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Müller H, Straßmann JK, Baier AS, von Bülow V, Stettler F, Hagen MJ, Schmidt FP, Tschuschner A, Schmid AR, Zahner D, Köhler K, Pons-Kühnemann J, Leufkens D, Glebe D, Kaur S, Möscheid MF, Haeberlein S, Grevelding CG, Weiskirchen R, El-Kassas M, Zalata K, Roeb E, Roderfeld M. Liver Fibrosis Is Enhanced by a Higher Egg Burden in Younger Mice Infected with S. mansoni. Cells 2024; 13:1643. [PMID: 39404406 PMCID: PMC11475498 DOI: 10.3390/cells13191643] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2024] [Revised: 09/25/2024] [Accepted: 10/01/2024] [Indexed: 10/19/2024] Open
Abstract
Schistosomiasis affects over 250 million people worldwide, with the highest prevalence at the age of 10-14 years. The influence of the host's age on the severity of liver damage is unclear. We infected male 8, 14, and 20-week-old mice with S. mansoni. Hepatic damage, inflammation, fibrosis, and metabolism were analyzed by RT-qPCR, Western blotting, ELISA, immunohistochemistry, and mechanistic transwell chamber experiments using S. mansoni eggs and human hepatic stellate cells (HSCs) or primary mouse hepatocytes. Major results were validated in human biopsies. We found that hepatosplenomegaly, granuloma size, egg load, inflammation, fibrosis, and glycogen stores all improved with the increasing age of the host. However, serum alanine transaminase (ALT) levels were lowest in young mice infected with S. mansoni. Hepatic carbohydrate exploitation was characterized by a shift towards Warburg-like glycolysis in S. mansoni-infected animals. Notably, S. mansoni eggs stimulated hepatic stellate cells to an alternatively activated phenotype (GFAP+/desmin+/αSMA-) that secretes IL-6 and MCP-1. The reduction of fibrosis in older age likely depends on the fine-tuning of regulatory and inflammatory cytokines, alternative HSC activation, and the age-dependent preservation of hepatic energy stores. The current results emphasize the significance of investigations on the clinical relevance of host age-dependent liver damage in patients with schistosomiasis.
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Affiliation(s)
- Heike Müller
- Department of Gastroenterology, Justus Liebig University, 35392 Giessen, Germany; (H.M.); (J.K.S.); (A.S.B.); (V.v.B.); (F.S.); (M.J.H.); (F.P.S.); (A.T.); (E.R.)
| | - Jan K. Straßmann
- Department of Gastroenterology, Justus Liebig University, 35392 Giessen, Germany; (H.M.); (J.K.S.); (A.S.B.); (V.v.B.); (F.S.); (M.J.H.); (F.P.S.); (A.T.); (E.R.)
| | - Anne S. Baier
- Department of Gastroenterology, Justus Liebig University, 35392 Giessen, Germany; (H.M.); (J.K.S.); (A.S.B.); (V.v.B.); (F.S.); (M.J.H.); (F.P.S.); (A.T.); (E.R.)
| | - Verena von Bülow
- Department of Gastroenterology, Justus Liebig University, 35392 Giessen, Germany; (H.M.); (J.K.S.); (A.S.B.); (V.v.B.); (F.S.); (M.J.H.); (F.P.S.); (A.T.); (E.R.)
| | - Frederik Stettler
- Department of Gastroenterology, Justus Liebig University, 35392 Giessen, Germany; (H.M.); (J.K.S.); (A.S.B.); (V.v.B.); (F.S.); (M.J.H.); (F.P.S.); (A.T.); (E.R.)
| | - Maximilian J. Hagen
- Department of Gastroenterology, Justus Liebig University, 35392 Giessen, Germany; (H.M.); (J.K.S.); (A.S.B.); (V.v.B.); (F.S.); (M.J.H.); (F.P.S.); (A.T.); (E.R.)
| | - Fabian P. Schmidt
- Department of Gastroenterology, Justus Liebig University, 35392 Giessen, Germany; (H.M.); (J.K.S.); (A.S.B.); (V.v.B.); (F.S.); (M.J.H.); (F.P.S.); (A.T.); (E.R.)
| | - Annette Tschuschner
- Department of Gastroenterology, Justus Liebig University, 35392 Giessen, Germany; (H.M.); (J.K.S.); (A.S.B.); (V.v.B.); (F.S.); (M.J.H.); (F.P.S.); (A.T.); (E.R.)
| | - Andreas R. Schmid
- Department of Internal Medicine III, Justus Liebig University, 35392 Giessen, Germany;
| | - Daniel Zahner
- Central Laboratory Animal Facility, Justus Liebig University, 35392 Giessen, Germany;
| | - Kernt Köhler
- Institute of Veterinary Pathology, Justus Liebig University, 35392, Germany;
| | - Jörn Pons-Kühnemann
- Institute of Medical Informatics, Justus Liebig University, 35392, Germany; (J.P.-K.); (D.L.)
| | - Daniel Leufkens
- Institute of Medical Informatics, Justus Liebig University, 35392, Germany; (J.P.-K.); (D.L.)
| | - Dieter Glebe
- Institute of Medical Virology, National Reference Center for Hepatitis B Viruses and Hepatitis D Viruses, German Center for Infection Research (DZIF; Partner Site Giessen-Marburg-Langen), Justus Liebig University, 35392 Giessen, Germany; (D.G.); (S.K.)
| | - Surmeet Kaur
- Institute of Medical Virology, National Reference Center for Hepatitis B Viruses and Hepatitis D Viruses, German Center for Infection Research (DZIF; Partner Site Giessen-Marburg-Langen), Justus Liebig University, 35392 Giessen, Germany; (D.G.); (S.K.)
| | - Max F. Möscheid
- Institute of Parasitology, BFS, Justus Liebig University, 35392 Giessen, Germany; (M.F.M.); (S.H.); (C.G.G.)
| | - Simone Haeberlein
- Institute of Parasitology, BFS, Justus Liebig University, 35392 Giessen, Germany; (M.F.M.); (S.H.); (C.G.G.)
| | - Christoph G. Grevelding
- Institute of Parasitology, BFS, Justus Liebig University, 35392 Giessen, Germany; (M.F.M.); (S.H.); (C.G.G.)
| | - Ralf Weiskirchen
- Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry (IFMPEGKC), RWTH University Hospital Aachen, 52074 Aachen, Germany;
| | - Mohamed El-Kassas
- Endemic Medicine Department, Faculty of Medicine, Helwan University, Cairo 11795, Egypt;
| | - Khaled Zalata
- Pathology Department, Faculty of Medicine, Mansoura University, Mansoura 35516, Egypt;
| | - Elke Roeb
- Department of Gastroenterology, Justus Liebig University, 35392 Giessen, Germany; (H.M.); (J.K.S.); (A.S.B.); (V.v.B.); (F.S.); (M.J.H.); (F.P.S.); (A.T.); (E.R.)
| | - Martin Roderfeld
- Department of Gastroenterology, Justus Liebig University, 35392 Giessen, Germany; (H.M.); (J.K.S.); (A.S.B.); (V.v.B.); (F.S.); (M.J.H.); (F.P.S.); (A.T.); (E.R.)
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Park HJ, Choi J, Kim H, Yang DY, An TH, Lee EW, Han BS, Lee SC, Kim WK, Bae KH, Oh KJ. Cellular heterogeneity and plasticity during NAFLD progression. Front Mol Biosci 2023; 10:1221669. [PMID: 37635938 PMCID: PMC10450943 DOI: 10.3389/fmolb.2023.1221669] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2023] [Accepted: 07/18/2023] [Indexed: 08/29/2023] Open
Abstract
Nonalcoholic fatty liver disease (NAFLD) is a progressive liver disease that can progress to nonalcoholic steatohepatitis (NASH), NASH-related cirrhosis, and hepatocellular carcinoma (HCC). NAFLD ranges from simple steatosis (or nonalcoholic fatty liver [NAFL]) to NASH as a progressive form of NAFL, which is characterized by steatosis, lobular inflammation, and hepatocellular ballooning with or without fibrosis. Because of the complex pathophysiological mechanism and the heterogeneity of NAFLD, including its wide spectrum of clinical and histological characteristics, no specific therapeutic drugs have been approved for NAFLD. The heterogeneity of NAFLD is closely associated with cellular plasticity, which describes the ability of cells to acquire new identities or change their phenotypes in response to environmental stimuli. The liver consists of parenchymal cells including hepatocytes and cholangiocytes and nonparenchymal cells including Kupffer cells, hepatic stellate cells, and endothelial cells, all of which have specialized functions. This heterogeneous cell population has cellular plasticity to adapt to environmental changes. During NAFLD progression, these cells can exert diverse and complex responses at multiple levels following exposure to a variety of stimuli, including fatty acids, inflammation, and oxidative stress. Therefore, this review provides insights into NAFLD heterogeneity by addressing the cellular plasticity and metabolic adaptation of hepatocytes, cholangiocytes, hepatic stellate cells, and Kupffer cells during NAFLD progression.
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Affiliation(s)
- Hyun-Ju Park
- Metabolic Regulation Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Republic of Korea
- Department of Functional Genomics, KRIBB School of Bioscience, University of Science and Technology (UST), Daejeon, Republic of Korea
| | - Juyong Choi
- Metabolic Regulation Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Republic of Korea
- Department of Functional Genomics, KRIBB School of Bioscience, University of Science and Technology (UST), Daejeon, Republic of Korea
| | - Hyunmi Kim
- Metabolic Regulation Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Republic of Korea
- Department of Functional Genomics, KRIBB School of Bioscience, University of Science and Technology (UST), Daejeon, Republic of Korea
| | - Da-Yeon Yang
- Metabolic Regulation Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Republic of Korea
- Department of Functional Genomics, KRIBB School of Bioscience, University of Science and Technology (UST), Daejeon, Republic of Korea
| | - Tae Hyeon An
- Metabolic Regulation Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Republic of Korea
- Department of Functional Genomics, KRIBB School of Bioscience, University of Science and Technology (UST), Daejeon, Republic of Korea
| | - Eun-Woo Lee
- Metabolic Regulation Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Republic of Korea
- Department of Functional Genomics, KRIBB School of Bioscience, University of Science and Technology (UST), Daejeon, Republic of Korea
| | - Baek-Soo Han
- Department of Functional Genomics, KRIBB School of Bioscience, University of Science and Technology (UST), Daejeon, Republic of Korea
- Biodefense Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Republic of Korea
| | - Sang Chul Lee
- Metabolic Regulation Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Republic of Korea
- Department of Functional Genomics, KRIBB School of Bioscience, University of Science and Technology (UST), Daejeon, Republic of Korea
| | - Won Kon Kim
- Metabolic Regulation Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Republic of Korea
- Department of Functional Genomics, KRIBB School of Bioscience, University of Science and Technology (UST), Daejeon, Republic of Korea
| | - Kwang-Hee Bae
- Metabolic Regulation Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Republic of Korea
- Department of Functional Genomics, KRIBB School of Bioscience, University of Science and Technology (UST), Daejeon, Republic of Korea
| | - Kyoung-Jin Oh
- Metabolic Regulation Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Republic of Korea
- Department of Functional Genomics, KRIBB School of Bioscience, University of Science and Technology (UST), Daejeon, Republic of Korea
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Maggi L, Camelo GMA, Rocha IC, Pereira Alves W, Moreira JMP, Almeida Pereira T, Tafuri WL, Rabelo ÉML, Correa A, Ecco R, Negrão-Corrêa DA. Role of the IL-33/ST2 Activation Pathway in the Development of the Hepatic Fibrosis Induced by Schistosoma mansoni Granulomas in Mice. Int J Mol Sci 2023; 24:10237. [PMID: 37373379 PMCID: PMC10299179 DOI: 10.3390/ijms241210237] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2023] [Revised: 06/06/2023] [Accepted: 06/13/2023] [Indexed: 06/29/2023] Open
Abstract
Schistosoma mansoni eggs retained in host tissues induce innate cytokine release, contributing to the induction of Type-2 immune responses and granuloma formation, important to restrain cytotoxic antigens, but leading to fibrosis. Interleukin(IL)-33 participates in experimental models of inflammation and chemically induced fibrosis, but its role in S. mansoni-induced fibrosis is still unknown. To explore the role of the IL-33/suppressor of the tumorigenicity 2 (ST2) pathway, serum and liver cytokine levels, liver histopathology, and collagen deposition were comparatively evaluated in S. mansoni-infected wild-type (WT) and IL-33-receptor knockout (ST2-/-) BALB/c mice. Our data show similar egg counts and hydroxyproline in the livers of infected WT and ST2-/- mice; however, the extracellular matrix in ST2-/- granulomas was loose and disorganised. Pro-fibrotic cytokines, such as IL-13 and IL-17, and the tissue-repairing IL-22 were significantly lower in ST2-/- mice, especially in chronic schistosomiasis. ST2-/- mice also showed decreased α-smooth muscle actin (α-SMA) expression in granuloma cells, in addition to reduced Col III and Col VI mRNA levels and reticular fibres. Therefore, IL-33/ST2 signalling is essential for tissue repairing and myofibroblast activation during S. mansoni infection. Its disruption results in inappropriate granuloma organisation, partly due to the reduced type III and VI collagen and reticular fibre formation.
