A.
      • Wang K.Y.
      • Nabeshima A.
      • Kitada S.
      • Noguchi H.
      • Kimura S.
      • Shimajiri S.
      • Kohno K.
      • Ichijo H.
      • Sasaguri Y.
      Apoptosis signal-regulating kinase 1 deficiency attenuates vascular injury-induced neointimal hyperplasia by suppressing apoptosis in smooth muscle cells.
      • Ding Y.
      • Yamada S.
      • Wang K.Y.
      • Shimajiri S.
      • Guo X.
      • Tanimoto A.
      • Murata Y.
      • Kitajima S.
      • Watanabe T.
      • Izumi H.
      • Kohno K.
      • Sasaguri Y.
      Overexpression of peroxiredoxin 4 protects against high-dose streptozotocin-induced diabetes by suppressing oxidative stress and cytokines in transgenic mice.
      • Guo X.
      • Yamada S.
      • Tanimoto A.
      • Ding Y.
      • Wang K.Y.
      • Shimajiri S.
      • Murata Y.
      • Kimura S.
      • Tasaki T.
      • Nabeshima A.
      • Watanabe T.
      • Kohno K.
      • Sasaguri Y.
      Overexpression of peroxiredoxin 4 attenuates atherosclerosis in apolipoprotein E knockout mice.
      • Nabeshima A.
      • Yamada S.
      • Guo X.
      • Tanimoto A.
      • Wang K.Y.
      • Shimajiri S.
      • Kimura S.
      • Tasaki T.
      • Noguchi H.
      • Kitada S.
      • Watanabe T.
      • Fujii J.
      • Kohno K.
      • Sasaguri Y.
      Peroxiredoxin 4 protects against nonalcoholic steatohepatitis and type 2 diabetes in a nongenetic mouse model.
      Liver sections were stained with a goat polyclonal anti-mouse cytokeratin 19 (CK19) antibody (1:50; Santa Cruz Biotechnology, Santa Cruz, CA) to highlight the lining of small and large bile ducts (small and large cholangiocytes, respectively). For various analyses, the bile ducts were separated according to size (small, <15-μm diameter; large, >15-μm diameter).
      • Alpini G.
      • Glaser S.
      • Ueno Y.
      • Pham L.
      • Podila P.V.
      • Caligiuri A.
      • LeSage G.
      • LaRusso N.F.
      Heterogeneity of the proliferative capacity of rat cholangiocytes after bile duct ligation.
      • Glaser S.
      • Gaudio E.
      • Rao A.
      • Pierce L.M.
      • Onori P.
      • Franchitto A.
      • Francis H.L.
      • Dostal D.E.
      • Venter J.K.
      • DeMorrow S.
      • Mancinelli R.
      • Carpino G.
      • Alvaro D.
      • Kopriva S.E.
      • Savage J.M.
      • Alpini G.
      Morphological and functional heterogeneity of the mouse intrahepatic biliary epithelium.
      We excluded tangentially sectioned bile ducts, where one cross-sectional diameter was three times larger than another diameter, from all analyses.
      • Alpini G.
      • Glaser S.
      • Ueno Y.
      • Pham L.
      • Podila P.V.
      • Caligiuri A.
      • LeSage G.
      • LaRusso N.F.
      Heterogeneity of the proliferative capacity of rat cholangiocytes after bile duct ligation.
      • Glaser S.
      • Gaudio E.
      • Rao A.
      • Pierce L.M.
      • Onori P.
      • Franchitto A.
      • Francis H.L.
      • Dostal D.E.
      • Venter J.K.
      • DeMorrow S.
      • Mancinelli R.
      • Carpino G.
      • Alvaro D.
      • Kopriva S.E.
      • Savage J.M.
      • Alpini G.
      Morphological and functional heterogeneity of the mouse intrahepatic biliary epithelium.
      The hyperplastic and proliferating bile ductules were identified in the interface areas of portal tracts per section. In addition, intrahepatic bile duct mass (IBDM) was measured as followed: area occupied by CK19+ bile ducts/total area × 100.
      • Mancinelli R.
      • Franchitto A.
      • Gaudio E.
      • Onori P.
      • Glaser S.
      • Francis H.
      • Venter J.
      • Demorrow S.
      • Carpino G.
      • Kopriva S.
      • White M.
      • Fava G.
      • Alvaro D.
      • Alpini G.
      After damage of large bile ducts by gamma-aminobutyric acid, small ducts replenish the biliary tree by amplification of calcium-dependent signaling and de novo acquisition of large cholangiocyte phenotypes.
      To evaluate the severity of BDL-induced cholestasis on day 3, we determined the intensity of inflammation using a polyclonal rabbit anti-human CD3 antibody (1:1; Dako Cytomation Co., Tokyo, Japan), a rat anti-mouse Mac-2 monoclonal antibody (1:500; Cedarlane Laboratories Ltd., Burlington, ON, Canada), or a rat anti-mouse Ly-6G antibody (Gr-1; 1:500; Birmingham, AL).
      • Gujral J.S.
      • Liu J.
      • Farhood A.
      • Jaeschke H.
      Reduced oncotic necrosis in Fas receptor-deficient C57BL/6J-lpr mice after bile duct ligation.
      • Nalapareddy P.D.
      • Schüngel S.
      • Hong J.Y.
      • Manns M.P.
      • Jaeschke H.
      • Vogel A.
      The BH3-only protein bid does not mediate death-receptor-induced liver injury in obstructive cholestasis.
      • Ding Y.
      • Yamada S.
      • Wang K.Y.
      • Shimajiri S.
      • Guo X.
      • Tanimoto A.
      • Murata Y.
      • Kitajima S.
      • Watanabe T.
      • Izumi H.
      • Kohno K.
      • Sasaguri Y.
      Overexpression of peroxiredoxin 4 protects against high-dose streptozotocin-induced diabetes by suppressing oxidative stress and cytokines in transgenic mice.
      • Guo X.
      • Yamada S.
      • Tanimoto A.
      • Ding Y.
      • Wang K.Y.
      • Shimajiri S.
      • Murata Y.
      • Kimura S.
      • Tasaki T.
      • Nabeshima A.
      • Watanabe T.
      • Kohno K.
      • Sasaguri Y.
      Overexpression of peroxiredoxin 4 attenuates atherosclerosis in apolipoprotein E knockout mice.
      • Nabeshima A.
      • Yamada S.
      • Guo X.
      • Tanimoto A.
      • Wang K.Y.
      • Shimajiri S.
      • Kimura S.
      • Tasaki T.
      • Noguchi H.
      • Kitada S.
      • Watanabe T.
      • Fujii J.
      • Kohno K.
      • Sasaguri Y.
      Peroxiredoxin 4 protects against nonalcoholic steatohepatitis and type 2 diabetes in a nongenetic mouse model.
      • Horlad H.
      • Fujiwara Y.
      • Takemura K.
      • Ohnishi K.
      • Ikeda T.
      • Tsukamoto H.
      • Mizuta H.
      • Nishimura Y.
      • Takeya M.
      • Komohara Y.
      Corosolic acid impairs tumor development and lung metastasis by inhibiting the immunosuppressive activity of myeloid-derived suppressor cells.
      We counted the number of positive T lymphocytes, macrophages (Kupffer cells), and neutrophils in 10 randomly selected fields of portal and interlobular areas per section, respectively (original magnification, ×400).
      • Nabeshima A.
      • Yamada S.
      • Guo X.
      • Tanimoto A.
      • Wang K.Y.
      • Shimajiri S.
      • Kimura S.
      • Tasaki T.
      • Noguchi H.
      • Kitada S.
      • Watanabe T.
      • Fujii J.
      • Kohno K.
      • Sasaguri Y.
      Peroxiredoxin 4 protects against nonalcoholic steatohepatitis and type 2 diabetes in a nongenetic mouse model.
      In addition, the number of neutrophils present in the sinusoids and extravasated into the parenchymal tissue were counted in 10 randomly selected high-power fields of interlobular and portal areas of similar size per section, respectively (original magnification, ×400). The sum of sinusoidal and extravasated neutrophils was expressed as the total neutrophil sequestration in the liver.
      • Gujral J.S.
      • Farhood A.
      • Bajt M.L.
      • Jaeschke H.
      Neutrophils aggravate acute liver injury during obstructive cholestasis in bile duct-ligated mice.
      Also, protein adducts of chlorotyrosine, as a useful marker of neutrophil-induced cytotoxicity, were detected by IHC using a rabbit antichlorotyrosine polyclonal antibody (1:50; Hycult Biotech, Uden, Netherlands).
      • Gujral J.S.
      • Farhood A.
      • Bajt M.L.
      • Jaeschke H.
      Neutrophils aggravate acute liver injury during obstructive cholestasis in bile duct-ligated mice.
      To assess the activation of peribiliary fibrogenic cells (ie, myofibroblasts) in BDL-injured portal tracts, we used a HistoMouse Plus Kit (Invitrogen Corp., Camarillo, CA) to block endogenous IgG and then stained tissues in mirror images of sequential sections with monoclonal mouse anti-human α-smooth muscle actin (α-SMA) (1:1000; Dako) and anti-human desmin (1:150; Dako) antibodies, as previously described.
      • Kinnman N.
      • Francoz C.
      • Barbu V.
      • Wendum D.
      • Rey C.
      • Hultcrantz R.
      • Poupon R.
      • Housset C.
      The myofibroblastic conversion of peribiliary fibrogenic cells distinct from hepatic stellate cells is stimulated by platelet-derived growth factor during liver fibrogenesis.
      The number of activated peribiliary fibrogenic cells (ie, myofibroblasts) was counted in 10 randomly selected fields of periportal or portal tracts per section, respectively (original magnification: ×400).
      • Nabeshima A.
      • Yamada S.
      • Guo X.
      • Tanimoto A.
      • Wang K.Y.
      • Shimajiri S.
      • Kimura S.
      • Tasaki T.
      • Noguchi H.
      • Kitada S.
      • Watanabe T.
      • Fujii J.
      • Kohno K.
      • Sasaguri Y.
      Peroxiredoxin 4 protects against nonalcoholic steatohepatitis and type 2 diabetes in a nongenetic mouse model.
      • Gujral J.S.
      • Farhood A.
      • Bajt M.L.
      • Jaeschke H.
      Neutrophils aggravate acute liver injury during obstructive cholestasis in bile duct-ligated mice.
      For double-immunofluorescence, injured livers at day 14 BDL were labeled with mouse monoclonal α-SMA (1:1000; Dako) and goat polyclonal CK19 antibodies (1:50; Santa Cruz Biotechnology) and visualized with goat anti-mouse IgG antibodies conjugated with Alexa Fluor Dyes (red stained) and donkey anti-goat IgG antibodies conjugated with Alexa Fluor Dyes (green stained) (Invitrogen, Carlsbad, CA), respectively.
      • Tasaki T.
      • Yamada S.
      • Guo X.
      • Tanimoto A.
      • Wang K.Y.
      • Nabeshima A.
      • Kitada S.
      • Noguchi H.
      • Kimura S.
      • Shimajiri S.
      • Kohno K.
      • Ichijo H.
      • Sasaguri Y.
      Apoptosis signal-regulating kinase 1 deficiency attenuates vascular injury-induced neointimal hyperplasia by suppressing apoptosis in smooth muscle cells.
      • Guo X.
      • Yamada S.
      • Tanimoto A.
      • Ding Y.
      • Wang K.Y.
      • Shimajiri S.
      • Murata Y.
      • Kimura S.
      • Tasaki T.
      • Nabeshima A.
      • Watanabe T.
      • Kohno K.
      • Sasaguri Y.
      Overexpression of peroxiredoxin 4 attenuates atherosclerosis in apolipoprotein E knockout mice.
      We applied the HistoMouse Plus Kit (Invitrogen) to block endogenous mouse IgG.
      • Yamada S.
      • Ding Y.
      • Tanimoto A.
      • Wang K.Y.
      • Guo X.
      • Li Z.
      • Tasaki T.
      • Nabesima A.
      • Murata Y.
      • Shimajiri S.
      • Kohno K.
      • Ichijo H.
      • Sasaguri Y.
      Apoptosis signal-regulating kinase 1 deficiency accelerates hyperlipidemia-induced atheromatous plaques via suppression of macrophage apoptosis.
      • Tasaki T.
      • Yamada S.
      • Guo X.
      • Tanimoto A.
      • Wang K.Y.
      • Nabeshima A.
      • Kitada S.
      • Noguchi H.
      • Kimura S.
      • Shimajiri S.
      • Kohno K.
      • Ichijo H.
      • Sasaguri Y.
      Apoptosis signal-regulating kinase 1 deficiency attenuates vascular injury-induced neointimal hyperplasia by suppressing apoptosis in smooth muscle cells.
      • Ding Y.
      • Yamada S.
      • Wang K.Y.
      • Shimajiri S.
      • Guo X.
      • Tanimoto A.
      • Murata Y.
      • Kitajima S.
      • Watanabe T.
      • Izumi H.
      • Kohno K.
      • Sasaguri Y.
      Overexpression of peroxiredoxin 4 protects against high-dose streptozotocin-induced diabetes by suppressing oxidative stress and cytokines in transgenic mice.
      • Guo X.
      • Yamada S.
      • Tanimoto A.
      • Ding Y.
      • Wang K.Y.
      • Shimajiri S.
      • Murata Y.
      • Kimura S.
      • Tasaki T.
      • Nabeshima A.
      • Watanabe T.
      • Kohno K.
      • Sasaguri Y.
      Overexpression of peroxiredoxin 4 attenuates atherosclerosis in apolipoprotein E knockout mice.
      • Nabeshima A.
      • Yamada S.
      • Guo X.
      • Tanimoto A.
      • Wang K.Y.
      • Shimajiri S.
      • Kimura S.
      • Tasaki T.
      • Noguchi H.
      • Kitada S.
      • Watanabe T.
      • Fujii J.
      • Kohno K.
      • Sasaguri Y.
      Peroxiredoxin 4 protects against nonalcoholic steatohepatitis and type 2 diabetes in a nongenetic mouse model.
      To analyze the proliferative activity of bile ducts, Ki67 (MIB-1; 1:2000; Epitomics, Cambridge, UK) rabbit monoclonal antibody was applied. For double-immunofluorescence of proliferating cholangiocytes, injured livers of day 3 BDL mice were labeled with rabbit monoclonal Ki67 (1:2000; Epitomics) and goat polyclonal CK19 antibodies (1:50; Santa Cruz Biotechnology) and visualized with goat anti-rabbit IgG antibodies conjugated with Alexa fluor Dyes (green stained) and donkey anti-goat IgG antibodies conjugated with Alexa fluor Dyes (red stained) (Invitrogen), respectively.
      • Tasaki T.
      • Yamada S.
      • Guo X.
      • Tanimoto A.
      • Wang K.Y.
      • Nabeshima A.
      • Kitada S.
      • Noguchi H.
      • Kimura S.
      • Shimajiri S.
      • Kohno K.
      • Ichijo H.
      • Sasaguri Y.
      Apoptosis signal-regulating kinase 1 deficiency attenuates vascular injury-induced neointimal hyperplasia by suppressing apoptosis in smooth muscle cells.
      • Guo X.
      • Yamada S.
      • Tanimoto A.
      • Ding Y.
      • Wang K.Y.
      • Shimajiri S.
      • Murata Y.
      • Kimura S.
      • Tasaki T.
      • Nabeshima A.
      • Watanabe T.
      • Kohno K.
      • Sasaguri Y.
      Overexpression of peroxiredoxin 4 attenuates atherosclerosis in apolipoprotein E knockout mice.
      More than 100 large or small cholangiocytes were counted in 10 randomly selected fields of portal or periportal tracts per section and viewed by confocal laser scanning microscopy (LSM5 Pascal Exciter; Carl Zeiss, Oberkochen, Germany) (original magnification, ×400).
      To evaluate ASK1 expression in BDL-injured or sham-operated livers, we used mouse monoclonal antibody to phosphorylated ASK1 (p-ASK1; 1:200; Wako Pure Chemical Co.).
      • Yamada S.
      • Ding Y.
      • Tanimoto A.
      • Wang K.Y.
      • Guo X.
      • Li Z.
      • Tasaki T.
      • Nabesima A.
      • Murata Y.
      • Shimajiri S.
      • Kohno K.
      • Ichijo H.
      • Sasaguri Y.
      Apoptosis signal-regulating kinase 1 deficiency accelerates hyperlipidemia-induced atheromatous plaques via suppression of macrophage apoptosis.
      • Tasaki T.
      • Yamada S.
      • Guo X.
      • Tanimoto A.
      • Wang K.Y.
      • Nabeshima A.
      • Kitada S.
      • Noguchi H.
      • Kimura S.
      • Shimajiri S.
      • Kohno K.
      • Ichijo H.
      • Sasaguri Y.
      Apoptosis signal-regulating kinase 1 deficiency attenuates vascular injury-induced neointimal hyperplasia by suppressing apoptosis in smooth muscle cells.
      For IHC or immunofluorescence studies, we examined one section from each of 5 or 10 mice per experimental group. All histologic and IHC slides were evaluated by two independent certified pathologists (Sohsuke Yamada and Shohei Shimajiri) who were masked to the physical outcome or other biological and pathological data for each sample. In case of disagreement, a consensus score was determined by a third board-certified pathologist (Yasuyuki Sasaguri). Agreement between observers was excellent (>0.9) for all sections investigated as measured by interclass correlation coefficient.