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Affiliation(s)
- Laura Maggi
- Laboratório de Esquistossomose e Imunohelmintologia, Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte 31270-901, MG, Brazil; (L.M.); (G.M.A.C.); (I.C.R.); (J.M.P.M.)
| | - Genil Mororó Araújo Camelo
- Laboratório de Esquistossomose e Imunohelmintologia, Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte 31270-901, MG, Brazil; (L.M.); (G.M.A.C.); (I.C.R.); (J.M.P.M.)
| | - Izabella Chrystina Rocha
- Laboratório de Esquistossomose e Imunohelmintologia, Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte 31270-901, MG, Brazil; (L.M.); (G.M.A.C.); (I.C.R.); (J.M.P.M.)
- Curso de Enfermagem, Instituto de Ciências Biológicas e Saúde, Universidade Federal de Mato Grosso, Barra do Garça 78698-000, MG, Brazil
| | - William Pereira Alves
- Laboratório de Parasitologia Molecular, Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte 31270-901, MG, Brazil; (W.P.A.); (É.M.L.R.)
| | - João Marcelo Peixoto Moreira
- Laboratório de Esquistossomose e Imunohelmintologia, Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte 31270-901, MG, Brazil; (L.M.); (G.M.A.C.); (I.C.R.); (J.M.P.M.)
| | - Thiago Almeida Pereira
- Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA;
| | - Wagner Luiz Tafuri
- Laboratório de Patologia das Leishmanioses, Departamento de Patologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte 31270-901, MG, Brazil;
| | - Élida Mara Leite Rabelo
- Laboratório de Parasitologia Molecular, Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte 31270-901, MG, Brazil; (W.P.A.); (É.M.L.R.)
| | - Ary Correa
- Laboratório de Micologia, Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte 31270-901, MG, Brazil;
| | - Roselene Ecco
- Setor de Patologia, Escola Veterinária, Universidade Federal de Minas Gerais, Belo Horizonte 31270-901, MG, Brazil;
| | - Deborah Aparecida Negrão-Corrêa
- Laboratório de Esquistossomose e Imunohelmintologia, Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte 31270-901, MG, Brazil; (L.M.); (G.M.A.C.); (I.C.R.); (J.M.P.M.)
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Activated Hepatic Stellate Cells Promote the M1 to M2 Macrophage Transformation and Liver Fibrosis by Elevating the Histone Acetylation Level. DISEASE MARKERS 2022; 2022:9883831. [PMID: 36133436 PMCID: PMC9484931 DOI: 10.1155/2022/9883831] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/18/2022] [Revised: 08/09/2022] [Accepted: 08/13/2022] [Indexed: 11/18/2022]
Abstract
Liver fibrosis results from the formation of fibrous scars of hepatic stellate cells by various chronic liver diseases. Considering that the liver is the most important metabolic organ in the human body, exploring the metabolic characteristics of liver fibrosis is expected to discover new markers and therapeutic targets. In this study, we first used mouse model to verify that both lactate content and histone acetylation levels were significantly increased in hepatic fibrosis mice. At the same time, it was confirmed that activated hepatic stellate cells (HSCs) cocultured with M1 macrophages can promote their transformation into M2 macrophages in hepatic stellate cell line and primary hepatic stellate cells. In addition, the addition of lactic acid to the medium in which M1 cells are cultured can promote their transformation into M2 macrophages. Therefore, we concluded that activated HSCs can promote the transformation of M1 to M2 macrophages through lactate accumulation, thereby causing liver fibrosis.
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Abstract
More than 27 yr ago, the vimentin knockout (Vim-/- ) mouse was reported to develop and reproduce without an obvious phenotype, implying that this major cytoskeletal protein was nonessential. Subsequently, comprehensive and careful analyses have revealed numerous phenotypes in Vim-/- mice and their organs, tissues, and cells, frequently reflecting altered responses in the recovery of tissues following various insults or injuries. These findings have been supported by cell-based experiments demonstrating that vimentin intermediate filaments (IFs) play a critical role in regulating cell mechanics and are required to coordinate mechanosensing, transduction, signaling pathways, motility, and inflammatory responses. This review highlights the essential functions of vimentin IFs revealed from studies of Vim-/- mice and cells derived from them.
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Affiliation(s)
- Karen M Ridge
- Division of Pulmonary and Critical Care Medicine, Northwestern University, Chicago, Illinois 60611, USA
- Department of Cell and Developmental Biology, Northwestern University, Chicago, Illinois 60611, USA
| | - John E Eriksson
- Cell Biology, Faculty of Science and Technology, Åbo Akademi University, FIN-20521 Turku, Finland
- Turku Bioscience Centre, University of Turku and Åbo Akademi University, FIN-20521 Turku, Finland
- Euro-Bioimaging European Research Infrastructure Consortium (ERIC), FIN-20521 Turku, Finland
| | - Milos Pekny
- Laboratory of Astrocyte Biology and CNS Regeneration, Center for Brain Repair, Department of Clinical Neuroscience, Institute of Neuroscience and Physiology, Sahlgrenska Academy at the University of Gothenburg, 413 90 Gothenburg, Sweden
- Florey Institute of Neuroscience and Mental Health, Parkville, Victoria 3052, Australia
- University of Newcastle, Newcastle, New South Wales 2300, Australia
| | - Robert D Goldman
- Division of Pulmonary and Critical Care Medicine, Northwestern University, Chicago, Illinois 60611, USA
- Department of Cell and Developmental Biology, Northwestern University, Chicago, Illinois 60611, USA
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Pessoa J, Teixeira J. Cytoskeleton alterations in non-alcoholic fatty liver disease. Metabolism 2022; 128:155115. [PMID: 34974078 DOI: 10.1016/j.metabol.2021.155115] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/19/2021] [Revised: 12/08/2021] [Accepted: 12/21/2021] [Indexed: 02/07/2023]
Abstract
BACKGROUND Due to its extremely high prevalence and severity, non-alcoholic fatty liver disease (NALFD) is a serious health and economic concern worldwide. Developing effective methods of diagnosis and therapy demands a deeper understanding of its molecular basis. One of the strategies in such an endeavor is the analysis of alterations in the morphology of liver cells. Such alterations, widely reported in NAFLD patients and disease models, are related to the cytoskeleton. Therefore, the fate of the cytoskeleton components is useful to uncover the molecular basis of NAFLD, to further design innovative approaches for its diagnosis and therapy. MAIN FINDINGS Several cytoskeleton proteins are up-regulated in liver cells of NAFLD patients. Under pathological conditions, keratin 18 is released from hepatocytes and its detection in the blood emerges as a non-invasive diagnosis tool. α-Smooth muscle actin is up-regulated in hepatic stellate cells and its down-regulation has been widely tested as a potential NALFD therapeutic approach. Other cytoskeleton proteins, such as vimentin, are also up-regulated. CONCLUSIONS NAFLD progression involves alterations in expression levels of proteins that build the liver cytoskeleton or associate with it. These findings provide a timely opportunity of developing novel approaches for NAFLD diagnosis and therapy.
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Affiliation(s)
- João Pessoa
- CNC - Center for Neuroscience and Cell Biology, CIBB - Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Coimbra, Portugal.
| | - José Teixeira
- CNC - Center for Neuroscience and Cell Biology, CIBB - Center for Innovative Biomedicine and Biotechnology, University of Coimbra, Coimbra, Portugal
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Dou SD, Zhang JN, Xie XL, Liu T, Hu JL, Jiang XY, Wang MM, Jiang HD. MitoQ inhibits hepatic stellate cell activation and liver fibrosis by enhancing PINK1/parkin-mediated mitophagy. Open Med (Wars) 2021; 16:1718-1727. [PMID: 34825063 PMCID: PMC8590110 DOI: 10.1515/med-2021-0394] [Citation(s) in RCA: 28] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2021] [Revised: 10/05/2021] [Accepted: 10/22/2021] [Indexed: 12/12/2022] Open
Abstract
Mitophagy affects the activation of hepatic stellate cells (HSCs). Mitochondria-targeted ubiquinone (MitoQ) is a mitochondria-targeted antioxidant that reduces the production of intracellular reactive oxygen species (ROS). However, its relationship with mitophagy remains unclear. This study evaluated mitophagy during HSC activation and the effects of MitoQ on mitophagy in cell culture and in an animal model of the activation of HSCs. We found that MitoQ reduced the activation of HSCs and alleviated hepatic fibrosis. PINK1 (PTEN-induced putative kinase 1) is a putative serine/threonine kinase located in the mitochondria’s outer membrane. While the activation of primary HSCs or LX-2 cells was associated with reduced PINK1/parkin-mediated mitophagy, MitoQ reduced intracellular ROS levels, enhanced PINK1/parkin-mediated mitophagy, and inhibited the activation of HSCs. After knocking down the key mitophagy-related protein, PINK1, in LX-2 cells to block mitophagy, MitoQ intervention failed to inhibit HSC activation. Our results showed that MitoQ inhibited the activation of HSCs and alleviated hepatic fibrosis by enhancing PINK1/parkin-mediated mitophagy.