       TUNEL and 5′-Bromo-2′-Deoxyuridine (BrdU) Staining

      TUNEL assays were performed using an In Situ Cell Death Detection Kit (POD; Roche Applied Science, Mannheim, Germany).
      • Yamada S.
      • Ding Y.
      • Tanimoto A.
      • Wang K.Y.
      • Guo X.
      • Li Z.
      • Tasaki T.
      • Nabesima A.
      • Murata Y.
      • Shimajiri S.
      • Kohno K.
      • Ichijo H.
      • Sasaguri Y.
      Apoptosis signal-regulating kinase 1 deficiency accelerates hyperlipidemia-induced atheromatous plaques via suppression of macrophage apoptosis.
      • Tasaki T.
      • Yamada S.
      • Guo X.
      • Tanimoto A.
      • Wang K.Y.
      • Nabeshima A.
      • Kitada S.
      • Noguchi H.
      • Kimura S.
      • Shimajiri S.
      • Kohno K.
      • Ichijo H.
      • Sasaguri Y.
      Apoptosis signal-regulating kinase 1 deficiency attenuates vascular injury-induced neointimal hyperplasia by suppressing apoptosis in smooth muscle cells.
      • Ding Y.
      • Yamada S.
      • Wang K.Y.
      • Shimajiri S.
      • Guo X.
      • Tanimoto A.
      • Murata Y.
      • Kitajima S.
      • Watanabe T.
      • Izumi H.
      • Kohno K.
      • Sasaguri Y.
      Overexpression of peroxiredoxin 4 protects against high-dose streptozotocin-induced diabetes by suppressing oxidative stress and cytokines in transgenic mice.
      • Guo X.
      • Yamada S.
      • Tanimoto A.
      • Ding Y.
      • Wang K.Y.
      • Shimajiri S.
      • Murata Y.
      • Kimura S.
      • Tasaki T.
      • Nabeshima A.
      • Watanabe T.
      • Kohno K.
      • Sasaguri Y.
      Overexpression of peroxiredoxin 4 attenuates atherosclerosis in apolipoprotein E knockout mice.
      • Nabeshima A.
      • Yamada S.
      • Guo X.
      • Tanimoto A.
      • Wang K.Y.
      • Shimajiri S.
      • Kimura S.
      • Tasaki T.
      • Noguchi H.
      • Kitada S.
      • Watanabe T.
      • Fujii J.
      • Kohno K.
      • Sasaguri Y.
      Peroxiredoxin 4 protects against nonalcoholic steatohepatitis and type 2 diabetes in a nongenetic mouse model.
      In addition, BDL-injured livers on day 3 were labeled with antifluorescein antibodies (brown stained) (TUNEL POD; Roche Applied Science) or fluorescein-conjugated TUNEL reaction mixture (green stained) (Roche Applied Science) and goat polyclonal anti-CK19 antibody (1:50; Santa Cruz Biotechnology) and, for the latter, visualized with donkey anti-goat IgG antibodies conjugated with Alexa Fluor Dyes (red stained) (Invitrogen) by confocal laser scanning microscopy (LSM5 Pascal Exciter) (original magnification, ×400).
      • Tasaki T.
      • Yamada S.
      • Guo X.
      • Tanimoto A.
      • Wang K.Y.
      • Nabeshima A.
      • Kitada S.
      • Noguchi H.
      • Kimura S.
      • Shimajiri S.
      • Kohno K.
      • Ichijo H.
      • Sasaguri Y.
      Apoptosis signal-regulating kinase 1 deficiency attenuates vascular injury-induced neointimal hyperplasia by suppressing apoptosis in smooth muscle cells.
      • Guo X.
      • Yamada S.
      • Tanimoto A.
      • Ding Y.
      • Wang K.Y.
      • Shimajiri S.
      • Murata Y.
      • Kimura S.
      • Tasaki T.
      • Nabeshima A.
      • Watanabe T.
      • Kohno K.
      • Sasaguri Y.
      Overexpression of peroxiredoxin 4 attenuates atherosclerosis in apolipoprotein E knockout mice.
      For quantitative analysis, we counted TUNEL+ hepatocytes and cholangiocytes (brown stained) in 10 randomly selected fields around the bile infarcts per section (original magnification, ×400).
      • Nabeshima A.
      • Yamada S.
      • Guo X.
      • Tanimoto A.
      • Wang K.Y.
      • Shimajiri S.
      • Kimura S.
      • Tasaki T.
      • Noguchi H.
      • Kitada S.
      • Watanabe T.
      • Fujii J.
      • Kohno K.
      • Sasaguri Y.
      Peroxiredoxin 4 protects against nonalcoholic steatohepatitis and type 2 diabetes in a nongenetic mouse model.
      To label proliferating large to small cholangiocytes in injured livers 3 days after BDL, BrdU (50 mg/kg body weight; Sigma, St. Louis, MO) was subcutaneously injected 2 hours before sacrifice. BrdU-incorporated cells were detected by IHC using a monoclonal mouse anti-BrdU antibody (Roche Applied Science, Lewes, UK).
      • Yamada S.
      • Ding Y.
      • Tanimoto A.
      • Wang K.Y.
      • Guo X.
      • Li Z.
      • Tasaki T.
      • Nabesima A.
      • Murata Y.
      • Shimajiri S.
      • Kohno K.
      • Ichijo H.
      • Sasaguri Y.
      Apoptosis signal-regulating kinase 1 deficiency accelerates hyperlipidemia-induced atheromatous plaques via suppression of macrophage apoptosis.
      • Tasaki T.
      • Yamada S.
      • Guo X.
      • Tanimoto A.
      • Wang K.Y.
      • Nabeshima A.
      • Kitada S.
      • Noguchi H.
      • Kimura S.
      • Shimajiri S.
      • Kohno K.
      • Ichijo H.
      • Sasaguri Y.
      Apoptosis signal-regulating kinase 1 deficiency attenuates vascular injury-induced neointimal hyperplasia by suppressing apoptosis in smooth muscle cells.
      • Guo X.
      • Yamada S.
      • Tanimoto A.
      • Ding Y.
      • Wang K.Y.
      • Shimajiri S.
      • Murata Y.
      • Kimura S.
      • Tasaki T.
      • Nabeshima A.
      • Watanabe T.
      • Kohno K.
      • Sasaguri Y.
      Overexpression of peroxiredoxin 4 attenuates atherosclerosis in apolipoprotein E knockout mice.

       High-Performance Liquid Chromatography Analysis of Lipoprotein

      Three days after BDL, the mice were fasted for 7 hours, and blood was collected from the inferior vena cava into a microtube that contained 5 μL of 0.5 mol/L EDTA. Samples were centrifuged for 12 minutes at 5500 × g at 4°C, and the resulting serum was stored at −80°C until assayed. Lipoproteins were analyzed by high-performance liquid chromatography using molecular sieve columns (Skylight Biotech Inc., Akita, Japan), as previously described.
      • Yamada S.
      • Ding Y.
      • Tanimoto A.
      • Wang K.Y.
      • Guo X.
      • Li Z.
      • Tasaki T.
      • Nabesima A.
      • Murata Y.
      • Shimajiri S.
      • Kohno K.
      • Ichijo H.
      • Sasaguri Y.
      Apoptosis signal-regulating kinase 1 deficiency accelerates hyperlipidemia-induced atheromatous plaques via suppression of macrophage apoptosis.
      • Guo X.
      • Yamada S.
      • Tanimoto A.
      • Ding Y.
      • Wang K.Y.
      • Shimajiri S.
      • Murata Y.
      • Kimura S.
      • Tasaki T.
      • Nabeshima A.
      • Watanabe T.
      • Kohno K.
      • Sasaguri Y.
      Overexpression of peroxiredoxin 4 attenuates atherosclerosis in apolipoprotein E knockout mice.
      • Nabeshima A.
      • Yamada S.
      • Guo X.
      • Tanimoto A.
      • Wang K.Y.
      • Shimajiri S.
      • Kimura S.
      • Tasaki T.
      • Noguchi H.
      • Kitada S.
      • Watanabe T.
      • Fujii J.
      • Kohno K.
      • Sasaguri Y.
      Peroxiredoxin 4 protects against nonalcoholic steatohepatitis and type 2 diabetes in a nongenetic mouse model.
      • Wang K.Y.
      • Tanimoto A.
      • Yamada S.
      • Guo X.
      • Ding Y.
      • Watanabe T.
      • Watanabe T.
      • Kohno K.
      • Hirano K.
      • Tsukada H.
      • Sasaguri Y.
      Histamine regulation in glucose and lipid metabolism via histamine receptors: model for nonalcoholic steatohepatitis in mice.

       Measurement of Serum TBARS Levels

      As a quantitative marker of oxidative stress closely associated with the development of cholestasis on day 3 after BDL surgery, we measured serum thiobarbituric acid reactive substance (TBARS) levels using a TBARS Assay Kit (Cayman Chemical Company, Ann Arbor, MI).
      • Guo X.
      • Yamada S.
      • Tanimoto A.
      • Ding Y.
      • Wang K.Y.
      • Shimajiri S.
      • Murata Y.
      • Kimura S.
      • Tasaki T.
      • Nabeshima A.
      • Watanabe T.
      • Kohno K.
      • Sasaguri Y.
      Overexpression of peroxiredoxin 4 attenuates atherosclerosis in apolipoprotein E knockout mice.
      • Nabeshima A.
      • Yamada S.
      • Guo X.
      • Tanimoto A.
      • Wang K.Y.
      • Shimajiri S.
      • Kimura S.
      • Tasaki T.
      • Noguchi H.
      • Kitada S.
      • Watanabe T.
      • Fujii J.
      • Kohno K.
      • Sasaguri Y.
      Peroxiredoxin 4 protects against nonalcoholic steatohepatitis and type 2 diabetes in a nongenetic mouse model.
      Results are expressed as nanomoles of malondialdehyde (MDA) per milligram of low-density lipoprotein (LDL) protein.

       RT-PCR and Real-Time RT-PCR

      Total RNAs were extracted with Trizol reagents (Invitrogen) from livers of mice sacrificed on day 3 or 14 after BDL injury, after careful removal of blood cells, using the sham-operated livers as controls. All procedures were performed as described previously.
      • Yamada S.
      • Ding Y.
      • Tanimoto A.
      • Wang K.Y.
      • Guo X.
      • Li Z.
      • Tasaki T.
      • Nabesima A.
      • Murata Y.
      • Shimajiri S.
      • Kohno K.
      • Ichijo H.
      • Sasaguri Y.
      Apoptosis signal-regulating kinase 1 deficiency accelerates hyperlipidemia-induced atheromatous plaques via suppression of macrophage apoptosis.
      • Tasaki T.
      • Yamada S.
      • Guo X.
      • Tanimoto A.
      • Wang K.Y.
      • Nabeshima A.
      • Kitada S.
      • Noguchi H.
      • Kimura S.
      • Shimajiri S.
      • Kohno K.
      • Ichijo H.
      • Sasaguri Y.
      Apoptosis signal-regulating kinase 1 deficiency attenuates vascular injury-induced neointimal hyperplasia by suppressing apoptosis in smooth muscle cells.
      • Yamada S.
      • Wang K.Y.
      • Tanimoto A.
      • Fan J.
      • Shimajiri S.
      • Kitajima S.
      • Morimoto M.
      • Tsutui M.
      • Watanabe T.
      • Yasumoto K.
      • Sasaguri Y.
      Matrix metalloproteinase 12 accelerates the initiation of atherosclerosis and stimulates the progression of fatty streaks to fibrous plaques in transgenic rabbits.
      • Ding Y.
      • Yamada S.
      • Wang K.Y.
      • Shimajiri S.
      • Guo X.
      • Tanimoto A.
      • Murata Y.
      • Kitajima S.
      • Watanabe T.
      • Izumi H.
      • Kohno K.
      • Sasaguri Y.
      Overexpression of peroxiredoxin 4 protects against high-dose streptozotocin-induced diabetes by suppressing oxidative stress and cytokines in transgenic mice.
      • Guo X.
      • Yamada S.
      • Tanimoto A.
      • Ding Y.
      • Wang K.Y.
      • Shimajiri S.
      • Murata Y.
      • Kimura S.
      • Tasaki T.
      • Nabeshima A.
      • Watanabe T.
      • Kohno K.
      • Sasaguri Y.
      Overexpression of peroxiredoxin 4 attenuates atherosclerosis in apolipoprotein E knockout mice.
      • Nabeshima A.
      • Yamada S.
      • Guo X.
      • Tanimoto A.
      • Wang K.Y.
      • Shimajiri S.
      • Kimura S.
      • Tasaki T.
      • Noguchi H.
      • Kitada S.
      • Watanabe T.
      • Fujii J.
      • Kohno K.
      • Sasaguri Y.
      Peroxiredoxin 4 protects against nonalcoholic steatohepatitis and type 2 diabetes in a nongenetic mouse model.
      • Wang K.Y.
      • Tanimoto A.
      • Yamada S.
      • Guo X.
      • Ding Y.
      • Watanabe T.
      • Watanabe T.
      • Kohno K.
      • Hirano K.
      • Tsukada H.
      • Sasaguri Y.
      Histamine regulation in glucose and lipid metabolism via histamine receptors: model for nonalcoholic steatohepatitis in mice.
      RNase-free conditions were used to prevent mRNA degradation. First-strand cDNA was synthesized with Superscript II RT (Invitrogen) using random primers, according to the manufacturer's instructions. Quantitative real-time RT-PCR was performed using the TaqMan fluorogenic probe method with a LightCycler 480 System II PCR machine (Roche Diagnostics K.K., Tokyo, Japan). Sequences of specific primers for RT-PCR are summarized in Table 1. Cycling conditions were as follows: 50°C for 2 minutes and 95°C for 10 minutes followed by 45 cycles of 95°C for 15 seconds and 60°C for 1 minute. The CT values were measured corresponding to the cycle number at which the fluorescent emission, monitored in real time, reached a threshold of 10 SDs above the mean baseline from cycles 1 to 15. Serial 1:10 dilutions of plasmid DNA that contained each target cDNA were analyzed and served as standard curves from which the rate of change of CT values was determined. Copy numbers of target cDNA were estimated by standard curves. All reactions for samples were performed in triplicate. Mean data were obtained from values of each reaction. To determine mRNA levels of various genes, an mRNA expression index was used where mRNA expression levels are standardized by 18s rRNA (Applied Biosystems, Foster City, CA). The mRNA expression index in arbitrary units (AU) was calculated as follows: mRNA expression index = (copy numbers of target gene mRNA/copy numbers of 18s rRNA) × 1 AU.
      Table 1Primers Used for Real-Time PCR and RT-PCR
      GenePrimer
      Real-time PCR primers
      ASK1
      Forward5′-ACGACCACCTCAGGGTCATTC-3′
      Reverse5′-TGGTCAGTTTCACAAAGTGTTTATATATCAG-3′
      Probe5′-AGACTGAAGACACCAGCGTGGTACCTCAAGT-3′
      Caspase-3
      Forward5′-AAGCCGAAACTCTTCATCATTCAG-3′
      Reverse5′-AGCCTCCACCGGTATCTTCTG-3′
      Probe5′-TGGACTGTGGCATTGAGACAGACAGTGG-3′
      Bak
      Forward5′-CTGGAACCCAACAGCATCTTG-3′
      Reverse5′-TGGTGAAGAGTTCGTAGGCATTC-3′
      Probe5′-CGGAGATGATATTAACCGGCGCTACGAC-3′
      Bax
      Forward5′-ATGGGCTGGACACTGGACTTC-3′
      Reverse5′-GAGGACTCCAGCCACAAAGATG-3′
      Probe5′-TGGCTGGGAAGGCCTCCTCTCCTACT-3′
      IL-1β
      Forward5′-TGCACTACAGGCTCCGAGATG-3′
      Reverse5′-GTACAAAGCTCATGGAGAATATCACTTG-3′
      Probe5′-TGTCGGACCCATATGAGCTGAAAGCTCTC-3′
      IL-1R1
      Forward5′-AGTTAAAAGCCAGTTTTATCGCTATCC-3′
      Reverse5′-CCCCCGATGAGGTAATTCTTG-3′
      Probe5′-AAATATTTTTGAGTCGGCGCATGTGCAGTTAAT-3′
      IL-6
      Forward5′-TTACACATGTTCTCTGGGAAATCG-3′
      Reverse5′-TTGGTAGCATCCATCATTTCTTTG-3′
      Probe5′-TGAGAAAAGAGTTGTGCAATGGCAATTCTGAT-3′
      NF-κB
      Forward5′-CTGACCCCTGTCCTCTCACATC-3′
      Reverse5′-CCGGTTTACTCGGCAGATCTT-3′
      Probe5′-TGATAACCGGGCCCCCAACACTG-3′
      TNF-α
      Forward5′-CCCAGACCCTCACACTCAGATC-3′
      Reverse5′-TGCTCCTCCACTTGGTGGTT-3′
      Probe5′-ATTCGAGTGACAAGCCTGTAGCCCACG-3′
      TNFR2
      Forward5′-TGGTCTGATTGTTGGAGTGACATC-3′
      Reverse5′-GGATTTCTCATCAGGCACATGAG-3′
      Probe5′-TGCATCATCCTGGTGCAGAGGAAAAAGA-3′
      TLR4
      Forward5′-CAGAACTTCAGTGGCTGGATTTATC-3′
      Reverse5′-TAGGGTTTCCTGTCAGTATCAAGTTTG-3′
      Probe5′-ACAAGGCATGGCATGGCTTACACCAC-3′
      IFN-γ
      Forward5′-CATTCATGAGTATTGCCAAGTTTGAG-3′
      Reverse5′-GCTTCCTGAGGCTGGATTCC-3′
      Probe5′-CCACAGGTCCAGCGCCAAGCATT-3′
      iNOS
      Forward5′-GCAGTGGAGAGATTTTGCATGAC-3′
      Reverse5′-ATGGACCCCAAGCAAGACTTG-3′
      Probe5′-CACCACAAGGCCACATCGGATTTCAC-3′
      TGFβ-1Mm00441724-m1 (Applied Biosystems Japan, Ltd., Tokyo, Japan).
      PRDX1
      Forward5′-TGTGGATTCTCACTTCTGTCATCTG-3′
      Reverse5′-GTGCGCTTGGGATCTGATATTAAG-3′
      Probe5′-CACACCCAAGAAACAAGGAGGATTGGGA-3′
      PRDX2
      Forward5′-CCCAGAATTACGGCGTGTTG-3′
      Reverse5′-CCCTTGGCATCGATGATAAAGA-3′
      Probe5′-AATGATGAGGGCATTGCTTACAGGG-3′
      PRDX3
      Forward5′-GGTGCTTTTCTTCTACCCTTTGG-3′
      Reverse5′-CACAGTTTACATCATGAAATTCATTGG-3′
      Probe5′-TTGTGTGTCCTACAGAAATTGTTGC-3′
      PRDX4
      Forward5′-TGGACGAGACACTGCGTTTG-3′
      Reverse5′-CTGGATCTGGGATTATTGTTTCACTAC-3′
      Probe5′-ACACTGACAAGCATGGAGAAGTCTGCCC-3′
      PRDX5
      Forward5′-GGAAGGCGACAGACTTATTATTGG-3′
      Reverse5′-CTCCACGTTCAGTGCCTTCAC-3′
      Probe5′-CGTCGGCTGAAAAGGTTCTCCATGGT-3′
      PRDX6
      Forward5′-GCCAAGAGGAATGTTAAGTTGATTG-3′
      Reverse5′-GTTTCACCATTGTAAGCATTGATGTC-3′
      Probe5′-CAATAGACAGTGTTGAGGATCATCATCTTGCCTGG-3′
      CYP7A1
      Forward5′-CGTGATCCTCTGGGCATCTC-3′
      Reverse5′-CTCTTGGCCAGCACTCTGTAATG-3′
      Probe5′-CAATGAAAGCAGCCTCTGAAGAAGTGAATGG-3′
      Catalase
      Forward5′-CCCTCCTCGTTCAGGATGTG-3′
      Reverse5′-TGATATCGTGGGTGACCTCAAAG-3′
      Probe5′-ACAGAGAGCGGATTCCTGAGAGAGTGGTACA-3′
      SOD1
      Forward5′-TGCAGGGAACCATCCACTTC-3′
      Reverse5′-CATGCTGGCCTTCAGTTAATCC-3′
      Probe5′-AAGCGGTGAACCAGTTGTGTTGTCAGGA-3′
      SOD2
      Forward5′-GCCTGCACTGAAGTTCAATGG-3′
      Reverse5′-ACCCAAAGTCACGCTTGATAGC-3′
      Probe5′-CTGGACAAACCTGAGCCCTAAGGGTGG-3′
      FXR
      Forward5′-CTGGAATATATGAACTCAGGCGTATG-3′
      Reverse5′-CCACAAACAACACACAGCTCATC-3′
      Probe5′-TAGCAGAGATGCCTGTAACAAAGAAGCCCC-3′
      SHP
      Forward5′-AAGGAGTATGCGTACCTGAAGG-3′
      Reverse5′-CTCCAAGACTTCACACAGTGC-3′
      Probe5′-CTCTTCAACCCAGATGTGCCAGGC-3′
      VCAM-1
      Forward5′-CTCATTCCCTGAAGATCCAGTAATTAA-3′
      Reverse5′-TCAAAGGGATACACATTAGGGACTGT-3′
      Probe5′-TGAGTGGGCCACTTGTGCATGGG-3′
      ICAM-1
      Forward5′-CAAACAGGAGATGAATGGTACATACG-3′
      Reverse5′-ACCAGAATGATTATAGTCCAGTTATTTTGAG-3′
      Probe5′-CCATGGGAATGTCACCAGGAATGTGTACC-3′
      LFA-1
      Forward5′-GGCCCCAGACTTTTGCTACTG-3′
      Reverse5′-GCAAGACCTGGTACCCAAAATG-3′
      Probe5′-CTACAACCTGGACACACGGCCTACGC-3′
      VLA-4
      Forward5′-CATCGTATATGAAATGAAAGGTAAAAAGC-3′
      Reverse5′-GATCTGAGAAGCCATCTGCATTG-3′
      Probe5′-GATCTGAGAAGCCATCTGCATTG-3′
      PDGF-BB
      Forward5′-AAGATCGAGATTGTGCGGAAGA-3′
      Reverse5′-AGCTGCCACTGTCTCACACTTG-3′
      Probe5′-AAGGCCACGGTGACGCTGGAAG-3′
      PDGFRB
      Forward5′-GCTCACGGTCTGAGCCATTC-3′
      Reverse5′-GACTCATAATCTTCAGCTCGGACATT-3′
      Probe5′-ATGCTGAAATCGACAGCCAGAAGTAGCGA-3′
      18s rRNATaqMan Ribosomal RNA Control Reagents VIC Probe (Applied Biosystems, Foster City, CA; Cat. No. 4308329)
      RT-PCR primers
      ASK1
      Forward5′-GCCGTGCTGGACCGTTTTTAC-3′
      Reverse5′-GTGAGGCGTGATGTAAATAGGAAGC-3′
      Neomycin
      Forward5′-CTAAAGCGCATGCTCCAGACTGCCTTG-3′
      Reverse5′-GTGAGGCGTGATGTAAATAGGAAGC-3′
      GAPDH
      Forward5′-AATGCATCCTGCACCACCAACTGC-3′
      Reverse5′-GGAGGCCATGTAGGCCATGAGGTCC-3′