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Affiliation(s)
- Shi-Ding Dou
- Department of Gastroenterology, The Second Hospital of Hebei Medical University, Hebei Key Laboratory of Gastroenterology, Hebei Institute of Gastroenterology, Hebei Clinical Research Center for Digestive Disease, Hebei, China.,Department of Infectious Diseases, The Second Hospital of Hebei Medical University, Shijiazhuang, Hebei, China
| | - Jiu-Na Zhang
- Department of Gastroenterology, The Second Hospital of Hebei Medical University, Hebei Key Laboratory of Gastroenterology, Hebei Institute of Gastroenterology, Hebei Clinical Research Center for Digestive Disease, Hebei, China
| | - Xiao-Li Xie
- Department of Gastroenterology, The Second Hospital of Hebei Medical University, Hebei Key Laboratory of Gastroenterology, Hebei Institute of Gastroenterology, Hebei Clinical Research Center for Digestive Disease, Hebei, China
| | - Ting Liu
- Department of Gastroenterology, The Second Hospital of Hebei Medical University, Hebei Key Laboratory of Gastroenterology, Hebei Institute of Gastroenterology, Hebei Clinical Research Center for Digestive Disease, Hebei, China
| | - Jun-Li Hu
- Emergency Department, The Second Hospital of Hebei Medical University, Shijiazhuang, Hebei, China
| | - Xiao-Yu Jiang
- Department of Gastroenterology, The Second Hospital of Hebei Medical University, Hebei Key Laboratory of Gastroenterology, Hebei Institute of Gastroenterology, Hebei Clinical Research Center for Digestive Disease, Hebei, China
| | - Miao-Miao Wang
- Department of Infectious Diseases, The Second Hospital of Hebei Medical University, Shijiazhuang, Hebei, China
| | - Hui-Ding Jiang
- Department of Gastroenterology, The Second Hospital of Hebei Medical University, Hebei Key Laboratory of Gastroenterology, Hebei Institute of Gastroenterology, Hebei Clinical Research Center for Digestive Disease, Hebei, China
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Wu X, Shu L, Zhang Z, Li J, Zong J, Cheong LY, Ye D, Lam KSL, Song E, Wang C, Xu A, Hoo RLC. Adipocyte Fatty Acid Binding Protein Promotes the Onset and Progression of Liver Fibrosis via Mediating the Crosstalk between Liver Sinusoidal Endothelial Cells and Hepatic Stellate Cells. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2021; 8:e2003721. [PMID: 34105268 PMCID: PMC8188197 DOI: 10.1002/advs.202003721] [Citation(s) in RCA: 43] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/30/2020] [Revised: 02/10/2021] [Indexed: 05/16/2023]
Abstract
Development of liver fibrosis results in drastic changes in the liver microenvironment, which in turn accelerates disease progression. Although the pathological function of various hepatic cells in fibrogenesis is identified, the crosstalk between them remains obscure. The present study demonstrates that hepatic expression of adipocyte fatty acid binding protein (A-FABP) is induced especially in the liver sinusoidal endothelial cells (LSECs) in mice after bile duct ligation (BDL). Genetic ablation and pharmacological inhibition of A-FABP attenuate BDL- or carbon tetrachloride-induced liver fibrosis in mice associating with reduced collagen accumulation, LSEC capillarization, and hepatic stellate cell (HSC) activation. Mechanistically, elevated A-FABP promotes LSEC capillarization by activating Hedgehog signaling, thus impairs the gatekeeper function of LSEC on HSC activation. LSEC-derived A-FABP also acts on HSCs in paracrine manner to potentiate the transactivation of transforming growth factor β1 (TGFβ1) by activating c-Jun N-terminal kinase (JNK)/c-Jun signaling. Elevated TGFβ1 subsequently exaggerates liver fibrosis. These findings uncover a novel pathological mechanism of liver fibrosis in which LSEC-derived A-FABP is a key regulator modulating the onset and progression of the disease. Targeting A-FABP may represent a potential approach against liver fibrosis.
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Affiliation(s)
- Xiaoping Wu
- State Key Laboratory of Pharmaceutical BiotechnologyLKS Faculty of Medicinethe University of Hong KongHong Kong999077China
- Department of Pharmacology and PharmacyLKS Faculty of Medicinethe University of Hong KongHong Kong999077China
| | - Lingling Shu
- State Key Laboratory of Pharmaceutical BiotechnologyLKS Faculty of Medicinethe University of Hong KongHong Kong999077China
- Department of MedicineLKS Faculty of Medicinethe University of Hong KongHong Kong999077China
- State Key Laboratory of Oncology in South ChinaCollaborative Innovation Center for Cancer MedicineSun Yat‐sen University Cancer CenterGuangzhou510060China
| | - Zixuan Zhang
- State Key Laboratory of Pharmaceutical BiotechnologyLKS Faculty of Medicinethe University of Hong KongHong Kong999077China
- Department of Pharmacology and PharmacyLKS Faculty of Medicinethe University of Hong KongHong Kong999077China
| | - Jingjing Li
- Department of Pharmacology and PharmacyLKS Faculty of Medicinethe University of Hong KongHong Kong999077China
| | - Jiuyu Zong
- State Key Laboratory of Pharmaceutical BiotechnologyLKS Faculty of Medicinethe University of Hong KongHong Kong999077China
- Department of Pharmacology and PharmacyLKS Faculty of Medicinethe University of Hong KongHong Kong999077China
| | - Lai Yee Cheong
- State Key Laboratory of Pharmaceutical BiotechnologyLKS Faculty of Medicinethe University of Hong KongHong Kong999077China
- Department of MedicineLKS Faculty of Medicinethe University of Hong KongHong Kong999077China
| | - Dewei Ye
- Joint Laboratory of Guangdong and Hong Kong on Metabolic DiseasesGuangdong Pharmaceutical UniversityGuangzhou510000China
| | - Karen S. L. Lam
- State Key Laboratory of Pharmaceutical BiotechnologyLKS Faculty of Medicinethe University of Hong KongHong Kong999077China
- Department of MedicineLKS Faculty of Medicinethe University of Hong KongHong Kong999077China
| | - Erfei Song
- Department of Metabolic and Bariatric SurgeryThe First Affiliated Hospital of Jinan UniversityGuangzhouGuangdong510630China
| | - Cunchuan Wang
- Department of Metabolic and Bariatric SurgeryThe First Affiliated Hospital of Jinan UniversityGuangzhouGuangdong510630China
| | - Aimin Xu
- State Key Laboratory of Pharmaceutical BiotechnologyLKS Faculty of Medicinethe University of Hong KongHong Kong999077China
- Department of Pharmacology and PharmacyLKS Faculty of Medicinethe University of Hong KongHong Kong999077China
- Department of MedicineLKS Faculty of Medicinethe University of Hong KongHong Kong999077China
| | - Ruby L. C. Hoo
- State Key Laboratory of Pharmaceutical BiotechnologyLKS Faculty of Medicinethe University of Hong KongHong Kong999077China
- Department of Pharmacology and PharmacyLKS Faculty of Medicinethe University of Hong KongHong Kong999077China
- HKU‐Shenzhen Institute of Research and Innovation (HKU‐SIRI)Shenzhen518057China
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9
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Rahman N, Kuramochi M, Izawa T, Kuwamura M, Yamate J. Characterization of Immature Myofibroblasts of Stellate Cell or Mesenchymal Cell Origin in D-Galactosamine-Induced Liver Injury in Rats. Vet Pathol 2021; 58:80-90. [PMID: 33054583 DOI: 10.1177/0300985820963097] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Lesions of D-galactosamine (D-GalN)-induced hepatotoxicity resemble those of human acute viral hepatitis. This study investigated hepatic mesenchymal cells including hepatic stellate cells (HSCs) and myofibroblasts in D-GalN-induced hepatotoxicity. Rats, injected with D-GalN (800 mg/kg body weight, once, intraperitoneally) were examined on post single injection (PSI) at 8 hours and days 1 to 5. Lesions consisting of hepatocyte necrosis and reparative fibrosis were present diffusely or focally within the hepatic lobules on PSI days 1 and 2, and then the injury recovered on PSI days 3 and 5. Myofibroblasts expressing vimentin, desmin, and α-smooth muscle actin (α-SMA) were present in the lesions. Double immunofluorescence showed that myofibroblasts reacted simultaneously to vimentin/α-SMA, desmin/α-SMA, and desmin/vimentin; furthermore, myofibroblasts reacting to vimentin, desmin, and α-SMA also co-expressed glial fibrillary acidic protein (GFAP), a marker of HSCs. Additionally, GFAP-expressing myofibroblasts reacted to nestin and A3 (both are markers of immature mesenchymal cells). Cells reacting to Thy-1, a marker for immature mesenchymal cells, also appeared in fibrotic lesions. In agreement with the myofibroblastic appearance, mRNAs of fibrosis-related factors (TGF-β1, PDGF-β, TNF-α, Timp2, and Mmp2) increased mainly on PSI days 1 and 2. Myofibroblasts with expression of various cytoskeletal proteins were present in diffuse or focal hepatic lesions, and they might be derived partly from immature HSCs and from immature mesenchymal cells.
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Affiliation(s)
- Nahid Rahman
- 13311Osaka Prefecture University, Izumisano City, Osaka, Japan
| | | | - Takeshi Izawa
- 13311Osaka Prefecture University, Izumisano City, Osaka, Japan
| | | | - Jyoji Yamate
- 13311Osaka Prefecture University, Izumisano City, Osaka, Japan
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10
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Liu J, Xie Y, Cui Z, Xia T, Wan L, Zhou H, Zhang P, Zhang Y, Guan F, Liu W, Shi C. Bnip3 interacts with vimentin, an intermediate filament protein, and regulates autophagy of hepatic stellate cells. Aging (Albany NY) 2020; 13:957-972. [PMID: 33290258 PMCID: PMC7834981 DOI: 10.18632/aging.202211] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2020] [Accepted: 09/09/2020] [Indexed: 04/21/2023]
Abstract
Bnip3, which is regulated by Hif-1 in cells under oxygen deprivation, is a death related protein associated with autophagy and apoptosis. Hif-1 was reported to regulate autophagy to activate hepatic stellate cells (HSCs), while the specific molecular mechanism is vague. The possible mechanism of Hif-1 regulating autophagy of HSCs via Bnip3 was explored in this study. Bnip3 was detected in fibrotic liver tissues from humans and mice. Hif-1 was inhibited by chemical inhibitor and Bnip3 was detected in activated HSCs. The co-localization of Bnip3 and LC3B was captured by confocal microscopy and autophagic flow was assessed in Bnip3 siRNA transfected cells. Bnip3 interacted proteins were screened with mass spectrometry. The interaction of Bnip3 and vimentin was detected with co-immunoprecipitation and confocal microscopy. The results showed that Bnip3 was increased in fibrotic liver tissues and activated HSCs. Hif-1 inhibition suppressed Bnip3 expression in activated HSCs. Bnip3 was partially co-localized with autophagosomes and Bnip3 inhibition suppessed autophagy in activated HSCs. Bnip3 interacted with vimentin and Bnip3 expression was inhibited as vimentin was inhibited in activated HSCs. Conclusively, this study indicated that Bnip3 promoted autophagy and activation of HSCs, via interacting with vimentin, an intermediate filament protein with highly abundant expression in HSCs.
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Affiliation(s)
- Jie Liu
- Department of Pathogen Biology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China
- Department of Hospital Infection Management, Wuhan Children’s Hospital (Wuhan Maternal and Child Healthcare Hospital), Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China
| | - Yuyu Xie
- Department of Pathogen Biology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China
| | - Zhangbo Cui
- Department of Pathogen Biology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China
| | - Tian Xia
- Department of Surgery, Wuhan Third Hospital, Wuhan, PR China
| | - Lu Wan
- Department of Pathogen Biology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China
- Department of Pathophysiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China
| | - Haifeng Zhou
- Department of Integrated Traditional Chinese and Western Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China
| | - Peng Zhang
- Department of Immunology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China
| | - Yijie Zhang
- Department of Pathogen Biology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China
| | - Fei Guan
- Department of Pathogen Biology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China
| | - Wenqi Liu
- Department of Pathogen Biology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China
| | - Chunwei Shi
- Department of Pathogen Biology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China
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11
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Morrow CS, Moore DL. Vimentin's side gig: Regulating cellular proteostasis in mammalian systems. Cytoskeleton (Hoboken) 2020; 77:515-523. [PMID: 33190414 DOI: 10.1002/cm.21645] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2020] [Revised: 11/05/2020] [Accepted: 11/09/2020] [Indexed: 02/06/2023]
Abstract
Intermediate filaments (IFs) perform a diverse set of well-known functions including providing structural support for the cell and resistance to mechanical stress, yet recent evidence has revealed unexpected roles for IFs as stress response proteins. Previously, it was shown that the type III IF protein vimentin forms cage-like structures around centrosome-associated proteins destined for degradation, structures referred to as aggresomes, suggesting a role for vimentin in protein turnover. However, vimentin's function at the aggresome has remained largely understudied. In a recent report, vimentin was shown to be dispensable for aggresome formation, but played a critical role in protein turnover at the aggresome through localizing proteostasis-related machineries, such as proteasomes, to the aggresome. Here, we review evidence for vimentin's function in proteostasis and highlight the organismal implications of these findings.
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Affiliation(s)
- Christopher S Morrow
- Department of Neuroscience, University of Wisconsin-Madison, Madison, Wisconsin, USA
| | - Darcie L Moore
- Department of Neuroscience, University of Wisconsin-Madison, Madison, Wisconsin, USA
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12
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Hypoxia sensing by hepatic stellate cells leads to VEGF-dependent angiogenesis and may contribute to accelerated liver regeneration. Sci Rep 2020; 10:4392. [PMID: 32152325 PMCID: PMC7062856 DOI: 10.1038/s41598-020-60709-9] [Citation(s) in RCA: 20] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2019] [Accepted: 02/05/2020] [Indexed: 02/06/2023] Open
Abstract
Portal vein ligation (PVL) induces liver growth prior to resection. Associating liver partition and portal vein ligation (PVL plus transection=ALPPS) or the addition of the prolyl-hydroxylase inhibitor dimethyloxalylglycine (DMOG) to PVL both accelerate growth via stabilization of HIF-α subunits. This study aims at clarifying the crosstalk of hepatocytes (HC), hepatic stellate cells (HSC) and liver sinusoidal endothelial cells (LSEC) in accelerated liver growth. In vivo, liver volume, HC proliferation, vascular density and HSC activation were assessed in PVL, ALPPS, PVL+DMOG and DMOG alone. Proliferation of HC, HSC and LSEC was determined under DMOG in vitro. Conditioned media experiments of DMOG-exposed cells were performed. ALPPS and PVL+DMOG accelerated liver growth and HC proliferation in comparison to PVL. DMOG alone did not induce HC proliferation, but led to increased vascular density, which was also observed in ALPPS and PVL+DMOG. Activated HSC were detected in ALPPS, PVL+DMOG and DMOG, again not in PVL. In vitro, DMOG had no proliferative effect on HC, but conditioned supernatant of DMOG-treated HSC induced VEGF-dependent proliferation of LSEC. Transcriptome analysis confirmed activation of proangiogenic factors in hypoxic HSC. Hypoxia signaling in HSC induces VEGF-dependent angiogenesis. HSC play a crucial role in the cellular crosstalk of rapid liver regeneration.
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13
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Danielsson F, Peterson MK, Caldeira Araújo H, Lautenschläger F, Gad AKB. Vimentin Diversity in Health and Disease. Cells 2018; 7:E147. [PMID: 30248895 PMCID: PMC6210396 DOI: 10.3390/cells7100147] [Citation(s) in RCA: 178] [Impact Index Per Article: 25.4] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2018] [Revised: 09/16/2018] [Accepted: 09/17/2018] [Indexed: 12/11/2022] Open
Abstract
Vimentin is a protein that has been linked to a large variety of pathophysiological conditions, including cataracts, Crohn's disease, rheumatoid arthritis, HIV and cancer. Vimentin has also been shown to regulate a wide spectrum of basic cellular functions. In cells, vimentin assembles into a network of filaments that spans the cytoplasm. It can also be found in smaller, non-filamentous forms that can localise both within cells and within the extracellular microenvironment. The vimentin structure can be altered by subunit exchange, cleavage into different sizes, re-annealing, post-translational modifications and interacting proteins. Together with the observation that different domains of vimentin might have evolved under different selection pressures that defined distinct biological functions for different parts of the protein, the many diverse variants of vimentin might be the cause of its functional diversity. A number of review articles have focussed on the biology and medical aspects of intermediate filament proteins without particular commitment to vimentin, and other reviews have focussed on intermediate filaments in an in vitro context. In contrast, the present review focusses almost exclusively on vimentin, and covers both ex vivo and in vivo data from tissue culture and from living organisms, including a summary of the many phenotypes of vimentin knockout animals. Our aim is to provide a comprehensive overview of the current understanding of the many diverse aspects of vimentin, from biochemical, mechanical, cellular, systems biology and medical perspectives.
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Affiliation(s)
- Frida Danielsson
- Science for Life Laboratory, Royal Institute of Technology, 17165 Stockholm, Sweden.
| | | | | | - Franziska Lautenschläger
- Campus D2 2, Leibniz-Institut für Neue Materialien gGmbH (INM) and Experimental Physics, NT Faculty, E 2 6, Saarland University, 66123 Saarbrücken, Germany.
| | - Annica Karin Britt Gad
- Centro de Química da Madeira, Universidade da Madeira, 9020105 Funchal, Portugal.
- Department of Medical Biochemistry and Microbiology, Uppsala University, 75237 Uppsala, Sweden.
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14
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Choi JH, Riew TR, Kim HL, Jin X, Lee MY. Desmin expression profile in reactive astrocytes in the 3-nitropropionic acid-lesioned striatum of rat: Characterization and comparison with glial fibrillary acidic protein and nestin. Acta Histochem 2017; 119:795-803. [PMID: 29054283 DOI: 10.1016/j.acthis.2017.10.003] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2017] [Revised: 10/12/2017] [Accepted: 10/13/2017] [Indexed: 02/07/2023]
Abstract
Desmin, a muscle-specific, type-III intermediate-filament protein, is reportedly expressed in astrocytes in the central nervous system. These cells become reactive astrocytes in response to brain injuries. To elucidate whether desmin is involved in this process, we examined the spatiotemporal expression profiles of desmin and their relationship with two astroglial intermediate filaments, glial fibrillary acidic protein (GFAP) and nestin, in the striatum of rats treated with the mitochondrial toxin 3-nitropropionic acid (3-NP). Weak, constitutive immunoreactivity for desmin was observed in astrocytes generally, and in reactive astrocytes in the peri-lesional area, its expression increased in parallel with that of GFAP over 3 d post-lesion and was maintained until at least day 28. Desmin, GFAP, and nestin showed characteristic time-dependent expression patterns in reactive astrocytes forming the astroglial scar; delayed and long-lasting induction of desmin and GFAP, and rapid but transient induction of nestin. In the lesion core, desmin was expressed in two categories of perivascular cells: nestin-negative and nestin-positive. These findings show that desmin, together with GFAP and nestin, is a dynamic component of intermediate filaments in activated astroglia, which may account for the dynamic structural changes seen in these cells in response to brain injuries.
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Affiliation(s)
- Jeong-Heon Choi
- Catholic Neuroscience Institute, Cell Death Disease Research Center, and Department of Anatomy, College of Medicine, The Catholic University of Korea, Republic of Korea
| | - Tae-Ryong Riew
- Catholic Neuroscience Institute, Cell Death Disease Research Center, and Department of Anatomy, College of Medicine, The Catholic University of Korea, Republic of Korea
| | - Hong Lim Kim
- Electron Microscopy Laboratory, Integrative Research Support Center, College of Medicine, The Catholic University of Korea, Republic of Korea
| | - Xuyan Jin
- Catholic Neuroscience Institute, Cell Death Disease Research Center, and Department of Anatomy, College of Medicine, The Catholic University of Korea, Republic of Korea
| | - Mun-Yong Lee
- Catholic Neuroscience Institute, Cell Death Disease Research Center, and Department of Anatomy, College of Medicine, The Catholic University of Korea, Republic of Korea.
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15
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Vilaseca M, García-Calderó H, Lafoz E, Ruart M, López-Sanjurjo CI, Murphy MP, Deulofeu R, Bosch J, Hernández-Gea V, Gracia-Sancho J, García-Pagán JC. Mitochondria-targeted antioxidant mitoquinone deactivates human and rat hepatic stellate cells and reduces portal hypertension in cirrhotic rats. Liver Int 2017; 37:1002-1012. [PMID: 28371136 DOI: 10.1111/liv.13436] [Citation(s) in RCA: 39] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/30/2017] [Accepted: 03/24/2017] [Indexed: 02/13/2023]
Abstract
BACKGROUND & AIMS In cirrhosis, activated hepatic stellate cells (HSC) play a major role in increasing intrahepatic vascular resistance and developing portal hypertension. We have shown that cirrhotic livers have increased reactive oxygen species (ROS), and that antioxidant therapy decreases portal pressure. Considering that mitochondria produce many of these ROS, our aim was to assess the effects of the oral mitochondria-targeted antioxidant mitoquinone on hepatic oxidative stress, HSC phenotype, liver fibrosis and portal hypertension. METHODS Ex vivo: Hepatic stellate cells phenotype was analysed in human precision-cut liver slices in response to mitoquinone or vehicle. In vitro: Mitochondrial oxidative stress was analysed in different cell type of livers from control and cirrhotic rats. HSC phenotype, proliferation and viability were assessed in LX2, and in primary human and rat HSC treated with mitoquinone or vehicle. In vivo: CCl4 - and thioacetamide-cirrhotic rats were treated with mitoquinone (5 mg/kg/day) or the vehicle compound, DecylTPP, for 2 weeks, followed by measurement of oxidative stress, systemic and hepatic haemodynamic, liver fibrosis, HSC phenotype and liver inflammation. RESULTS Mitoquinone deactivated human and rat HSC, decreased their proliferation but with no effects on viability. In CCl4 -cirrhotic rats, mitoquinone decreased hepatic oxidative stress, improved HSC phenotype, reduced intrahepatic vascular resistance and diminished liver fibrosis. These effects were associated with a significant reduction in portal pressure without changes in arterial pressure. These results were further confirmed in the thioacetamide-cirrhotic model. CONCLUSION We propose mitochondria-targeted antioxidants as a novel treatment approach against portal hypertension and cirrhosis.
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Affiliation(s)
- Marina Vilaseca
- Barcelona Hepatic Hemodynamic Laboratory, Liver Unit, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Hospital Clínic de Barcelona, University of Barcelona Medical School, Barcelona, Spain
| | - Héctor García-Calderó
- Barcelona Hepatic Hemodynamic Laboratory, Liver Unit, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Hospital Clínic de Barcelona, University of Barcelona Medical School, Barcelona, Spain.,Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Barcelona, Spain
| | - Erica Lafoz
- Barcelona Hepatic Hemodynamic Laboratory, Liver Unit, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Hospital Clínic de Barcelona, University of Barcelona Medical School, Barcelona, Spain
| | - Maria Ruart
- Barcelona Hepatic Hemodynamic Laboratory, Liver Unit, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Hospital Clínic de Barcelona, University of Barcelona Medical School, Barcelona, Spain
| | - Cristina Isabel López-Sanjurjo
- Barcelona Hepatic Hemodynamic Laboratory, Liver Unit, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Hospital Clínic de Barcelona, University of Barcelona Medical School, Barcelona, Spain.,Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Barcelona, Spain
| | | | - Ramon Deulofeu
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Barcelona, Spain.,Department of Biochemistry and Chromatography, Hospital Clinic de Barcelona, Barcelona, Spain
| | - Jaume Bosch
- Barcelona Hepatic Hemodynamic Laboratory, Liver Unit, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Hospital Clínic de Barcelona, University of Barcelona Medical School, Barcelona, Spain.,Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Barcelona, Spain
| | - Virginia Hernández-Gea
- Barcelona Hepatic Hemodynamic Laboratory, Liver Unit, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Hospital Clínic de Barcelona, University of Barcelona Medical School, Barcelona, Spain.,Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Barcelona, Spain
| | - Jordi Gracia-Sancho
- Barcelona Hepatic Hemodynamic Laboratory, Liver Unit, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Hospital Clínic de Barcelona, University of Barcelona Medical School, Barcelona, Spain.,Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Barcelona, Spain
| | - Juan Carlos García-Pagán
- Barcelona Hepatic Hemodynamic Laboratory, Liver Unit, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Hospital Clínic de Barcelona, University of Barcelona Medical School, Barcelona, Spain.,Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Barcelona, Spain
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16
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Nielsen MFB, Mortensen MB, Detlefsen S. Identification of markers for quiescent pancreatic stellate cells in the normal human pancreas. Histochem Cell Biol 2017; 148:359-380. [PMID: 28540429 DOI: 10.1007/s00418-017-1581-5] [Citation(s) in RCA: 34] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 05/13/2017] [Indexed: 12/16/2022]
Abstract
Pancreatic stellate cells (PSCs) play a central role as source of fibrogenic cells in pancreatic cancer and chronic pancreatitis. In contrast to quiescent hepatic stellate cells (qHSCs), a specific marker for quiescent PSCs (qPSCs) that can be used in formalin-fixed and paraffin embedded (FFPE) normal human pancreatic tissue has not been identified. The aim of this study was to identify a marker enabling the identification of qPSCs in normal human FFPE pancreatic tissue. Immunohistochemical (IHC), double-IHC, immunofluorescence (IF) and double-IF analyses were carried out using a tissue microarray consisting of cores with normal human pancreatic tissue. Cores with normal human liver served as control. Antibodies directed against adipophilin, α-SMA, CD146, CRBP-1, cytoglobin, desmin, GFAP, nestin, S100A4 and vinculin were examined, with special emphasis on their expression in periacinar cells in the normal human pancreas and perisinusoidal cells in the normal human liver. The immunolabelling capacity was evaluated according to a semiquantitative scoring system. Double-IF of the markers of interest together with markers for other periacinar cells was performed. Moreover, the utility of histochemical stains for the identification of human qPSCs was examined, and their ultrastructure was revisited by electron microscopy. Adipophilin, CRBP-1, cytoglobin and vinculin were expressed in qHSCs in the liver, whereas cytoglobin and adipophilin were expressed in qPSCs in the pancreas. Adipophilin immunohistochemistry was highly dependent on the preanalytical time interval (PATI) from removal of the tissue to formalin fixation. Cytoglobin, S100A4 and vinculin were expressed in periacinar fibroblasts (FBs). The other examined markers were negative in human qPSCs. Our data indicate that cytoglobin and adipophilin are markers of qPSCs in the normal human pancreas. However, the use of adipophilin as a qPSC marker may be limited due to its high dependence on optimal PATI. Cytoglobin, on the other hand, is a sensitive marker for qPSCs but is expressed in FBs as well.