       Microarray Studies and Data Analysis

      DNA microarray analysis was performed using 3D-Gene software (Toray Industries, Kamakura, Kanagawa, Japan), as previously described.
      • Yasuniwa Y.
      • Izumi H.
      • Wang K.Y.
      • Shimajiri S.
      • Sasaguri Y.
      • Kawai K.
      • Kasai H.
      • Shimada T.
      • Miyake K.
      • Kashiwagi E.
      • Hirano G.
      • Kidani A.
      • Akiyama M.
      • Han B.
      • Wu Y.
      • Ieiri I.
      Circadian disruption accelerates tumor growth and angio/stromagenesis through a Wnt signaling pathway.
      High-quality RNA samples (1 μg each) prepared from injured livers of WT and ASK1−/− mice sacrificed on day 3 after BDL were amplified and labeled with cyanine 3 and cyanine 5 CTP (Amersham Biosciences Corp., Piscataway, NJ) to produce labeled cRNA using Agilent low-RNA-input fluorescent linear amplification kits following the manufacturer's protocol. After amplification and labeling, the dye-incorporation ratio was determined using a Nanodrop spectrophotometer, and ratios were within 10 to 20 pM per microgram of cRNA. For hybridization, 750 ng of cyanine 3– and 750 ng of cyanine 5–labeled cRNA was fragmented and hybridized to an Agilent Technologies Human 1A (V2) Gene Expression Microarray using the Agilent Gene Expression hybridization kit as described in the Two-Color Microarray-Based Gene Expression Analysis Version 4.0 manual. After hybridization, microarrays were washed and scanned with the Agilent dual-laser DNA microarray scanner. Scans were converted to data files with Agilent Feature Extraction software (version 8.5). Data were deposited in Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo; accession no. GSE23969) and analyzed with Microsoft Access version 2010 and Spotfire version 7.0 (Spotfire, Somerville, MA). Arrays were scanned by Agilent dual-laser DNA microarray scanner using SureScan technology, extracted by feature Extraction software version 7.1, and analyzed by Rosetta Resolver software version 3.2, build (3.2.2.0.33) (Rosetta Biosoftware, Kirkland, WA). Three replicate samples were used for each experiment. Genes were selected as significant using criteria of greater than twofold (up- or down-regulated) changes.

       Preparation of Liver Hepatocytes and Analysis of ASK1 Expression

      Liver hepatocytes were isolated from 8-week-old male ASK1−/− and WT mice fed a normal chow diet, as previously described.
      • Nabeshima A.
      • Yamada S.
      • Guo X.
      • Tanimoto A.
      • Wang K.Y.
      • Shimajiri S.
      • Kimura S.
      • Tasaki T.
      • Noguchi H.
      • Kitada S.
      • Watanabe T.
      • Fujii J.
      • Kohno K.
      • Sasaguri Y.
      Peroxiredoxin 4 protects against nonalcoholic steatohepatitis and type 2 diabetes in a nongenetic mouse model.
      All mice were anesthetized by i.p. injection of ketamine (Daiichi Sankyo Co.) and medetamidine (Meiji Yakuhin Co.). The abdominal cavity was opened, and the mesenteric and bilateral renal arteries were ligated. A ligature was placed around the portal vein at a site distal to the bifurcation and another around the inferior vena cava at a site proximal to the mesenteric artery. A tapered polyethylene cannula connected to a perfusion apparatus was inserted into the portal vein and was ligated in place. An 18-G needle was also inserted into the vena cava and ligated in place. Then liver perfusion medium (GIBCO17701-038; Gibco, Grand Island, NY) and collagenase type II solution medium (Gibco) with an oxygen and carbon dioxide mixture (95:5) at 37°C were injected into the cannula (4.5 mL/min). The collected cells were washed with Dulbecco's modified Eagle's medium; cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin (Gibco), and 2 mmol/L glutamine (Gibco) at a density of 2 to 3 × 105 cells/cm2; and placed onto six-well glass slides in an incubator for >3 days. Adherent cells (hepatocytes) were maintained in the medium.
      For p-ASK1 staining, hepatocytes were fixed in 95% acetone for 30 seconds at room temperature and permeabilized in 0.1% Triton X-100 for 2 minutes at 4°C. Cells were then stained with a mouse monoclonal antibody to p-ASK1 (1:100; Wako Pure Chemical Co.) for 1 hour, washed with PBS, and reacted with Alexa Fluor Dyes (red stained)–conjugated goat anti-mouse IgG antibodies (Invitrogen). Isolated hepatocytes were observed and images immediately captured with a Nikon ECLIPSE E600 inverted fluorescence microscope (Nikon, Tokyo, Japan). We applied the HistoMouse Plus Kit (Invitrogen) to block endogenous mouse IgG.

       Western Blot Analysis

      Proteins (40 μg) isolated from BDL-injured livers of WT and ASK1−/− mice were separated by SDS-PAGE and transferred to Immun-Blot polyvinylidene difluoride membranes (Bio-Rad Laboratories, K.K., Tokyo, Japan) using a semidry blotter.
      • Yamada S.
      • Ding Y.
      • Tanimoto A.
      • Wang K.Y.
      • Guo X.
      • Li Z.
      • Tasaki T.
      • Nabesima A.
      • Murata Y.
      • Shimajiri S.
      • Kohno K.
      • Ichijo H.
      • Sasaguri Y.
      Apoptosis signal-regulating kinase 1 deficiency accelerates hyperlipidemia-induced atheromatous plaques via suppression of macrophage apoptosis.
      • Tasaki T.
      • Yamada S.
      • Guo X.
      • Tanimoto A.
      • Wang K.Y.
      • Nabeshima A.
      • Kitada S.
      • Noguchi H.
      • Kimura S.
      • Shimajiri S.
      • Kohno K.
      • Ichijo H.
      • Sasaguri Y.
      Apoptosis signal-regulating kinase 1 deficiency attenuates vascular injury-induced neointimal hyperplasia by suppressing apoptosis in smooth muscle cells.
      • Yamada S.
      • Wang K.Y.
      • Tanimoto A.
      • Fan J.
      • Shimajiri S.
      • Kitajima S.
      • Morimoto M.
      • Tsutui M.
      • Watanabe T.
      • Yasumoto K.
      • Sasaguri Y.
      Matrix metalloproteinase 12 accelerates the initiation of atherosclerosis and stimulates the progression of fatty streaks to fibrous plaques in transgenic rabbits.
      • Ding Y.
      • Yamada S.
      • Wang K.Y.
      • Shimajiri S.
      • Guo X.
      • Tanimoto A.
      • Murata Y.
      • Kitajima S.
      • Watanabe T.
      • Izumi H.
      • Kohno K.
      • Sasaguri Y.
      Overexpression of peroxiredoxin 4 protects against high-dose streptozotocin-induced diabetes by suppressing oxidative stress and cytokines in transgenic mice.
      • Guo X.
      • Yamada S.
      • Tanimoto A.
      • Ding Y.
      • Wang K.Y.
      • Shimajiri S.
      • Murata Y.
      • Kimura S.
      • Tasaki T.
      • Nabeshima A.
      • Watanabe T.
      • Kohno K.
      • Sasaguri Y.
      Overexpression of peroxiredoxin 4 attenuates atherosclerosis in apolipoprotein E knockout mice.
      • Nabeshima A.
      • Yamada S.
      • Guo X.
      • Tanimoto A.
      • Wang K.Y.
      • Shimajiri S.
      • Kimura S.
      • Tasaki T.
      • Noguchi H.
      • Kitada S.
      • Watanabe T.
      • Fujii J.
      • Kohno K.
      • Sasaguri Y.
      Peroxiredoxin 4 protects against nonalcoholic steatohepatitis and type 2 diabetes in a nongenetic mouse model.
      • Wang K.Y.
      • Tanimoto A.
      • Yamada S.
      • Guo X.
      • Ding Y.
      • Watanabe T.
      • Watanabe T.
      • Kohno K.
      • Hirano K.
      • Tsukada H.
      • Sasaguri Y.
      Histamine regulation in glucose and lipid metabolism via histamine receptors: model for nonalcoholic steatohepatitis in mice.
      The blotted membranes were treated with 5% (w/v) skimmed milk in 10 mmol/L Tris, 150 mmol/L NaCl, and 0.2% (v/v) Tween-20 and incubated for 1 hour at room temperature with primary antibody. The following antibodies and dilutions were used: mouse monoclonal antibodies to phosphorylated JNK (p-JNK), JNK, phosphorylated p38 (p-p38), p38, proliferating cell nuclear antigen (PCNA) (1:200; Santa Cruz Biotechnology), or p-ASK1 (1:1000; Wako Pure Chemical Industries); goat polyclonal antibody to caspase-3 precursor (procaspase-3; 32 kDa) and caspase-3 subunit (active fragment; 11 kDa) (1:200; Santa Cruz Biotechnology)
      • Gujral J.S.
      • Liu J.
      • Farhood A.
      • Jaeschke H.
      Reduced oncotic necrosis in Fas receptor-deficient C57BL/6J-lpr mice after bile duct ligation.
      ; rabbit monoclonal antibody to ASK1 (1:2000; Abcam, Cambridge, UK); rabbit polyclonal antibodies to Bax (1:50; Santa Cruz Biotechnology); inducible nitric oxide synthase (iNOS: 1:200; BD Biosciences, San Jose, CA); or transforming growth factor β1 (activated TGF-β1: 25 kDa; 1:200; Santa Cruz Biotechnology) and 1:5000 dilution of anti–β-actin (Santa Cruz Biotechnology). The membranes were then incubated for 45 minutes at room temperature with a peroxidase-conjugated secondary antibody and visualized using an ECL kit (GE Healthcare Bio-Science, Buckinghamshire, England, UK). Bands on Western blots were analyzed densitometrically using Scion Image software (version 4.0.2; Scion Corp., Frederick, MD).

       ELISA for TNF-α and IL-1β

      Levels of serum TNF-α and IL-1β in the BDL-injury model on day 3 were measured using an enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions (R&D Systems, Minneapolis, MN).
      • Yamada S.
      • Ding Y.
      • Tanimoto A.
      • Wang K.Y.
      • Guo X.
      • Li Z.
      • Tasaki T.
      • Nabesima A.
      • Murata Y.
      • Shimajiri S.
      • Kohno K.
      • Ichijo H.
      • Sasaguri Y.
      Apoptosis signal-regulating kinase 1 deficiency accelerates hyperlipidemia-induced atheromatous plaques via suppression of macrophage apoptosis.
      • Tasaki T.
      • Yamada S.
      • Guo X.
      • Tanimoto A.
      • Wang K.Y.
      • Nabeshima A.
      • Kitada S.
      • Noguchi H.
      • Kimura S.
      • Shimajiri S.
      • Kohno K.
      • Ichijo H.
      • Sasaguri Y.
      Apoptosis signal-regulating kinase 1 deficiency attenuates vascular injury-induced neointimal hyperplasia by suppressing apoptosis in smooth muscle cells.
      • Ding Y.
      • Yamada S.
      • Wang K.Y.
      • Shimajiri S.
      • Guo X.
      • Tanimoto A.
      • Murata Y.
      • Kitajima S.
      • Watanabe T.
      • Izumi H.
      • Kohno K.
      • Sasaguri Y.
      Overexpression of peroxiredoxin 4 protects against high-dose streptozotocin-induced diabetes by suppressing oxidative stress and cytokines in transgenic mice.
      • Guo X.
      • Yamada S.
      • Tanimoto A.
      • Ding Y.
      • Wang K.Y.
      • Shimajiri S.
      • Murata Y.
      • Kimura S.
      • Tasaki T.
      • Nabeshima A.
      • Watanabe T.
      • Kohno K.
      • Sasaguri Y.
      Overexpression of peroxiredoxin 4 attenuates atherosclerosis in apolipoprotein E knockout mice.
      • Nabeshima A.
      • Yamada S.
      • Guo X.
      • Tanimoto A.
      • Wang K.Y.
      • Shimajiri S.
      • Kimura S.
      • Tasaki T.
      • Noguchi H.
      • Kitada S.
      • Watanabe T.
      • Fujii J.
      • Kohno K.
      • Sasaguri Y.
      Peroxiredoxin 4 protects against nonalcoholic steatohepatitis and type 2 diabetes in a nongenetic mouse model.

       Electron Microscopy

      At day 14 after BDL surgery, injured livers from ASK1−/− and WT mice were fixed with 2.5% glutaraldehyde, and small specimens were cut (approximately 1 × 1 mm), including large cholangiocytes, and were immersed in 2% osmium tetroxide and dehydrated in ethanol. For transmission electron microscopy, dehydrated tissue specimens were embedded in epoxy resin. Silver-gold sections produced with a diamond knife were transferred to copper grids and stained with uranyl acetate for viewing on a transmission electron microscopy (JEM-1200EX; JEOL Ltd., Tokyo, Japan).