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Affiliation(s)
- Michael Friberg Bruun Nielsen
- Department of Pathology, Odense University Hospital, J.B. Winsløws Vej 15, 5000, Odense C, Denmark.,Department of Clinical Research, University of Southern Denmark, J.B. Winsløws Vej 19, 5000, Odense C, Denmark
| | - Michael Bau Mortensen
- Department of Clinical Research, University of Southern Denmark, J.B. Winsløws Vej 19, 5000, Odense C, Denmark.,Department of Surgery, HPB Section, Odense University Hospital, Sdr. Boulevard 29, 5000, Odense C, Denmark
| | - Sönke Detlefsen
- Department of Pathology, Odense University Hospital, J.B. Winsløws Vej 15, 5000, Odense C, Denmark. .,Department of Clinical Research, University of Southern Denmark, J.B. Winsløws Vej 19, 5000, Odense C, Denmark.
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17
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Non-alcoholic fatty liver disease, vascular inflammation and insulin resistance are exacerbated by TRAIL deletion in mice. Sci Rep 2017; 7:1898. [PMID: 28507343 PMCID: PMC5432513 DOI: 10.1038/s41598-017-01721-4] [Citation(s) in RCA: 38] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2016] [Accepted: 03/06/2017] [Indexed: 12/15/2022] Open
Abstract
Non-alcoholic fatty liver disease (NAFLD) incorporates steatosis, non-alcoholic steato-hepatitis (NASH) and liver cirrhosis, associating with diabetes and cardiovascular disease (CVD). TNF-related apoptosis-inducing ligand (TRAIL) is protective of CVD. We aimed to determine whether TRAIL protects against insulin resistance, NAFLD and vascular injury. Twelve-week high fat diet (HFD)-fed Trail−/− mice had increased plasma cholesterol, insulin and glucose compared to wildtype. Insulin tolerance was impaired with TRAIL-deletion, with reduced p-Akt, GLUT4 expression and glucose uptake in skeletal muscle. Hepatic triglyceride content, inflammation and fibrosis were increased with TRAIL-deletion, with elevated expression of genes regulating lipogenesis and gluconeogenesis. Moreover, Trail−/− mice exhibited reduced aortic vasorelaxation, impaired insulin signaling, and >20-fold increased mRNA expression for IL-1β, IL-6, and TNF-α. In vitro, palmitate treatment of hepatocytes increased lipid accumulation, inflammation and fibrosis, with TRAIL mRNA significantly reduced. TRAIL administration inhibited palmitate-induced hepatocyte lipid uptake. Finally, patients with NASH had significantly reduced plasma TRAIL compared to control, simple steatosis or obese individuals. These findings suggest that TRAIL protects against insulin resistance, NAFLD and vascular inflammation. Increasing TRAIL levels may be an attractive therapeutic strategy, to reduce features of diabetes, as well as liver and vascular injury, so commonly observed in individuals with NAFLD.
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18
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Manzanares MÁ, Usui A, Campbell DJ, Dumur CI, Maldonado GT, Fausther M, Dranoff JA, Sirica AE. Transforming Growth Factors α and β Are Essential for Modeling Cholangiocarcinoma Desmoplasia and Progression in a Three-Dimensional Organotypic Culture Model. THE AMERICAN JOURNAL OF PATHOLOGY 2017; 187:1068-1092. [PMID: 28315313 DOI: 10.1016/j.ajpath.2017.01.013] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/02/2016] [Revised: 12/19/2016] [Accepted: 01/17/2017] [Indexed: 12/12/2022]
Abstract
To gain insight into the cellular and molecular interactions mediating the desmoplastic reaction and aggressive malignancy of mass-forming intrahepatic cholangiocarcinoma (ICC), we modeled ICC desmoplasia and progression in vitro. A unique three-dimensional (3D) organotypic culture model was established; within a dilute collagen-type I hydrogel, a novel clonal strain of rat cancer-associated myofibroblasts (TDFSM) was co-cultured with a pure rat cholangiocarcinoma cell strain (TDECC) derived from the same ICC type as TDFSM. This 3D organotypic culture model reproduced key features of desmoplastic reaction that closely mimicked those of the in situ tumor, as well as promoted cholangiocarcinoma cell growth and progression. Our results supported a resident liver mesenchymal cell origin of the TDFSM cells, which were not neoplastically transformed. Notably, 3D co-culturing of TDECC cells with TDFSM cells provoked the formation of a dense fibrocollagenous stroma in vitro that was associated with significant increases in both proliferative TDFSM myofibroblastic cells and TDECC cholangiocarcinoma cells accumulating within the gel matrix. This dramatic desmoplastic ICC-like phenotype, which was not observed in the TDECC or TDFSM controls, was highly dependent on transforming growth factor (TGF)-β, but not promoted by TGF-α. However, TGF-α was determined to be a key factor for promoting cholangiocarcinoma cell anaplasia, hyperproliferation, and higher malignant grading in this 3D culture model of desmoplastic ICC.
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Affiliation(s)
- Miguel Á Manzanares
- Division of Cellular and Molecular Pathogenesis, Department of Pathology, Virginia Commonwealth University, Richmond, Virginia
| | - Akihiro Usui
- Division of Cellular and Molecular Pathogenesis, Department of Pathology, Virginia Commonwealth University, Richmond, Virginia
| | - Deanna J Campbell
- Division of Cellular and Molecular Pathogenesis, Department of Pathology, Virginia Commonwealth University, Richmond, Virginia
| | - Catherine I Dumur
- Division of Cellular and Molecular Pathogenesis, Department of Pathology, Virginia Commonwealth University, Richmond, Virginia
| | - Gabrielle T Maldonado
- Division of Cellular and Molecular Pathogenesis, Department of Pathology, Virginia Commonwealth University, Richmond, Virginia
| | - Michel Fausther
- Division of Gastroenterology and Hepatology, University of Arkansas for Medical Sciences, Little Rock, Arkansas
| | - Jonathan A Dranoff
- Division of Gastroenterology and Hepatology, University of Arkansas for Medical Sciences, Little Rock, Arkansas
| | - Alphonse E Sirica
- Division of Cellular and Molecular Pathogenesis, Department of Pathology, Virginia Commonwealth University, Richmond, Virginia.
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Jalan R, De Chiara F, Balasubramaniyan V, Andreola F, Khetan V, Malago M, Pinzani M, Mookerjee RP, Rombouts K. Ammonia produces pathological changes in human hepatic stellate cells and is a target for therapy of portal hypertension. J Hepatol 2016; 64:823-33. [PMID: 26654994 DOI: 10.1016/j.jhep.2015.11.019] [Citation(s) in RCA: 65] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/08/2015] [Revised: 11/09/2015] [Accepted: 11/11/2015] [Indexed: 12/14/2022]
Abstract
BACKGROUND & AIMS Hepatic stellate cells (HSCs) are vital to hepatocellular function and the liver response to injury. They share a phenotypic homology with astrocytes that are central in the pathogenesis of hepatic encephalopathy, a condition in which hyperammonemia plays a pathogenic role. This study tested the hypothesis that ammonia modulates human HSC activation in vitro and in vivo, and evaluated whether ammonia lowering, by using l-ornithine phenylacetate (OP), modifies HSC activation in vivo and reduces portal pressure in a bile duct ligation (BDL) model. METHODS Primary human HSCs were isolated and cultured. Proliferation (BrdU), metabolic activity (MTS), morphology (transmission electron, light and immunofluorescence microscopy), HSC activation markers, ability to contract, changes in oxidative status (ROS) and endoplasmic reticulum (ER) were evaluated to identify effects of ammonia challenge (50 μM, 100 μM, 300 μM) over 24-72 h. Changes in plasma ammonia levels, markers of HSC activation, portal pressure and hepatic eNOS activity were quantified in hyperammonemic BDL animals, and after OP treatment. RESULTS Pathophysiological ammonia concentrations caused significant and reversible changes in cell proliferation, metabolic activity and activation markers of hHSC in vitro. Ammonia also induced significant alterations in cellular morphology, characterised by cytoplasmic vacuolisation, ER enlargement, ROS production, hHSC contraction and changes in pro-inflammatory gene expression together with HSC-related activation markers such as α-SMA, myosin IIa, IIb, and PDGF-Rβ. Treatment with OP significantly reduced plasma ammonia (BDL 199.1 μmol/L±43.65 vs. BDL+OP 149.27 μmol/L±51.1, p<0.05) and portal pressure (BDL 14±0.6 vs. BDL+OP 11±0.3 mmHg, p<0.01), which was associated with increased eNOS activity and abrogation of HSC activation markers. CONCLUSIONS The results show for the first time that ammonia produces deleterious morphological and functional effects on HSCs in vitro. Targeting ammonia with the ammonia lowering drug OP reduces portal pressure and deactivates hHSC in vivo, highlighting the opportunity for evaluating ammonia lowering as a potential therapy in cirrhotic patients with portal hypertension.
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Affiliation(s)
- Rajiv Jalan
- Liver Failure Group, Institute for Liver & Digestive Health, University College of London, Royal Free, London, UK
| | - Francesco De Chiara
- Liver Failure Group, Institute for Liver & Digestive Health, University College of London, Royal Free, London, UK
| | - Vairappan Balasubramaniyan
- Liver Failure Group, Institute for Liver & Digestive Health, University College of London, Royal Free, London, UK
| | - Fausto Andreola
- Liver Failure Group, Institute for Liver & Digestive Health, University College of London, Royal Free, London, UK
| | - Varun Khetan
- Liver Failure Group, Institute for Liver & Digestive Health, University College of London, Royal Free, London, UK
| | - Massimo Malago
- Division of Surgery, University College London, Royal Free, London, UK
| | - Massimo Pinzani
- Regenerative Medicine & Fibrosis Group, Institute for Liver & Digestive Health, University College London, Royal Free, London, UK
| | - Rajeshwar P Mookerjee
- Liver Failure Group, Institute for Liver & Digestive Health, University College of London, Royal Free, London, UK.
| | - Krista Rombouts
- Regenerative Medicine & Fibrosis Group, Institute for Liver & Digestive Health, University College London, Royal Free, London, UK.