       Statistical Analysis

      Results are expressed as means ± SEM. Significant differences were analyzed using Student's t-test, Welch's t-test, or one-way analysis of variance where appropriate. In all cases when the analysis of variance method was used for nonparametric data, Tukey's multiple comparison post hoc test was used.
      • Yamada S.
      • Ding Y.
      • Tanimoto A.
      • Wang K.Y.
      • Guo X.
      • Li Z.
      • Tasaki T.
      • Nabesima A.
      • Murata Y.
      • Shimajiri S.
      • Kohno K.
      • Ichijo H.
      • Sasaguri Y.
      Apoptosis signal-regulating kinase 1 deficiency accelerates hyperlipidemia-induced atheromatous plaques via suppression of macrophage apoptosis.
      • Tasaki T.
      • Yamada S.
      • Guo X.
      • Tanimoto A.
      • Wang K.Y.
      • Nabeshima A.
      • Kitada S.
      • Noguchi H.
      • Kimura S.
      • Shimajiri S.
      • Kohno K.
      • Ichijo H.
      • Sasaguri Y.
      Apoptosis signal-regulating kinase 1 deficiency attenuates vascular injury-induced neointimal hyperplasia by suppressing apoptosis in smooth muscle cells.
      • Guo X.
      • Yamada S.
      • Tanimoto A.
      • Ding Y.
      • Wang K.Y.
      • Shimajiri S.
      • Murata Y.
      • Kimura S.
      • Tasaki T.
      • Nabeshima A.
      • Watanabe T.
      • Kohno K.
      • Sasaguri Y.
      Overexpression of peroxiredoxin 4 attenuates atherosclerosis in apolipoprotein E knockout mice.
      • Nabeshima A.
      • Yamada S.
      • Guo X.
      • Tanimoto A.
      • Wang K.Y.
      • Shimajiri S.
      • Kimura S.
      • Tasaki T.
      • Noguchi H.
      • Kitada S.
      • Watanabe T.
      • Fujii J.
      • Kohno K.
      • Sasaguri Y.
      Peroxiredoxin 4 protects against nonalcoholic steatohepatitis and type 2 diabetes in a nongenetic mouse model.
      P < 0.05 was considered statistically significant.

      Results

       Expression of ASK1, p-ASK1, p-JNK, and p-p38 Is Absent or Decreased in Sham-Operated and/or BDL-Injured Livers of ASK1−/− Mice

      Liver mRNA and protein expression levels of ASK1 and/or p-ASK1 were detected by RT-PCR and Western blotting, respectively. PCR clearly revealed no expression of ASK1 in the livers of ASK1−/− mice with or without BDL on day 3 (Figure 1A). ASK1 was only detected in WT mice, and its expression was significantly greater in BDL WT mice than in sham-operated WT mice (Figure 1A). In addition, immunofluorescence studies found that hepatocytes obtained from WT mice, but not those from ASK1−/− mice, expressed intracellular p-ASK1 (Figure 1B, inset). However, biochemical and morphologic (ratio of liver to body weight) studies revealed no differences in livers of ASK1−/− and WT mice under basal conditions (data not shown).
      Figure thumbnail gr1
      Figure 1Analysis of ASK1 mRNA and protein expression in mice. A: RT-PCR of ASK1 expression in livers of ASK1−/− and WT mice subjected to BDL surgery (n = 10 mice per group) and of controls (sham operated) (n = 5 mice per group). B: IHC of p-ASK1 at day 3 after BDL surgery (n = 10 mice per group). p-ASK1 is present in many hepatocytes and large but not small (inset) cholangiocytes throughout the livers of WT mice, but not in ASK1−/− mice, and sham-operated animals. Immunofluorescence staining of p-ASK1 (red stained) in cultured hepatocytes harvested from WT and ASK1−/− mice (inset). C: Western blot analysis of p-ASK1, ASK1, p-JNK, and p-p38 in day 3 BDL livers of ASK1−/− and WT mice (n = 10 mice per group) and control sham-operated mouse livers (n = 5 mice per group). Scale bar = 25 μm. Values were normalized for GAPDH expression (RT-PCR) (A) and for β-actin expression (Western blotting) (C).
      In mice subjected to day 3 BDL surgery, IHC (Figure 1B) and Western blotting (Figure 1C) confirmed that p-ASK1 and ASK1 protein was specifically expressed throughout the livers of WT mice (Figure 1, B and C), especially in cholangiocytes lining large but not small (Figure 1B, inset) bile ducts and hepatocytes around bile infarcts, but was not present in ASK1−/− mice and sham-operated animals (Figure 1B). Moreover, Western blotting analysis revealed that the expression of p-JNK and p-p38 in BDL-injured livers in ASK1−/− mice on day 3 was also significantly reduced compared with WT mice (Figure 1C).

       Deletion of ASK1 Suppresses Acute Cholestatic Liver Injury

      The serum levels of ALT and AST were significantly increased in WT mice after 3 days of BDL compared with ASK1−/− mice (Figure 2A), indicative of more extensive hepatocyte necrosis and cell death (apoptosis). Similarly, total plasma and conjugated bilirubin (Figure 2B), plasma bile acid concentration (Figure 2C), hepatic levels of bile acids (Figure 2C), and cholesterol, especially very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) (data not shown), were significantly elevated in WT mice compared with ASK1−/− mice on day 3 after BDL surgery and control mice (P < 0.05 or P < 0.001, P < 0.001, and P < 0.0001, respectively). These in vivo bile acid levels <300 μmol/L (Figure 2C) in the BDL models are insufficient to not only directly cause liver cells death but also trigger inflammatory mediators formation, as described in the recent review paper.
      • Woolbright B.L.
      • Jaeschke H.
      Novel insight into mechanisms of cholestatic liver injury.
      Accumulation of conjugated bilirubin contributed to increased plasma bilirubin levels in both groups of mice with BDL (Figure 2B). The serum levels of alkaline phosphatase were markedly higher in the day 3 post–BDL surgery mice groups (2444 ± 491 U/L for the WT mice versus 2529 ± 322 U/L for the ASK1−/− mice) compared with sham-operated animals (165 ± 10 U/L for the WT mice versus 135 ± 21 U/L for the ASK1−/− mice); however, no significant difference was found between ASK1−/− and WT mice at day 3 after BDL surgery or controls, respectively (data not shown). According to the serum cholesterol profiles by high-performance liquid chromatography in mice at day 3 after BDL surgery, no significant difference was found in total cholesterol, high-density lipoprotein, or triglyceride levels between the two groups (data not shown). Both day 3 post–BDL surgery WT and ASK1−/− mice developed hypercholesterolemia and hypotriglyceridemia, with significantly increased levels of total cholesterol, VLDL, and LDL but decreased high-density lipoprotein cholesterol and triglyceride levels compared with control animals (data not shown). In addition, BDL resulted in increased urine bilirubin levels in WT and ASK1−/− mice. A similar pattern was observed without statistical significant between the two groups of mice at day 3 after BDL surgery (Figure 2D). No statistical differences between genotypes were observed in sham-operated animals for any of the above parameters (Figure 2).
      Figure thumbnail gr2
      Figure 2Effects of ASK1 deletion on plasma aminotransferases, bilirubin, bile acid levels, and hepatic bile acid levels in the murine BDL model. A: Plasma AST and ALT levels in day 3 BDL WT and ASK1−/− mice (n = 10 mice per group) and control (sham-operated) mice (n = 5 mice per group) (P < 0.0001). B: Total and conjugated bilirubin levels in day 3 BDL WT and ASK1−/− mice (n = 10 mice per group) and control mice (n = 5 mice per group) (P < 0.0001). C: Levels of plasma and hepatic bile acids in day 3 BDL WT and ASK1−/− mice (n = 10 mice per group) and control mice (n = 5 mice per group). D: Urine bilirubin levels in day 3 BDL WT and ASK1−/− mice (n = 10 mice per group) and sham-operated animals (n = 5 mice per group). Values are means ± SEM. P < 0.05, ∗∗P < 0.001, and ∗∗∗P < 0.0001.

       ASK1-Deficient Hepatocytes Are More Resistant to Acute Cholestatic Liver Injury but Delay Regeneration of BDL-Injured Livers

      Corresponding to the above AST, ALT, or bilirubin data, the area of bile infarcts was significantly more extensive in WT mice than in ASK1−/− mice at day 3 after BDL surgery (8.51% ± 0.60% for WT mice versus 2.64% ± 0.58% for ASK1−/− mice; P < 0.0001) and control mice (P < 0.0001), respectively (Figure 3, A and B). Although liver histologic analysis in WT mice at day 14 after BDL surgery revealed significantly faster repair processes of acute cholestatic injury and smaller infarcted areas compared with ASK1−/− mice (2.15% ± 0.40% for WT mice versus 3.87% ± 0.54% for ASK1−/− mice; P < 0.05) (Figure 3, A and B). No significant difference was found between areas of bile infarcts at day 3 and 14 after BDL surgery in ASK1−/− mice, unlike in WT mice (P < 0.0001; Figure 3B). In addition, no difference was found in the ratios of liver to body weight at day 14 after BDL surgery between the two groups of mice (data not shown). However, significantly decreased cholestatic complications and mortality were observed in ASK1−/− mice 21 days after BDL surgery compared with WT mice (overall survival: 50.8% ± 5.6% for WT mice versus 77.8% ± 5.7% for ASK1−/− mice; P < 0.0001) (Supplemental Figure S1), whereas all control animals were free of complications and did not die during the study period.
      Figure thumbnail gr3
      Figure 3Histologic analysis of BDL-induced bile infarcts. A: Representative H&E-stained liver sections of WT and ASK1−/− mice 3 or 14 days after BDL surgery (n = 10 mice per group) are shown. Sham-operated livers from both groups of mice served as controls (n = 5 mice per group). B: Quantitative analysis of day 3 and day 14 post–BDL surgery ASK1−/− and WT mice (confluent necrotic foci of hepatocytes, indicated by yellow arrows). Injured livers in WT mice at day 14 after BDL surgery displayed a significantly smaller amount of infarcted areas, indicative of a faster repair process to acute cholestatic injury, compared with ASK1−/− mice. Values are means ± SEM. ∗∗P < 0.001 and ∗∗∗P < 0.0001. Scale bar = 200 μm. C, central vein; P, portal vein.

       Deletion of ASK1 Does Not Affect Apoptotic Activity of Both Hepatocytes and Large Cholangiocytes after BDL

      On the basis of morphologic analysis, such as cell shrinkage, chromatin condensation and margination, and formation of apoptotic bodies, the numbers of apoptotic liver cells 3 days after BDL surgery (0.04% ± 0.01% in WT mice versus 0.03% ± 0.01% in ASK1−/− mice of all hepatocytes; approximately 0.4 cells per 10 fields, respectively) (0.01% ± 0.01% in WT mice versus 0.01% ± 0.01% in ASK1−/− mice of all large cholangiocytes) were negligible (Supplemental Figure S2A) and not significantly increased compared with sham-operated controls (0.02% ± 0.02% of all hepatocytes) (0.01% ± 0.01% of all large cholangiocytes) in the H&E sections. Consistent with these findings, no significant differences were found in caspase-3 activities and caspase-3 processing (Supplemental Figure S2B) or Bax expression (data not shown) in Western blotting analysis between the respective groups (WT versus ASK1−/− mice and BDL versus sham surgery).
      In striking contrast, the number of TUNEL+ interlobular hepatocytes around bile infarcts was significantly lower in ASK1−/− mice compared with WT mice (45.9 ± 2.4 per 10 fields in WT mice versus 39.6 ± 2.6 per 10 fields in ASK1−/− mice; P < 0.05) (data not shown). However, staining pattern was diffuse over the entire cell and did not remain confined to the nucleus as was seen in true apoptotic hepatocytes. Also, these oncotic hepatocytes were swollen with total nuclear fragmentation or cell lysis (ie, characteristics of necrosis). Therefore, no morphologically apoptotic characteristics were identified in most of the TUNEL+ hepatocytes. Furthermore, the number of TUNEL+ large cholangiocytes was significantly lower in ASK1−/− mice than in WT mice (7.6 ± 0.5 per 10 fields in WT mice versus 5.8 ± 0.4 per 10 fields in ASK1−/− mice; P < 0.05) (data not shown) but that of TUNEL+ small cholangiocytes was not (8.2 ± 0.7 per 10 fields in WT mice versus 8.1 ± 0.7 per 10 fields in ASK1−/− mice) (data not shown). Similarly, the morphologic characteristics of apoptosis were not noted in the TUNEL+ cholangiocytes. In fact, double-immunofluorescence staining (data not shown) revealed that there were no apoptotic (green stained) CK19+ large cholangiocytes (red stained). Livers of sham-operated control mice revealed no injury and no TUNEL+ cells (data not shown).