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20
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Liu M, Peng P, Wang J, Wang L, Duan F, Jia D, Ruan Y, Gu J. RACK1-mediated translation control promotes liver fibrogenesis. Biochem Biophys Res Commun 2015; 463:255-61. [PMID: 26002467 DOI: 10.1016/j.bbrc.2015.05.040] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2015] [Accepted: 05/03/2015] [Indexed: 01/12/2023]
Abstract
Activation of quiescent hepatic stellate cells (HSCs) is the central event of liver fibrosis. The translational machinery is an optimized molecular network that affects cellular homoeostasis and diseases, whereas the role of protein translation in HSCs activation and liver fibrosis is little defined. Our previous report suggests that up-regulation of receptor for activated C-kinase 1(RACK1) in HSCs is critical for liver fibrogenesis. In this study, we found that RACK1 promoted macrophage conditioned medium (MCM)-induced assembly of eIF4F and phosphorylation of eIF4E in primary HSCs. RACK1 enhanced the translation and expression of pro-fibrogenic factors collagen 1α1, snail and cyclin E1 induced by MCM. Administration of PP242 or knock-down of eIF4E suppressed RACK1-stimulated collagen 1α1 production, proliferation and migration in primary HSCs. In addition, depletion of eIF4E attenuated thioacetamide (TAA)-induced liver fibrosis in vivo. Our data suggest that RACK1-mediated stimulation of cap-dependent translation plays crucial roles in HSCs activation and liver fibrogenesis, and targeting translation initiation could be a promising strategy for the treatment of liver fibrosis.
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Affiliation(s)
- Min Liu
- Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China
| | - Peike Peng
- Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China
| | - Jiajun Wang
- Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China
| | - Lan Wang
- Institute of Biomedical Science, Fudan University, Shanghai 200032, China
| | - Fangfang Duan
- Institute of Biomedical Science, Fudan University, Shanghai 200032, China
| | - Dongwei Jia
- Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China.
| | - Yuanyuan Ruan
- Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China.
| | - Jianxin Gu
- Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China; Institute of Biomedical Science, Fudan University, Shanghai 200032, China
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21
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Dusabineza AC, Najimi M, van Hul N, Legry V, Khuu DN, van Grunsven LA, Sokal E, Leclercq IA. Hepatic Stellate Cells Improve Engraftment of Human Primary Hepatocytes: A Preclinical Transplantation Study in an Animal Model. Cell Transplant 2015; 24:2557-71. [PMID: 25706818 DOI: 10.3727/096368915x686788] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
Human hepatocytes are used for liver cell therapy, but the small number of engrafting cells limits the benefit of cell transplantation. We tested whether cotransplantation of hepatocytes with hepatic stellate cells (HSCs) could improve hepatocyte engraftment in vivo. Human primary hepatocytes were transplanted into SCID mice either alone or in a mixture with HSCs (quiescent or after culture activation) or LX-2 cells (ratio 20:1). Four weeks after transplantation into mouse livers, human albumin-positive (huAlb(+)) hepatocytes were found scattered. When cotransplanted in a mixture with HSCs or LX-2 cells, huAlb(+) hepatocytes formed clusters and were more numerous occupying 2- to 5.9-fold more surface on the tissue section than in livers transplanted with hepatocytes alone. Increased huAlb mRNA expression in livers transplanted with the cell mixtures confirmed those results. The presence of HSCs increased the number of hepatocytes entrapped in the host liver at an early time point posttransplantation but not their proliferation in situ as assessed by cumulative incorporation of BrdU. Importantly, 4 weeks posttransplantation, we found no accumulation of αSMA(+)-activated HSCs or collagen deposition. To follow the fate of transplanted HSCs, HSCs derived from GFP(+) mice were injected into GFP(-) littermates: 17 h posttransplant, GFP(+) HSCs were found in the sinusoids, without proliferating or actively producing ECM; they were undetectable at later time points. Coculture with HSCs improved the number of adherent hepatocytes, with best attachment obtained when hepatocytes were seeded in contact with activated HSCs. In vivo, cotransplantation of hepatocytes with HSCs into a healthy liver recipient does not generate fibrosis, but significantly improves the engraftment of hepatocytes, probably by ameliorating cell homing.
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Affiliation(s)
- Ange-Clarisse Dusabineza
- Laboratory of Hepato-Gastroenterology, Institut de Recherche Expérimentale et Clinique (IREC), Université catholique de Louvain, UCL, Brussels, Belgium
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22
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Elaboration of tubules with active hedgehog drives parenchymal fibrogenesis in gestational alloimmune liver disease. Hum Pathol 2015; 46:84-93. [DOI: 10.1016/j.humpath.2014.09.010] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/08/2014] [Revised: 09/17/2014] [Accepted: 09/19/2014] [Indexed: 12/28/2022]
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23
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Mezaki Y, Morii M, Hebiguchi T, Yoshikawa K, Yamaguchi N, Miura M, Imai K, Yoshino H, Senoo H. Differential increases in the expression of intermediate filament proteins and concomitant morphological changes of transdifferentiating rat hepatic stellate cells observed in vitro. Acta Histochem Cytochem 2013; 46:137-43. [PMID: 24194627 PMCID: PMC3813821 DOI: 10.1267/ahc.13007] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2013] [Accepted: 08/30/2013] [Indexed: 01/17/2023] Open
Abstract
The primary function of hepatic stellate cells (HSCs) is the storage of vitamin A. However, they are also responsible for liver fibrosis and are therapeutic targets for treatment of liver cirrhosis. Among the many molecular markers that define quiescent or activated states of HSCs, the characteristics of type III intermediate filaments are of particular interest. Whereas vimentin and desmin are upregulated in activated HSCs, glial fibrillary acidic protein is downregulated in activated HSCs. The functional differences between vimentin and desmin are poorly understood. By time-course quantifications of several molecular markers for HSC activation, we observed that the expression of vimentin preceded that of desmin during the transdifferentiation of HSCs. The immunoreactivity of vimentin in transdifferentiated HSCs was more intense in perinuclear regions compared to that of desmin. We propose that the delayed expression of desmin following the expression of vimentin and the peripheral localization of desmin compared to vimentin are both related to the more extended phenotype of transdifferentiating HSCs observed in vitro.
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Affiliation(s)
- Yoshihiro Mezaki
- Department of Cell Biology and Morphology, Akita University Graduate School of Medicine
| | - Mayako Morii
- Department of Pediatric Surgery, Akita University Graduate School of Medicine
| | - Taku Hebiguchi
- Department of Pediatric Surgery, Akita University Graduate School of Medicine
| | - Kiwamu Yoshikawa
- Department of Cell Biology and Morphology, Akita University Graduate School of Medicine
| | - Noriko Yamaguchi
- Department of Cell Biology and Morphology, Akita University Graduate School of Medicine
| | - Mitsutaka Miura
- Department of Cell Biology and Morphology, Akita University Graduate School of Medicine
| | - Katsuyuki Imai
- Department of Cell Biology and Morphology, Akita University Graduate School of Medicine
| | - Hiroaki Yoshino
- Department of Pediatric Surgery, Akita University Graduate School of Medicine
| | - Haruki Senoo
- Department of Cell Biology and Morphology, Akita University Graduate School of Medicine
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24
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Van Rossen E, Liu Z, Blijweert D, Eysackers N, Mannaerts I, Schroyen B, El Taghdouini A, Edwards B, Davies KE, Sokal E, Najimi M, Reynaert H, van Grunsven LA. Syncoilin is an intermediate filament protein in activated hepatic stellate cells. Histochem Cell Biol 2013; 141:85-99. [PMID: 24043511 DOI: 10.1007/s00418-013-1142-5] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 09/05/2013] [Indexed: 02/06/2023]
Abstract
Hepatic stellate cells (HSCs) play an important role in several (patho)physiologic conditions in the liver. In response to chronic injury, HSCs are activated and change from quiescent to myofibroblast-like cells with contractile properties. This shift in phenotype is accompanied by a change in expression of intermediate filament (IF) proteins. HSCs express a broad, but variable spectrum of IF proteins. In muscle, syncoilin was identified as an alpha-dystrobrevin binding protein with sequence homology to IF proteins. We investigated the expression of syncoilin in mouse and human HSCs. Syncoilin expression in isolated and cultured HSCs was studied by qPCR, Western blotting, and fluorescence immunocytochemistry. Syncoilin expression was also evaluated in other primary liver cell types and in in vivo-activated HSCs as well as total liver samples from fibrotic mice and cirrhotic patients. Syncoilin mRNA was present in human and mouse HSCs and was highly expressed in in vitro- and in vivo-activated HSCs. Syncoilin protein was strongly upregulated during in vitro activation of HSCs and undetectable in hepatocytes and liver sinusoidal endothelial cells. Syncoilin mRNA levels were elevated in both CCl4- and common bile duct ligation-treated mice. Syncoilin immunocytochemistry revealed filamentous staining in activated mouse HSCs that partially colocalized with α-smooth muscle actin, β-actin, desmin, and α-tubulin. We show that in the liver, syncoilin is predominantly expressed by activated HSCs and displays very low-expression levels in other liver cell types, making it a good marker of activated HSCs. During in vitro activation of mouse HSCs, syncoilin is able to form filamentous structures or at least to closely interact with existing cellular filaments.
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Affiliation(s)
- E Van Rossen
- Liver Cell Biology Lab, Department of Cell Biology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090, Brussels, Belgium,
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25
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Tennakoon AH, Izawa T, Wijesundera KK, Golbar HM, Tanaka M, Ichikawa C, Kuwamura M, Yamate J. Characterization of glial fibrillary acidic protein (GFAP)-expressing hepatic stellate cells and myofibroblasts in thioacetamide (TAA)-induced rat liver injury. ACTA ACUST UNITED AC 2013; 65:1159-71. [PMID: 23806769 DOI: 10.1016/j.etp.2013.05.008] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2013] [Accepted: 05/28/2013] [Indexed: 01/14/2023]
Abstract
Hepatic stellate cells (HSCs), which can express glial fibrillary acidic protein (GFAP) in normal rat livers, play important roles in hepatic fibrogenesis through the conversion into myofibroblasts (MFs). Cellular properties and possible derivation of GFAP-expressing MFs were investigated in thioacetamide (TAA)-induced rat liver injury and subsequent fibrosis. Seven-week-old male F344 rats were injected with TAA (300mg/kg BW, once, intraperitoneally), and were examined on post single injection (PSI) days 1-10 by the single and double immunolabeling with MF and stem cell marker antibodies. After hepatocyte injury in the perivenular areas on PSI days 1 and 2, the fibrotic lesion consisting of MF developed at a peak on PSI day 3, and then recovered gradually by PSI day 10. MFs expressed GFAP, and also showed co-expressions such cytoskeletons (MF markers) as vimentin, desmin and α-SMA in varying degrees. Besides MFs co-expressing vimentin/desmin, desmin/α-SMA or α-SMA/vimentin, some GFAP positive MFs co-expressed with nestin or A3 (both, stem cell markers), and there were also MFs co-expressing nestin/A3. However, there were no GFAP positive MFs co-expressing RECA-1 (endothelial marker) or Thy-1 (immature mesenchymal cell marker). GFAP positive MFs showed the proliferating activity, but they did not undergo apoptosis. However, α-SMA positive MFs underwent apoptosis. These findings indicate that HSCs can proliferate and then convert into MFs with co-expressing various cytoskeletons for MF markers, and that the converted MFs may be derived partly from the stem cell lineage. Additionally, well-differentiated MFs expressing α-SMA may disappear by apoptosis for healing. These findings shed some light on the pathogenesis of chemically induced hepatic fibrosis.