       Deletion of ASK1 Represses Inflammatory Reactions in Interlobular and Portal Tracts after BDL Surgery

      Gr-1 staining revealed significantly more decreasing numbers of accumulated neutrophils not only per injured interlobular areas in sinusoids or extravasated into the parenchymal tissue (total: 139.6 ± 4.1 per 10 fields in WT mice versus 105.8 ± 4.1 per 10 fields in ASK1−/− mice; P < 0.0001) (extravasated: 91.7 ± 2.6 per 10 fields in WT mice versus 61.5 ± 3.0 per 10 fields in ASK1−/− mice; P < 0.0001) (Figure 4A), but also per injured portal tracts (69.8 ± 3.6 per 10 fields in WT mice versus 61.5 ± 2.9 per 10 fields in ASK1−/− mice; P < 0.05) (Figure 4B) in ASK1−/− livers than in WT livers at day 3 after BDL surgery. Approximately 66% of these neutrophils were extravasated into the injured interlobular areas of WT livers, whereas 58% of those were located in the parenchyma in ASK1−/− livers (P < 0.05) (Figure 4A). Moreover, significantly extensive positive staining for chlorotyrosine protein adducts was recognized, especially in these interlobular areas around confluent bile infracts of day 3 post–BDL surgery WT livers, indicating that the extravasated neutrophils were cytotoxically active.
      • Gujral J.S.
      • Farhood A.
      • Bajt M.L.
      • Jaeschke H.
      Neutrophils aggravate acute liver injury during obstructive cholestasis in bile duct-ligated mice.
      Only <5 neutrophils per 10 high-power fields were noted in sham-operated controls of both WT and ASK1−/− mice, respectively (data not shown), and no neutrophils were observed extravasated into the parenchyma.
      Figure thumbnail gr4
      Figure 4Analysis of infiltrating neutrophils and adhesion molecule expression in BDL-induced cholestatic livers of mice. A: IHC reveals the number of accumulated neutrophils per injured interlobular areas in sinusoids or extravasated into the parenchymal tissue of ASK1−/− and WT mice at day 3 after BDL surgery (n = 10 mice per group). Approximately 66% of these neutrophils were extravasated into the injured interlobular areas of WT livers, whereas significantly fewer (58%) of those were located in the parenchyma in ASK1−/− livers. Positivity for chlorotyrosine protein adducts was recognized, especially in these interlobular areas around confluent bile infarcts of day 3 post-BDL WT livers. Fewer than 5 neutrophils were noted in sham-operated controls of both WT and ASK1−/− mice (n = 5 mice per group), and no neutrophils extravasated into the parenchyma. B: There were few accumulated neutrophils per injured portal tracts in ASK1−/− and WT livers at day 3 after BDL surgery (n = 10 mice per group). C: Real-time RT-PCR of adhesion molecules, such as ICAM-1, VCAM-1, VLA-4, and LFA-1, in the injured livers of WT and ASK1−/− mice (n = 10 mice per group) at day 3 after BDL surgery and control mice (n = 5 mice per group). Values are means ± SEM and were normalized for 18s rRNA expression (real-time RT-PCR). P < 0.05, ∗∗P < 0.001, and ∗∗∗P < 0.0001. Scale bar = 25 μm.
      Furthermore, IHC for CD3 revealed that modestly injured livers in ASK1−/− mice contained significantly fewer infiltrating T lymphocytes, especially around large bile ducts in the portal tracts, compared with WT mice at day 3 after BDL surgery (132.0 ± 10.1 per 10 fields in WT mice versus 100.2 ± 6.9 per 10 fields in ASK1−/− mice; P < 0.05) (Figure 5A). Mac-2 staining also revealed significantly fewer macrophages, including Kupffer cells, per injured portal tract in ASK1−/− livers than in WT livers (149.3 ± 4.9 per 10 fields in WT mice versus 137.5 ± 3.1 per 10 fields in ASK1−/− mice; P < 0.05) (Figure 5A). Neither CD3-positive T lymphocytes nor Mac-2–positive macrophages in interlobular areas around bile infarcts had significant differences in numbers between the two groups of day 3 post–BDL surgery mice (CD3: 143.7 ± 6.9 per 10 fields in WT mice versus 136.0 ± 7.6 per 10 fields in ASK1−/− mice) (Mac-2: 162.9 ± 2.8 per 10 fields in WT mice versus 160.1 ± 5.6 per 10 fields in ASK1−/− mice). Sham-operated animals had markedly fewer inflammatory cells (Figure 5A).
      Figure thumbnail gr5
      Figure 5Analysis of infiltrating T lymphocytes or Kupffer cells and proinflammatory reactions in BDL-induced cholestatic livers of mice. A: IHC of infiltrating CD3+ T lymphocytes and Mac-2+ macrophages (Kupffer cells) around large bile ducts in the portal tracts of day 3 BDL WT mice compared with ASK1−/− mice (n = 10 mice per group) and control mice (n = 5 mice per group). B and C: Real-time RT-PCR of TNF-α (B) and IL-1β (C) expression in the injured livers of WT mice compared with ASK1−/− mice (n = 10 mice per group) and control mice (n = 5 mice per group). ELISA confirmed gene expression. D: Real-time RT-PCR and Western blotting of iNOS in day 3 post-BDL livers of WT and ASK1−/− mice (n = 10 mice per group) and control mice (n = 5 mice per group). Values are means ± SEM and were normalized for 18s rRNA expression (real-time RT-PCR) or β-actin expression (Western blotting), respectively. P < 0.05, ∗∗P < 0.001, and ∗∗∗P < 0.0001. Scale bar = 25 μm.
      Real-time RT-PCR revealed that the expression levels of several adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), very late antigen-4 (VLA-4), and lymphocyte function-associated antigen-1 (LFA-1) (Figure 4C), and many proinflammatory signaling factors, such as TNF-α (Figure 5B), TNF receptor (TNFR) 2 (Supplemental Figure S3), IL-1β (Figure 5C), IL-1 receptor (IL-1R) 1, IL-6, nuclear factor-kappa B (NF-κB), interferon-γ (IFN-γ), Toll-like receptor 4 (TLR4) (Supplemental Figure S3), or iNOS (Figure 5D), were significantly down-regulated in the livers of day 3 post–BDL surgery ASK1−/− mice compared with WT and control mice (P < 0.05, P < 0.001, and P < 0.0001, respectively). Corresponding to these data, ELISA analysis found that serum TNF-α (Figure 5B) and IL-1β (Figure 5C) levels were significantly lower in ASK1−/− mice than in WT mice 3 days after BDL ligation (TNF-α: 6.79 ± 0.38 pg/mL in WT mice versus 5.61 ± 0.56 pg/mL in ASK1−/− mice, P < 0.05; IL-1β: 3.70 ± 0.45 pg/mL in WT mice versus 1.44 ± 1.30 pg/mL in ASK1−/− mice, P < 0.05). Moreover, the iNOS data were confirmed by Western blotting analysis (Figure 5D). No statistical differences between genotypes were observed in sham-operated animals for any the above parameters.

       Deletion of ASK1 Decreases Proliferating Activity of Hepatocytes and Large Cholangiocytes after BDL Surgery

      It has been found that the proliferation of interlobular hepatocytes and portal and periportal (interface) cholangiocytes peaks during the first 5 days after BDL surgery.
      • Woolbright B.L.
      • Jaeschke H.
      Novel insight into mechanisms of cholestatic liver injury.
      At day 3 after BDL surgery, the numbers of hepatocytes around bile infarcts (48.2 ± 1.9 per 10 fields in WT mice versus 35.1 ± 0.8 per 10 fields in ASK1−/− mice; P < 0.001) (Figure 6A) and Ki67+ large cholangiocytes (43.2 ± 1.4 per 10 fields in WT mice versus 35.1 ± 0.8 per 10 fields I ASK1−/− mice; P < 0.001) (Figure 6B) were significantly reduced in ASK1−/− mice compared with WT mice. In contrast, the number of Ki67+ small cholangiocytes was relatively small and not statistically different between the two groups of mice (14.6 ± 1.0 per 10 fields in WT mice versus 14.3 ± 0.6 per 10 fields in ASK1−/− mice). Double-immunofluorescence staining (Figure 6B) confirmed that the proliferating cells (green stained) were partly CK19+ large cholangiocytes (red stained). Similarly, the proliferation activity of BrdU+ cholangiocytes in large bile ducts 3 days after BDL surgery was significantly different between the two groups of mice (Figure 6B). Interestingly, a substantial number of Ki67+ or BrdU+ oval-to-spindle myofibroblast-like cells were also noted in portal tracts of WT mice at day 3 after BDL surgery (Figure 6B). In addition, some of the BrdU-retaining spindle cells were located within large bile ducts (Figure 6B).
      Figure thumbnail gr6
      Figure 6Analysis of proliferating liver cells in BDL-induced cholestasis. A: The number of Ki67+ hepatocytes (indicated by yellow arrowheads) was significantly reduced in ASK1−/− compared with WT mice, measurements of Ki67+ hepatocytes around bile infarcts, at day 3 after BDL surgery (n = 10 mice per group). B: Double-immunofluorescence staining revealed that proliferating cells in portal tracts (Ki67+, green stained) were large cholangiocytes (CK19+, red stained). The number of Ki67+ and CK19+ proliferating large cholangiocytes (white arrowheads) was markedly decreased in the portal tracts of day 3 post-BDL ASK1−/− mice than in WT mice (n = 10 mice per group). BrdU incorporation at day 3 after BDL surgery confirmed significant differences in proliferating large cholangiocytes between the two groups of mice. Interestingly, a substantial number of Ki67+ or BrdU+ oval to spindle myofibroblast-like cells are noted in the portal tracts of WT mice. Some BrdU-retaining spindle cells are located within large bile ducts (red arrowheads). C: Western blotting demonstrated that the hepatic expression of PCNA was significantly up-regulated in day 3 post-BDL WT and ASK1−/− mice (n = 10 mice per group) and control sham-operated mice (n = 5 mice per group). Values are means ± SEM (A and B) and were normalized for β-actin expression (Western blot analysis), respectively. ∗∗P < 0.001. Scale bar = 25 μm.
      Corresponding to the above histologic data, Western blotting analysis revealed that the expression of PCNA in modestly injured livers of ASK1−/− mice at day 3 after BDL surgery was significantly decreased compared with WT mice (Figure 6C). Sham-operated mice had no significant histologic or immunohistochemical differences between the two groups for any of the above parameters.

       Deletion of ASK1 Does Not Affect the Antioxidant State after BDL Surgery

      Unexpectedly, serum TBARS levels were low in both ASK1−/− and WT mice at day 3 after BDL surgery and without statistical difference (0.87 ± 0.02 nmol MDA per milligram of LDL protein in WT mice versus 0.86 ± 0.04 nmol MDA per milligrams of LDL protein in ASK1−/− mice). Real-time RT-PCR revealed that the antioxidant family gene expressions of peroxiredoxin (PRDX) 1 to 6, catalase, or Cu, Zn-superoxide dismutase (SOD) 1 to 2 were not significantly different between the two groups (data not shown).

       Deletion of ASK1 Represses Activation of Peribiliary Fibrogenic Cells in or around Large Bile Ducts without Significant Proliferation after BDL Surgery

      IBDM composed of CK19+ large-to-small bile ducts was significantly decreased in ASK1−/− mice at day 14 after BDL surgery compared with WT mice (10.3% ± 0.6% in WT mice versus 8.8% ± 0.7% in ASK1−/− mice; P < 0.05) (Figure 7, A and B), indicating that deletion of ASK1 could delay regenerative hyperplastic or proliferating responses of bile ducts against BDL injury. Similarly, IHC of day 14 BDL ASK1−/− mice livers revealed significantly lower specific and linear expression of α-SMA along the hyperplastic bile ductules and the hepatic sinusoids, in the portal and periportal tracts, compared with WT mice (5.9% ± 0.12% in WT mice versus 4.3% ± 0.1% in ASK1−/− mice; P < 0.001) (Figure 7, A and B). In mirror images of these sequential sections, α-SMA+ but desmin spindle to oval cells were easily detected in WT mice. These were consistent with the overt activation of peribiliary fibrogenic cells (ie, myofibroblasts), especially in WT mice, as previously described.
      • Gujral J.S.
      • Farhood A.
      • Bajt M.L.
      • Jaeschke H.
      Neutrophils aggravate acute liver injury during obstructive cholestasis in bile duct-ligated mice.
      Similarly, real-time RT-PCR revealed that the expression levels of platelet-derived growth factor-BB (PDGF-BB) and PDGF receptor β (PDGFRB) (Supplemental Figure S3) were significantly down-regulated in the livers of day 3 BDL ASK1−/− mice compared with WT and control mice (P < 0.05 and P < 0.0001, respectively). Double-immunofluorescence staining (Figure 7C) revealed that a small number of hyperplastic CK19+ cholangiocytes (green-stained) concomitantly had a phenotype of α-SMA+ myofibroblasts (red-stained) in WT mice at day 14 after BDL surgery, which was rarely observed in ASK1−/− mice.
      Figure thumbnail gr7
      Figure 7Measurements of IBDM and activated peribiliary fibrogenic cells in the BDL-induced cholestatic livers of mice. A and B: IHC For CK19, α-SMA, and desmin in portal and periportal tracts of day 14 BDL WT and ASK1−/− mice (n = 10 mice per group) and control sham-operated mice (n = 5 mice per group). C: Double immunofluorescence staining of hyperplastic CK19+ cholangiocytes (green-stained) and α-SMA+ activated peribiliary fibrogenic cells (red stained) in WT and ASK1−/− mice at day 14 after BDL surgery (yellow arrowheads). Values are means ± SEM. P < 0.05 and ∗∗P < 0.001. Scale bar = 25 μm. P, portal vein.

       Ultrastructural Analysis of Portal Tracts in BDL-Injured Livers

      Electron microscopy was performed to determine the phenotypes of ASK1-deficient stellate cells or large cholangiocytes in the portal and periportal tracts of day 14 BDL injured livers from ASK1−/− mice. Peribiliary fibrogenic cell phenotypes in the injured WT livers mainly had synthetic myofibroblast-like cells that contained significantly large amounts of cytoplasmic organelles, such as rough ER or lysosomes, compared with ASK1−/− mice (Supplemental Figure S4). ER and lysosome counts per peribiliary fibrogenic cell revealed statistically significant differences between the two groups of mice (7.6 ± 1.9 in WT mice versus 3.8 ± 0.9 in ASK1−/− mice; P < 0.05).
      However, large cholangiocytes in day 14 BDL livers of ASK1−/− mice had a significantly higher density of microvilli for each cell, which characterized a phenotype of large or mature cholangiocytes
      • Gradilone S.A.
      • Masyuk A.I.
      • Splinter P.L.
      • Banales J.M.
      • Huang B.Q.
      • Tietz P.S.
      • Masyuk T.V.
      • Larusso N.F.
      Cholangiocyte cilia express TRPV4 and detect changes in luminal tonicity inducing bicarbonate secretion.
      compared with WT mice (Supplemental Figure S4). Indeed, the morphologic features of large cholangiocytes in day 14 BDL injured livers of WT mice were not well preserved, displaying a significant decrease in density of microvilli per large cholangiocytes (10.5 ± 3.5 in WT mice versus 21.0 ± 1.6 in ASK1−/− mice; P < 0.05) (Supplemental Figure S4).