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Affiliation(s)
- Anusha Hemamali Tennakoon
- Laboratory of Veterinary Pathology, Division of Veterinary Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-58, Rinku-ourai-kita, Izumisano City, Osaka 598-8531, Japan
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26
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Non-cell-autonomous tumor suppression by p53. Cell 2013; 153:449-60. [PMID: 23562644 PMCID: PMC3702034 DOI: 10.1016/j.cell.2013.03.020] [Citation(s) in RCA: 622] [Impact Index Per Article: 51.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2012] [Revised: 01/16/2013] [Accepted: 03/14/2013] [Indexed: 12/15/2022]
Abstract
The p53 tumor suppressor can restrict malignant transformation by triggering cell-autonomous programs of cell-cycle arrest or apoptosis. p53 also promotes cellular senescence, a tumor-suppressive program that involves stable cell-cycle arrest and secretion of factors that modify the tissue microenvironment. In the presence of chronic liver damage, we show that ablation of a p53-dependent senescence program in hepatic stellate cells increases liver fibrosis and cirrhosis associated with reduced survival and enhances the transformation of adjacent epithelial cells into hepatocellular carcinoma. p53-expressing senescent stellate cells release factors that skew macrophage polarization toward a tumor-inhibiting M1-state capable of attacking senescent cells in culture, whereas proliferating p53-deficient stellate cells secrete factors that stimulate polarization of macrophages into a tumor-promoting M2-state and enhance the proliferation of premalignant cells. Hence, p53 can act non-cell autonomously to suppress tumorigenesis by promoting an antitumor microenvironment, in part, through secreted factors that modulate macrophage function.
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27
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Up-regulation of RACK1 by TGF-β1 promotes hepatic fibrosis in mice. PLoS One 2013; 8:e60115. [PMID: 23555900 PMCID: PMC3612079 DOI: 10.1371/journal.pone.0060115] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2012] [Accepted: 02/21/2013] [Indexed: 01/18/2023] Open
Abstract
Liver fibrosis represents the consequences of a sustained wound healing response to chronic liver injury, and activation of quiescent hepatic stellate cells (HSCs) into a myofibroblast-like phenotype is considered as the central event of liver fibrosis. RACK1, the receptor for activated C-kinase 1, is a classical scaffold protein implicated in numerous signaling pathways and cellular processes; however, the role of RACK1 in liver fibrosis is little defined. Herein, we report that RACK1 is up-regulated in activated HSCs in transforming growth factor beta 1 (TGF-β1)-dependent manner both in vitro and in vivo, and TGF-β1 stimulates the expression of RACK1 through NF-κB signaling. Moreover, RACK1 promotes TGF-β1 and platelet-derived growth factor (PDGF)-mediated activation of pro-fibrogenic pathways as well as the differentiation, proliferation and migration of HSCs. Depletion of RACK1 suppresses the progression of TAA-induced liver fibrosis in vivo. In addition, the expression of RACK1 in fibrogenic cells also positively correlates well with the stage of liver fibrosis in clinical cases. Our results suggest RACK1 as a downstream target gene of TGF-β1 involved in the modulation of liver fibrosis progression in vitro and in vivo, and propose a strategy to target RACK1 for liver fibrosis treatment.
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Proteomic analysis of the effect of fuzheng huayu recipe on fibrotic liver in rats. EVIDENCE-BASED COMPLEMENTARY AND ALTERNATIVE MEDICINE 2013; 2013:972863. [PMID: 23431353 PMCID: PMC3569931 DOI: 10.1155/2013/972863] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/19/2012] [Accepted: 12/04/2012] [Indexed: 01/07/2023]
Abstract
Hepatic fibrosis is a common pathological process of chronic liver diseases and would lead to cirrhosis, and Fuzheng Huayu (FZHY) is an effective Chinese herbal product against liver fibrosis. This study observes FZHY influence on proteome of fibrotic liver with differential proteomic approach and aims to understand FZHY multiple action mechanisms on liver fibrosis.
The liver fibrosis models were induced with intraperitoneal injection of dimethylnitrosamine for 4 weeks in rats and divided into model control (model) and FZHY-treated (FZHY) groups, while normal rats were used as normal control (normal). After model establishment, rats in FZHY groups were administered 4 g/kg wt of FZHY for 4 weeks, and normal and model groups were given the same volume of saline. The liver proteins in the above 3 groups were separated by two-dimensional gel electrophoresis (2-DE), the differentially expressed spots were analyzed and compared between normal and model or model and FZHY groups, and then the proteins were identified with mass spectrum analysis and validated partially with western blot and real-time PCR. 1000~1200 spots were displayed on each 2D gel, and a total of 61 protein spots were found with significant intensity difference between normal control or FZHY and model control. 23 most obviously differential spots were excised, and in-gel digestion and 21 peptide mass fingerprints (PMF) were obtained with MALDI-TOF MS analysis, and 14 proteins were identified through protein database searching. Among 14 differentially expressed proteins, 8 proteins in normal and FZHY groups had the same tendency of differential expression compared with the ones in model group. And one of them, vimentin, was validated by western blot and real-time PCR analyses. Our study reveals 12 proteins responsible for fibrogenesis induced by DMN in rats, and among them, 8 proteins in fibrotic liver were regulated by FZHY, including aldehyde dehydrogenase, vimentin isoform (CRA_b), gamma-actin, vimentin, fructose-bisphosphate aldolase B, aldo-keto reductase, S-adenosylhomocysteine hydrolase isoform, and HSP90. It indicates that the action mechanism of FZHY antiliver fibrosis may be associated with modulation of proteins associated with metabolism and stress response, as well as myofibroblast activation. The study provides new insights and data for exploring the liver fibrogenesis pathophysiology and FZHY action mechanism against liver fibrosis.
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Kuang P, Zhao W, Su W, Zhang Z, Zhang L, Liu J, Ren G, Yin Z, Wang X. 18β-glycyrrhetinic acid inhibits hepatocellular carcinoma development by reversing hepatic stellate cell-mediated immunosuppression in mice. Int J Cancer 2012; 132:1831-41. [PMID: 22991231 DOI: 10.1002/ijc.27852] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2012] [Accepted: 09/05/2012] [Indexed: 01/16/2023]
Abstract
Hepatic stellate cells (HSCs) have immunosuppressive capabilities and contribute to the occurrence and development of hepatocellular carcinoma (HCC). Thus, activated HSCs may be a suitable target for HCC therapy. Our study used mixed leukocyte reactions (MLR) in vitro to demonstrate that 18β-glycyrrhetinic acid (GA) could reverse HSC-mediated immunosuppression by reducing T-cell apoptosis and regulatory T (Treg) cells expression, thereby enhancing the ability of T cells to attack tumor cells and attenuating HCC cell invasiveness. Moreover, we established a HCC orthotopic implantation model in immunocompetent C57BL/6 mice, which suggested that GA played a protective role in HCC development by reducing immunosuppression mediated by HSCs in the tumor microenvironment.
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Affiliation(s)
- Penghao Kuang
- Department of Hepatobiliary Surgery, Zhongshan Hospital, Xiamen University, Xiamen, Fujian, China
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Avraham Y, Amer J, Doron S, Abu-Tair L, Mahamid M, Khatib A, Berry EM, Safadi R. The direct profibrotic and indirect immune antifibrotic balance of blocking the cannabinoid 2 receptor. Am J Physiol Gastrointest Liver Physiol 2012; 302:G1364-72. [PMID: 22517772 DOI: 10.1152/ajpgi.00191.2011] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Cannabinoid 2 (CB2) receptors expressed on immune cells are considered to be antifibrogenic. Hepatic stellate cells (HSCs) directly interact with phagocytosis lymphocytes, but the nature of this interaction is obscure. We aimed to study the effects of CB2 receptors on hepatic fibrosis via their role in mediating immunity. Hepatic fibrosis was induced by carbon-tetrachloride (CCl(4)) administration in C57BL/6 wild-type (WT) and CB2 knockout (CB2(-/-)) mice. Irradiated animals were reconstituted with WT or CB2(-/-) lymphocytes. Lymphocytes from naïve/fibrotic WT animals and healthy/cirrhotic hepatitis C virus were preincubated in vitro with or without CB2 antagonist, evaluated for proliferation and apoptosis, and then cocultured with primary mouse HSCs or a human HSC line (LX2), respectively. Lymphocyte phagocytosis was then evaluated. Following CCl(4)-administration, CB2(-/-) mice developed significant hepatic fibrosis but less necroinflammation. WT mice harbored decreased liver CD4(+) and NK(+) cells but increased CD8(+) subsets. Naïve CB2(-/-) mice had significantly decreased T cell subsets. Adoptive transfer of CB2(-/-) lymphocytes led to decreased fibrosis in the irradiated WT recipient compared with animals receiving WT lymphocytes. Moreover, necroinflammation also tended to decrease. In vitro, a CB2-antagonist directly increased human HSC activation and increased apoptosis and decreased proliferation of mice/human T cells (healthy/fibrotic) and their phagocytosis. We concluded that CB2(-/-) lymphocytes exert an antifibrotic activity, whereas lack of CB2 receptor in HSCs promotes fibrosis. These findings broaden our understanding of cannabinoid signaling in hepatic fibrosis beyond their activity solely in HSCs.
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Affiliation(s)
- Yosefa Avraham
- Department of Human Nutrition and Metabolism, Braun School of Public Health, Faculty of Medicine, Hebrew University-Hadassah Medical School, Jerusalem, Israel
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Zhao W, Zhang L, Yin Z, Su W, Ren G, Zhou C, You J, Fan J, Wang X. Activated hepatic stellate cells promote hepatocellular carcinoma development in immunocompetent mice. Int J Cancer 2011; 129:2651-61. [PMID: 21213212 DOI: 10.1002/ijc.25920] [Citation(s) in RCA: 96] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2010] [Accepted: 12/22/2010] [Indexed: 12/29/2022]
Abstract
Activated hepatic stellate cells (HSCs) play a central role in the hepatic fibrosis and cirrhosis. Recently, HSCs were reported to have strong immune modulatory activities. However, the role of HSCs in hepatocellular carcinoma (HCC) remains unclear. In this study, we showed that HSCs could promote HCC growth both in vitro and in vivo. We examined the HSC-mediated inhibition of T-cell proliferation and the ability of conditioned medium from activated HSCs to promote the growth of murine HCC cell lines in vitro. We also assessed the immune suppression by HSCs during the development of HCC in immunocompetent mice. Cotransplantation of HSCs promoted HCC growth and progression by enhancing tumor angiogenesis and tumor cell proliferation and by creating an immunosuppressed microenvironment. Cotransplanted HSCs inhibited the lymphocyte infiltration in tumors and the spleens of mice bearing tumors, induced apoptosis of infiltrating mononuclear cells, and enhanced the expression of B7H1 and CD4(+) CD25(+) Treg cells. The immune modulation by HSCs seemed to be systemic. In conclusion, our data provide new information to support an integral role for HSCs in promoting HCC progression in part via their immune regulatory activities, and suggest that HSCs may serve as a therapeutic target in HCC.