       Deletion of ASK1 Suppresses Peribiliary Fibrosis or Fibrogenesis after BDL

      Corresponding to the above IBDM or α-SMA IHC findings (Figure 7), picrosirius red staining revealed evidence of significantly greater peribiliary fibrosis in WT mice at day 14 after BDL surgery than in ASK1−/− mice (4.6% ± 0.2% in WT mice versus 2.0% ± 0.1% in ASK1−/− mice; P < 0.0001) and control mice (P < 0.0001) (Figure 8A). Similarly, Masson's trichrome staining in injured livers 14 days after BDL revealed significant differences between the two groups of mice (data not shown). Furthermore, real-time RT-PCR and Western blotting analyses revealed that the expression level of activated TGF-β1 (Figure 8B) was significantly down-regulated in the livers of day 14 BDL ASK1−/− mice compared with WT and control mice (P < 0.05).
      Figure thumbnail gr8
      Figure 8Analysis of peribiliary fibrosis or fibrogenesis in the BDL-induced cholestatic livers of mice. A: Picrosirius red staining reveals areas of peribiliary fibrosis or fibrogenesis in the cholestatic livers of WT and ASK1−/− mice at day 14 after BDL surgery (n = 10 mice per group) and sham-operated control mice (n = 5 mice per group). Under polarized light, the red-stained fibers expressed collagen type I, as shown by yellow birefringence. B: Real-time RT-PCR and Western blotting of activated TGF-β1 in day 14 post-BDL livers of WT and ASK1−/− mice (n = 10 mice per group) and control mice (n = 5 mice per group). Values are means ± SEM and were normalized for 18s rRNA expression (real-time RT-PCR) or β-actin expression (Western blotting), respectively. P < 0.05 and ∗∗∗P < 0.0001. Scale bar = 25 μm. P, portal vein.
      No significant differences between genotypes were observed in sham-operated animals for any of the above parameters. Both groups of control mice had markedly less fibrosis and fibrogenesis compared with experimental groups (Figures 7A and 8A).

       Microarray and Real-Time RT-PCR Analyses Reveal Variable Changes in Gene Expression Patterns, including Regulatory Genes of Bile Acid Synthesis, in BDL-Induced Cholestatic Models because of ASK1 Deficiency

      To identify new mechanisms involved in the suppression of liver necroinflammation and fibrosis or fibrogenesis observed in ASK1−/− mice, cDNA expression array analyses were performed comparing injured liver tissue samples obtained from ASK1−/− and ASK1+/+ WT mice 3 days after BDL surgery. A total of 86 genes revealed a twofold change (58 up-regulated and 28 down-regulated genes) in ASK1−/− mice. The top up-regulated and down-regulated genes are listed in Supplementary Table S1. They include the up-regulation of the cytochrome P450 families, such as cholesterol 7α-hydroxylase (CYP7A1) or sterol 12α-hydroxylase (CYP8B1), components of bile acid metabolism,
      • Georgiev P.
      • Jochum W.
      • Heinrich S.
      • Jang J.H.
      • Nocito A.
      • Dahm F.
      • Clavien P.A.
      Characterization of time-related changes after experimental bile duct ligation.
      and the down-regulation of IL-1 receptor type II (IL-1R2), a proinflammatory mediator, as well as ASK1. Real-time RT-PCR confirmed that the expression level of CYP7A1 (Supplemental Figure S3) was significantly up-regulated in the livers of day 3 BDL ASK1−/− mice compared with WT and control mice (P < 0.05 or P < 0.0001, respectively). Conversely, the expression levels of small heterodimer partner (SHP), but not farnesoid X receptor (FXR) (Supplemental Figure S3), were significantly down-regulated in the modestly injured livers of day 3 post–BDL surgery ASK1−/− mice compared with WT mice (P < 0.05).

      Discussion

      The present study reveals, for the first time to our knowledge, a critical protective role of depleted ASK1 in a BDL injury–induced murine cholestasis model. Cholestasis-induced necroinflammation and fibrosis or fibrogenesis were significantly reduced, and the survival rate was increased in ASK1−/− mice having no expression of ASK1 compared with WT mice that expressed higher levels of p-ASK1. ASK1−/− mice demonstrated potential anti-inflammatory profiles, especially in the acute phase of day 3 after BDL surgery, including manifestations of reduced bile infarcts, suppressed extensive inflammatory and proinflammatory reactions, lowered local (intrahepatic) and systemic (circulating) levels of bile acids, and decreased proliferation, but not apoptosis, in hepatocytes and large cholangiocytes. These effects also improved subsequent peribiliary fibrosis or fibrogenesis in ASK1-deleted mice with antifibrogenic profiles in the subacute (to chronic) phase of day 14 post-BDL injury. More recently, we found that ASK1−/− mice also had antiatherogenic (anti-inflammatory and/or antifibrogenic) features, especially in mechanical injury (carotid artery ligation)–induced vascular remodeling, manifesting as acute to subacute inflammatory disease (atherosclerosis).
      • Tasaki T.
      • Yamada S.
      • Guo X.
      • Tanimoto A.
      • Wang K.Y.
      • Nabeshima A.
      • Kitada S.
      • Noguchi H.
      • Kimura S.
      • Shimajiri S.
      • Kohno K.
      • Ichijo H.
      • Sasaguri Y.
      Apoptosis signal-regulating kinase 1 deficiency attenuates vascular injury-induced neointimal hyperplasia by suppressing apoptosis in smooth muscle cells.
      In this context, ASK1 deficiency would reveal significant protective effects against acute to subacute inflammatory injury. From other viewpoints, because ASK1−/− mice manifested as a delay of healing (ie, delayed repair process) in day 14 BDL livers, deletion of ASK1 might partly display a detrimental profile in the subacute to chronic phase after BDL. Figure 9 summarizes the diverse roles of ASK1 in the murine BDL model. Furthermore, we proposed that BDL-induced oncotic necrosis and proliferation, but not apoptosis, are closely correlated with the severity of the inflammatory responses and that the pathogenic roles of ASK1-mediated necroinflammation and proliferation of not only hepatocytes but also large cholangiocytes are crucial in cholestasis-induced fibrosis or fibrogenesis.
      Figure thumbnail gr9
      Figure 9The critical roles of ASK1 in BDL-induced cholestasis. This diagram depicts the diverse, key roles of ASK1 in the current BDL-induced murine cholestatic model.
      After establishing the BDL model, we detected the specific expression of ASK1 and/or activated p-ASK1 in the injured livers of WT mice by RT-PCR, IHC, and Western blotting (Figure 1) but not in ASK1−/− mice. Interestingly, IHC (Figure 1B) revealed that there would be heterogeneity of p-ASK1 expression profiles in large versus small cholangiocytes, almost parallel to the severity of BDL-induced cholestatic injury, which might partly support the previous findings regarding the functional heterogeneities in large to small cholangiocytes.
      • Alpini G.
      • Glaser S.
      • Ueno Y.
      • Pham L.
      • Podila P.V.
      • Caligiuri A.
      • LeSage G.
      • LaRusso N.F.
      Heterogeneity of the proliferative capacity of rat cholangiocytes after bile duct ligation.
      • LeSage G.
      • Glaser S.
      • Marucci L.
      • Benedetti A.
      • Phinizy J.L.
      • Rodgers R.
      • Caligiuri A.
      • Papa E.
      • Tretjak Z.
      • Jezequel A.M.
      • Holcomb L.A.
      • Alpini G.
      Acute carbon tetrachloride feeding induces damage of large but not small cholangiocytes from BDL rat liver.
      • Glaser S.
      • Gaudio E.
      • Rao A.
      • Pierce L.M.
      • Onori P.
      • Franchitto A.
      • Francis H.L.
      • Dostal D.E.
      • Venter J.K.
      • DeMorrow S.
      • Mancinelli R.
      • Carpino G.
      • Alvaro D.
      • Kopriva S.E.
      • Savage J.M.
      • Alpini G.
      Morphological and functional heterogeneity of the mouse intrahepatic biliary epithelium.
      The MAP3K family is likely stimulated by distinct mechanisms during the progression of cholestatic liver injury in response to various immune system mediators, such as TNF-α, IL-1β, or iNOS (Figure 6 and Supplemental Figure S3), and/or unknown ER and oxidative stressors. This subsequently increases the expression of activated downstream p-p38 and p-JNK. Indeed, the expression of these MAPK family members was significantly higher in WT livers than the modestly injured livers of ASK1−/− mice after BDL surgery (Figure 1C). However, only <0.1% of hepatocytes or large cholangiocytes were apoptotic even in both of the two groups at day 3 after BDL surgery, respectively (Supplemental Figure S2A), using morphologic criteria (ie, the gold standard to detect liver cells undergoing apoptosis after BDL, according to much of the recent data in the hepatology study).
      • Schoemaker M.H.
      • Gommans W.M.
      • Conde de la Rosa L.
      • Homan M.
      • Klok P.
      • Trautwein C.
      • van Goor H.
      • Poelstra K.
      • Haisma H.J.
      • Jansen P.L.
      • Moshage H.
      Resistance of rat hepatocytes against bile acid-induced apoptosis in cholestatic liver injury is due to nuclear factor-kappa B activation.
      • Gujral J.S.
      • Liu J.
      • Farhood A.
      • Jaeschke H.
      Reduced oncotic necrosis in Fas receptor-deficient C57BL/6J-lpr mice after bile duct ligation.
      • Fickert P.
      • Trauner M.
      • Fuchsbichler A.
      • Zollner G.
      • Wagner M.
      • Marschall H.U.
      • Zatloukal K.
      • Denk H.
      Oncosis represents the main type of cell death in mouse models of cholestasis.
      • Nalapareddy P.D.
      • Schüngel S.
      • Hong J.Y.
      • Manns M.P.
      • Jaeschke H.
      • Vogel A.
      The BH3-only protein bid does not mediate death-receptor-induced liver injury in obstructive cholestasis.
      • Mitchell C.
      • Mahrouf-Yorgov M.
      • Mayeuf A.
      • Robin M.A.
      • Mansouri A.
      • Fromenty B.
      • Gilgenkrantz H.
      Overexpression of Bcl-2 in hepatocytes protects against injury but does not attenuate fibrosis in a mouse model of chronic cholestatic liver disease.
      Moreover, in particular, TUNEL+ hepatocytes had no apoptotic morphologic features because both of the nucleus and cytosol were stained, which is known to be characteristic of early necrosis.
      • Grasl-Kraupp B.
      • Ruttkay-Nedecky B.
      • Koudelka H.
      • Bukowska K.
      • Bursch W.
      • Schulte-Hermann R.
      In situ detection of fragmented DNA (TUNEL assay) fails to discriminate among apoptosis, necrosis, and autolytic cell death: a cautionary note.
      Thus, these findings indicate that apoptosis plays little role in BDL-induced cholestasis and that ASK1−/− mice could be a good model for determining the net effects of blocked ASK1 signaling pathway in cholestatic necroinflammation and fibrosis.
      Our data are in agreement with various previous observations from several other laboratories. Hepatic inflammation that involves the accumulation and recruitment of neutrophils, Kupffer cells, or T lymphocytes (Figures 4 and 5), all of which are cellular components of the innate to adapted immune system, plays a critical role in contributing to a large fraction of oncotic necrosis and subsequent fibrosis or fibrogenesis during murine BDL models.
      • Gujral J.S.
      • Liu J.
      • Farhood A.
      • Jaeschke H.
      Reduced oncotic necrosis in Fas receptor-deficient C57BL/6J-lpr mice after bile duct ligation.
      • Nalapareddy P.D.
      • Schüngel S.
      • Hong J.Y.
      • Manns M.P.
      • Jaeschke H.
      • Vogel A.
      The BH3-only protein bid does not mediate death-receptor-induced liver injury in obstructive cholestasis.
      • Mitchell C.
      • Mahrouf-Yorgov M.
      • Mayeuf A.
      • Robin M.A.
      • Mansouri A.
      • Fromenty B.
      • Gilgenkrantz H.
      Overexpression of Bcl-2 in hepatocytes protects against injury but does not attenuate fibrosis in a mouse model of chronic cholestatic liver disease.
      • Gujral J.S.
      • Farhood A.
      • Bajt M.L.
      • Jaeschke H.
      Neutrophils aggravate acute liver injury during obstructive cholestasis in bile duct-ligated mice.
      Moreover, it is accompanied by not only the stimulation of various adhesion molecules and proinflammatory cascades
      • Gujral J.S.
      • Liu J.
      • Farhood A.
      • Jaeschke H.
      Reduced oncotic necrosis in Fas receptor-deficient C57BL/6J-lpr mice after bile duct ligation.
      • Nalapareddy P.D.
      • Schüngel S.
      • Hong J.Y.
      • Manns M.P.
      • Jaeschke H.
      • Vogel A.
      The BH3-only protein bid does not mediate death-receptor-induced liver injury in obstructive cholestasis.
      • Mitchell C.
      • Mahrouf-Yorgov M.
      • Mayeuf A.
      • Robin M.A.
      • Mansouri A.
      • Fromenty B.
      • Gilgenkrantz H.
      Overexpression of Bcl-2 in hepatocytes protects against injury but does not attenuate fibrosis in a mouse model of chronic cholestatic liver disease.
      • Gujral J.S.
      • Farhood A.
      • Bajt M.L.
      • Jaeschke H.
      Neutrophils aggravate acute liver injury during obstructive cholestasis in bile duct-ligated mice.
      but also significant up-regulation of TGF-β1.
      • Nalapareddy P.D.
      • Schüngel S.
      • Hong J.Y.
      • Manns M.P.
      • Jaeschke H.
      • Vogel A.
      The BH3-only protein bid does not mediate death-receptor-induced liver injury in obstructive cholestasis.
      • Mitchell C.
      • Mahrouf-Yorgov M.
      • Mayeuf A.
      • Robin M.A.
      • Mansouri A.
      • Fromenty B.
      • Gilgenkrantz H.
      Overexpression of Bcl-2 in hepatocytes protects against injury but does not attenuate fibrosis in a mouse model of chronic cholestatic liver disease.
      Interestingly, iNOS was markedly expressed in injured liver cells of WT mice (Figure 5D) in response to hepatic activation through various inflammatory and proinflammatory cytokines, including TNF-α or IL-1β (Figure 5, B and C and Supplemental Figure S3), and stimulated p38 MAPK-mediated signaling (Figure 1C), similar to mouse astrocytes or bovine chondrocytes, as previously described.
      • Da Silva J.
      • Pierrat B.
      • Mary J.L.
      • Lesslauer W.
      Blockade of p38 mitogen-activated protein kinase pathway inhibits inducible nitric-oxide synthase expression in mouse astrocytes.
      • Badger A.M.
      • Cook M.N.
      • Lark M.W.
      • Newman-Tarr T.M.
      • Swift B.A.
      • Nelson A.H.
      • Barone F.C.
      • Kumar S.
      SB 203580 inhibits p38 mitogen-activated protein kinase, nitric oxide production, and inducible nitric oxide synthase in bovine cartilage-derived chondrocytes.
      Furthermore, corresponding to Yes-associated protein–deficient mice demonstrating suppressed proliferation of large cholangiocytes in day 5 BDL livers with following increased bile infarcts at day 15 after BDL surgery,
      • Bai H.
      • Zhang N.
      • Xu Y.
      • Chen Q.
      • Khan M.
      • Potter J.J.
      • Nayar S.K.
      • Cornish T.
      • Alpini G.
      • Bronk S.
      • Pan D.
      • Anders R.A.
      Yes-associated protein regulates the hepatic response after bile duct ligation.
      ASK1−/− mice had significantly fewer proliferating hepatocytes and large cholangiocytes and reduced PCNA expression at day 3 after BDL surgery (Figure 5), together with greater necrotic foci in day 14 BDL livers (Figure 3). On the one hand, on the basis of a vast amount of results, this is just a consequence of reduced early-phase liver injury (ie, necroinflammation) in ASK1−/− mice rather than ASK1 directly affecting proliferation. On the other hand, because ASK1 is situated in an important upstream position for many signal transduction pathways, ASK1 deficiency suppresses many signals required for not only inflammation but also cell survival or proliferation.
      • Ichijo H.
      • Nishida E.
      • Irie K.
      • Digike P.T.
      • Saitoh M.
      • Moriguchi T.
      • Takagi M.
      • Matsumoto K.
      • Miyazono K.
      • Gotoh Y.
      Induction of apoptosis by ASK1, mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways.
      • Yamada S.
      • Ding Y.
      • Tanimoto A.
      • Wang K.Y.
      • Guo X.
      • Li Z.
      • Tasaki T.
      • Nabesima A.
      • Murata Y.
      • Shimajiri S.
      • Kohno K.
      • Ichijo H.
      • Sasaguri Y.
      Apoptosis signal-regulating kinase 1 deficiency accelerates hyperlipidemia-induced atheromatous plaques via suppression of macrophage apoptosis.
      • Tasaki T.
      • Yamada S.
      • Guo X.
      • Tanimoto A.
      • Wang K.Y.
      • Nabeshima A.
      • Kitada S.
      • Noguchi H.
      • Kimura S.
      • Shimajiri S.
      • Kohno K.
      • Ichijo H.
      • Sasaguri Y.
      Apoptosis signal-regulating kinase 1 deficiency attenuates vascular injury-induced neointimal hyperplasia by suppressing apoptosis in smooth muscle cells.
      Therefore, the ASK1 pathway might have a close relationship with PCNA-induced proliferation signaling in liver cells, supported by the reported data that inhibition of the JNK pathway induces diminished proliferative responses to partial hepatectomy together with decreased PCNA expression.
      • Schwabe R.F.
      • Bradham C.A.
      • Uehara T.
      • Hatano E.
      • Bennett B.L.
      • Schoonhoven R.
      • Brenner D.A.
      c-Jun-N-terminal kinase drives cyclin D1 expression and proliferation during liver regeneration.
      Nevertheless, further studies are needed to clarify those issues because identifying promising key factors and pathways related to the MAP3K family member, ASK1, would help control hepatic responses against cholestasis-induced liver injury.
      The present pathologic or molecular findings should be remarkable for other reasons. First, double-immunofluorescence staining with CK19 and α-SMA indicated a small number of hyperplastic cells (within IBDM) having phenotypes of both cholangiocytes and peribiliary fibrogenic cells, especially in WT mice at day 14 after BDL surgery, but which were absent or very rare in ASK1−/− mice (Figure 7C). The double-stained immature and precursor-like cells should play a key role in the repair process of injured cholangiocytes during cholestasis because hyperplastic or proliferating cholangiocytes could originate from pleiotropic hepatic stem or progenitor cells.
      • Cantz T.
      • Manns M.P.
      • Ott M.
      Stem cells in liver regeneration and therapy.
      Second, ultrastructural analysis confirmed that hyperplastic large cholangiocytes in WT mice at day 14 after BDL surgery manifested as a phenotype of immature cholangiocytes (ie, differentiation of small into large cholangiocytes).
      • Alpini G.
      • Glaser S.
      • Ueno Y.
      • Pham L.
      • Podila P.V.
      • Caligiuri A.
      • LeSage G.
      • LaRusso N.F.
      Heterogeneity of the proliferative capacity of rat cholangiocytes after bile duct ligation.
      • LeSage G.
      • Glaser S.
      • Marucci L.
      • Benedetti A.
      • Phinizy J.L.
      • Rodgers R.
      • Caligiuri A.
      • Papa E.
      • Tretjak Z.
      • Jezequel A.M.
      • Holcomb L.A.
      • Alpini G.
      Acute carbon tetrachloride feeding induces damage of large but not small cholangiocytes from BDL rat liver.
      • Glaser S.
      • Gaudio E.
      • Rao A.
      • Pierce L.M.
      • Onori P.
      • Franchitto A.
      • Francis H.L.
      • Dostal D.E.
      • Venter J.K.
      • DeMorrow S.
      • Mancinelli R.
      • Carpino G.
      • Alvaro D.
      • Kopriva S.E.
      • Savage J.M.
      • Alpini G.
      Morphological and functional heterogeneity of the mouse intrahepatic biliary epithelium.
      These cholangiocytes had significantly fewer microvilli, compared with ASK1−/− mice, and displayed overtly well-preserved morphologic features (Supplemental Figure S4). Furthermore, electron microscopy revealed that peribiliary fibrogenic cells from WT mice also had a dedifferentiated (immature) synthetic myofibroblast-like phenotype, containing a significantly larger number of cell organelles (Supplemental Figure S4). This is similar to the migrating intimal smooth muscle cells in injured arteries, as previously described by us.
      • Tasaki T.
      • Yamada S.
      • Guo X.
      • Tanimoto A.
      • Wang K.Y.
      • Nabeshima A.
      • Kitada S.
      • Noguchi H.
      • Kimura S.
      • Shimajiri S.
      • Kohno K.
      • Ichijo H.
      • Sasaguri Y.
      Apoptosis signal-regulating kinase 1 deficiency attenuates vascular injury-induced neointimal hyperplasia by suppressing apoptosis in smooth muscle cells.
      The activated p38 MAPK pathway plays a crucial role in myofibroblastic dedifferentiation of not only smooth muscle cells
      • Tasaki T.
      • Yamada S.
      • Guo X.
      • Tanimoto A.
      • Wang K.Y.
      • Nabeshima A.
      • Kitada S.
      • Noguchi H.
      • Kimura S.
      • Shimajiri S.
      • Kohno K.
      • Ichijo H.
      • Sasaguri Y.
      Apoptosis signal-regulating kinase 1 deficiency attenuates vascular injury-induced neointimal hyperplasia by suppressing apoptosis in smooth muscle cells.
      • Hayashi K.
      • Takahashi M.
      • Kimura K.
      • Nishida W.
      • Saga H.
      • Sobue K.
      Changes in the balance of phosphoinositide 3-kinase/protein kinase B (Akt) and the mitogen-activated protein kinases (ERK/p38 MAPK) determine a phenotype of visceral and vascular smooth muscle cells.
      but also possibly peribiliary fibrogenic cells. These features are in line with significantly increased collagen production in day 14 BDL livers from WT mice (Figure 8). Third, cDNA expression array analyses revealed the significant up-regulation of cytochrome P450 families in ASK1−/− mice livers at day 3 after BDL surgery, including CYP7A1 or CYP8B1, which are key regulated enzymes in bile acid synthesis (Supplemental Table S1).
      • Lefebvre P.
      • Cariou B.
      • Lien F.
      • Kuipers F.
      • Staels B.
      Role of bile acids and bile acid receptors in metabolic regulation.
      Because significantly lower levels of bile acids in the ASK1−/− livers (Figure 2C) possibly activate FXR less and subsequently up-regulate SHP less (Supplemental Figure S3), involving the inhibition of SHP-mediated CYP7A1 repression,
      • Georgiev P.
      • Jochum W.
      • Heinrich S.
      • Jang J.H.
      • Nocito A.
      • Dahm F.
      • Clavien P.A.
      Characterization of time-related changes after experimental bile duct ligation.
      these data might be consistent with the potential anticholestatic profile, especially in the acute phase of the BDL model.
      In conclusion, on the basis of our collected data, we postulate that ASK1 deficiency plays critical roles in ameliorating cholestatic liver injury together with i) decreased accumulation and recruitment of various inflammatory cells into interlobular and portal areas accompanied by down-regulated expression of various adhesion molecules and proinflammatory mediators, ii) reduced hepatocellular necrosis (bile infarcts), iii) suppressed proliferation of hepatocytes and large cholangiocytes, iv) inactivation and rare myofibroblastic dedifferentiation in peribiliary fibrogenic cells, and v) repressed synthesis of collagen-rich extracellular matrix by the above mechanisms, as summarized in Figure 9. Hence, we can conclude that depletion of ASK1 prevents the development of acute necroinflammation and subsequent peribiliary fibrosis or fibrogenesis, suppressing aberrant regenerative (proliferating) responses and exerting beneficial effects in BDL injury–induced cholestasis. All these features indicate that a specific ASK1 pathway blocker could offer a therapeutic strategy against the progression of human cholestatic liver disease.

      Acknowledgments

      We thank Hiroko Isagai, Hana Nishimura, and Naoko Une for their expert technical assistance.

      Supplemental Data

      • Supplemental Figure S1

        Kaplan-Meier curves of overall survival in ASK1−/− and WT mice undergoing BDL surgery (n = 58 mice per group) and ASK1−/− and WT mice undergoing sham surgery (n = 12 mice per group). ∗∗P < 0.001.

      • Supplemental Figure S2

        Analysis of apoptotic activity on liver cells in BDL-induced cholestasis. A: ASK1−/− and WT mice undergoing BDL surgery (n = 10 mice per group) and sham-operated controls (n = 5 mice per group). B: Western blotting for caspase-3 activities and caspase-3 processing between the respective groups (WT versus ASK1−/− mice and BDL versus sham surgery). Scale bar = 25 μm.

      • Supplemental Figure S3

        Real-time RT-PCR analyses of inflammatory signaling, growth factors, and bile acid metabolism gene expression in the livers of BDL mice. Real-time RT-PCR analyses of the inflammatory signaling molecules TNFR2, IL-1R, IL-6, NF-κB, IFN-γ, and TLR4 and the growth factor PDGF-BB and its receptor (PDGFRB) in the injured livers of WT mice at day 3 after BDL surgery and compared with ASK1−/− mice (n = 10 mice per group) and control mice (n = 5 mice per group). The expression levels of CYP7A1, SHP, and FXR were also assessed in day 3 BDL ASK1−/− mice livers and compared with WT and control mice. Values are means ± SE and normalized for 18s rRNA expression. P < 0.05, ∗∗P < 0.001, and ∗∗∗P < 0.0001.

      • Supplemental Figure S4

        Ultrastructural analysis of portal tracts in injured livers of BDL mice. Electron microscopy of ASK1-deficient stellate cells phenotypes in day 14 BDL livers of ASK1−/− and WT mice. The phenotypes of peribiliary fibrogenic cells in WT mice were predominantly synthetic myofibroblast-like cells that contained significantly larger amounts of cytoplasmic organelles (arrows), such as rough ER or lysosome, compared with ASK1−/− mice (n = 4 mice per group). Large cholangiocytes in day 14 BDL livers of ASK1−/− mice had a significantly higher density of microvilli (arrowheads) per cell, suggestive of a large or mature cholangiocyte phenotype, compared with WT mice, which had poorly preserved morphologic features (n = 4 mice per group). Scale bar = 2 μm. Original magnification: ×4000.

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