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Affiliation(s)
- Wenxiu Zhao
- Department of Hepatobiliary Surgery, Zhongshan Hospital Xiamen University, Research Institute of Digestive Disease, Xiamen, Fujian, China
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Ehnert S, Knobeloch D, Blankenstein A, Müller A, Böcker U, Gillen S, Friess H, Thasler WE, Dooley S, Nussler AK. Neohepatocytes from alcoholics and controls express hepatocyte markers and display reduced fibrogenic TGF-ß/Smad3 signaling: advantage for cell transplantation? Alcohol Clin Exp Res 2010; 34:708-718. [PMID: 20102559 DOI: 10.1111/j.1530-0277.2009.01140.x] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
BACKGROUND Liver transplantation is the only definitive treatment for end stage liver disease. Donor organ scarcity raises a growing interest in new therapeutic options. Recently, we have shown that injection of monocyte-derived NeoHepatocytes can increase survival in rats with extended liver resection. In order to apply this technology in humans with chronic liver diseases in an autologous setting, we generated NeoHepatocytes from patients with alcoholic liver disease and healthy controls and compared those to human hepatocytes. METHODS We generated NeoHepatocytes from alcoholics with Child A and B cirrhosis and healthy controls. Hepatocytes marker expression and transforming growth factor (TGF)-beta signaling was investigated by RT-PCR, Western blot, immunofluorescent staining, and adenoviral reporter assays. Glucose and urea was measured photometrically. Phase I and II enzyme activities were measured using fluorogenic substrates. Neutral lipids were visualized by Oil Red O staining. RESULTS There was no significant difference in generation and yield of NeoHepatocytes from alcoholics and controls. Hepatocyte markers, e.g., cytokeratin18 and alcohol dehydrogenase 1, increased significantly throughout differentiation. Glucose and urea production did not differ between alcoholics and controls and was comparable to human hepatocytes. During differentiation, phase I and II enzyme activities increased, however remained significantly lower than in human hepatocytes. Fat accumulation was induced by treatment with insulin, TGF-beta and ethanol only in differentiated cells and hepatocytes. TGF-beta signaling, via Smad transcription factors, critically required for progression of chronic liver disease, was comparable among the investigated cell types, merely expression of Smad1 and -3 was reduced (approximately 30 and approximately 60%) in monocytes, programmable cells of monocytic origin, and NeoHepatocytes. Subsequently, expression of TGF-beta regulated pro-fibrogenic genes, e.g., connective tissue growth factor and fibronectin was reduced. CONCLUSIONS Generation of NeoHepatocytes from alcoholics, displaying several features of human hepatocytes, offers new perspectives for cell therapeutic approaches, as cells can be obtained repeatedly in a noninvasive manner. Furthermore, the autologous setting reduces the need for immunosuppressants, which may support recovery of patients which are declined for liver transplantation.
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Affiliation(s)
- Sabrina Ehnert
- Klinikum rechts der Isar, Department of Traumatology, MRI, TechnischeUniversität München, München, Germany.
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Kara B, Daglioglu K, Doran F, Akkiz H, Sandikci M, Kara I. Expression of Mesenchymal, Hematopoietic, and Biliary Cell Markers in Adult Rat Hepatocytes After Partial Hepatectomy. Transplant Proc 2009; 41:4401-4. [DOI: 10.1016/j.transproceed.2009.09.082] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2009] [Accepted: 09/29/2009] [Indexed: 12/31/2022]
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Nagatsuma K, Hayashi Y, Hano H, Sagara H, Murakami K, Saito M, Masaki T, Lu T, Tanaka M, Enzan H, Aizawa Y, Tajiri H, Matsuura T. Lecithin: retinol acyltransferase protein is distributed in both hepatic stellate cells and endothelial cells of normal rodent and human liver. Liver Int 2009; 29:47-54. [PMID: 18544127 DOI: 10.1111/j.1478-3231.2008.01773.x] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/13/2023]
Abstract
BACKGROUND To determine the extent to which hepatic stellate cell (HSC) activation contributes to liver fibrosis, it was found necessary to develop an alternative structural and functional stellate cell marker for in situ studies. Although several HSC markers have been reported, none of those are associated with particular HSC functions. AIM The present study was undertaken to examine whether lecithin:retinol acyltransferase (LRAT), the physiological retinol esterification enzyme of the liver, is a potential and relevant tissue marker for HSC. METHODS An antibody specific to mouse and human LRAT was prepared based on the amino acid sequences. Antibodies to LRAT were used for immunohistochemical studies to assess the distribution of LRAT-positive cells in the liver with the aid of fluorescence and immunogold electron microscopy. RESULTS LRAT-positive cells were found to be confined in the space of Disse, corresponding with the location of desmin-positive HSC in rodent liver, also in human liver. Interestingly, LRAT-positive staining was also observed along the liver sinusoidal endothelial lining. Furthermore, immune electron microscopic studies revealed that LRAT was mainly distributed in HSC within the rough-endoplasmic reticulum (RER) and multivesicular bodies, whereas LRAT staining within the endothelial cells was largely confined to the perinuclear area and to some extent to the RER. CONCLUSION Evidence has been accumulated that LRAT might serve as an excellent alternative HSC marker for future structural and functional studies. Furthermore, the presence of LRAT in endothelial cells might suggest a currently unknown function of this enzyme in liver endothelial biology.
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Affiliation(s)
- Keisuke Nagatsuma
- Department of Internal Medicine, Division of Gastroenterology and Hepatology, The Jikei University School of Medicine, Tokyo, Japan
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Vinculin and cellular retinol-binding protein-1 are markers for quiescent and activated hepatic stellate cells in formalin-fixed paraffin embedded human liver. Histochem Cell Biol 2008; 131:313-25. [PMID: 19052772 DOI: 10.1007/s00418-008-0544-2] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 11/13/2008] [Indexed: 12/15/2022]
Abstract
Hepatic stellate cells (HSCs) have important roles in the pathogenesis of liver fibrosis and cirrhosis. As response to chronic injury HSCs are activated and change from quiescent into myofibroblast-like cells. Several HSC-specific markers have been described in rat or mouse models. The aim of our work was to identify the best marker(s) for human HSCs. To this end we used the automated high throughput NexES IHC staining device (Ventana Medical Systems) to incubate sections under standardized conditions. Formalin fixed paraffin embedded (FFPE) normal and diseased human livers were studied. With immunohistochemistry we examined the expression of synemin, desmin, vimentin, vinculin, neurotrophin-3 (NT-3), alpha-smooth muscle actin (alpha-SMA), cellular retinol-binding protein-1 (CRBP-1), glial fibrillary acidic protein (GFAP), cysteine- and glycine-rich protein 2 (CRP2), and cytoglobin/stellate cell activation-associated protein (cygb/STAP). This is the first study in which a series of HSC markers is compared on serial FFPE human tissues. CRBP-1 clearly stains lobular HSCs without reacting with smooth muscle cells (SMCs) and shows variable cholangiocyte positivity. Vinculin has a similar staining pattern as CRBP-1 but additionally stains SMCs, and (myo)fibroblasts. In conclusion, we therefore propose to use CRBP-1 and/or vinculin to stain HSCs in human liver tissues.
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Abdeen SM, Olusi SO, Askar HA, Thalib L, Al-Azemi A, George S. The predictive value of CD38 positive hepatic stellate cell count for assessing disease activity and fibrosis in patients with chronic hepatitis. Acta Histochem 2008; 111:520-30. [PMID: 18829073 DOI: 10.1016/j.acthis.2008.04.008] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2008] [Revised: 04/03/2008] [Accepted: 04/03/2008] [Indexed: 01/19/2023]
Abstract
The activation of hepatic stellate cells (HSCs) is a critical event in hepatic fibrosis. The objectives of this study were to find out if cluster of differentiation 38 (CD38) can be demonstrated immunohistochemically on HSCs in liver biopsies from patients with chronic liver disease and if CD38 immunopositive HSC count is correlated with METAVIR inflammatory and fibrosis scores. Immunohistochemical labelling for CD38 was performed on 100 liver biopsies from patients with chronic liver disease. The CD38 immunopositive HSCs were identified and counted. The CD38 immunopositive HSC count was found to be associated with both the METAVIR score and the fibrosis scores. The CD38 immunopositive HSC count was able to discriminate between no fibrosis and stages 2, 3 or 4 fibrosis, but could not discriminate between no fibrosis and stage 1 fibrosis. Using receiver operating characteristic (ROC) curves, a cut-off point of 10 HSCs per 10 high power field (hpf), or 25 per 100 hepatocytes, is 80% sensitive and 70% specific for predicting fibrosis. The specificity rose to 100% in patients with hepatitis C viral (HCV) infection. We conclude that CD38 positive HSCs can be demonstrated immunohistochemically and that the count is highly predictive of moderate to severe hepatic fibrosis.
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Dudas J, Mansuroglu T, Batusic D, Ramadori G. Thy-1 is expressed in myofibroblasts but not found in hepatic stellate cells following liver injury. Histochem Cell Biol 2008; 131:115-27. [PMID: 18797914 DOI: 10.1007/s00418-008-0503-y] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 08/31/2008] [Indexed: 01/19/2023]
Abstract
Thy-1 (CD90) is an adhesion molecule induced in fibroblast populations associated with wound healing and fibrosis. In this study the question whether Thy-1-gene-expression can be induced in hepatic stellate cells (HSC) in vivo, under conditions of liver injury or liver regeneration was addressed. Acute and chronic rat liver injury was induced by the administration of CCl4. For comparison, cirrhotic human liver, and rat 67% partial hepatectomy (PH) was studied as well. Thy-1-gene-expression was examined also in isolated human liver myofibroblasts. Thy-1-mRNA expression was significantly upregulated in chronic liver injury. Thy-1+ cells were detected in the periportal area of rat liver specimens in normal-, injured- and regenerative-conditions. In chronic human and rat liver injury, Thy-1+ cells were located predominantly in scar tissue. In the pericentral necrotic zone after CCl4-treatment, no induction of Thy-1 was found. Gremlin and Thy-1 showed comparable localization in the periportal areas. Thy-1 was not detected in either normal or capillarized sinusoids, in isolated rat HSC, and was neither inducible by inflammatory cytokines in isolated HSC, nor upregulated in treated myofibroblasts. Based upon these data Thy-1 is not a marker of "activated" sinusoidal HSC, but it is a marker of "activated" (myo)fibroblasts found in portal areas and in scar tissue.
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Affiliation(s)
- Jozsef Dudas
- Department of Internal Medicine, Section of Gastroenterology and Endocrinology, Georg-August-University Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany
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38
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Carotti S, Morini S, Corradini SG, Burza MA, Molinaro A, Carpino G, Merli M, De Santis A, Muda AO, Rossi M, Attili AF, Gaudio E. Glial fibrillary acidic protein as an early marker of hepatic stellate cell activation in chronic and posttransplant recurrent hepatitis C. Liver Transpl 2008; 14:806-14. [PMID: 18508359 DOI: 10.1002/lt.21436] [Citation(s) in RCA: 69] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Activated alpha-smooth muscle actin (alpha-SMA)-positive hepatic stellate cells (HSCs) are pericytes responsible for fibrosis in chronic liver injury. The glial fibrillary acidic protein (GFAP), commonly expressed by astrocytes in the central nervous system, is expressed in vivo in the liver in a subpopulation of quiescent stellate cells. In the rat, increased GFAP expression in the acute response to injury and down-regulation in the chronic response have been observed, whereas reports concerning GFAP expression in human liver are still conflicting. We investigated the utility of GFAP compared to alpha-SMA as an immunohistochemical marker of early activated HSCs in chronic and posttransplant recurrent hepatitis C and correlated GFAP expression with vascular remodeling and fibrosis progression. With immunohistochemistry and a semiquantitative scoring system, the expression of GFAP and alpha-SMA in HSCs and the microvessel density were analyzed in biopsies from normal livers obtained from cadaveric donors [donor liver (DL); n = 21] and from livers from posttransplant hepatitis C virus recurrent hepatitis (HCV-PTR) patients (n = 19), hepatitis C virus chronic hepatitis (HCV-CH) patients, (n = 12), and hepatitis C virus cirrhosis (HCV-C) patients (n = 16). The percentage of alpha-SMA-positive HSCs was significantly higher in the HCV-PTR, HCV-CH, and HCV-C groups compared to the DL group (P < 0.01). The percentage of GFAP-positive HSCs was significantly higher in the HCV-PTR group compared to the DL, HCV-C (P < 0.01), and HCV-CH (P < 0.05) groups and in the HCV-CH group compared to the DL group (P < 0.01), inversely correlating with the extent of fibrosis and microvessel density (P < 0.01). In the HCV-PTR group, the percentage of GFAP-positive HSCs correlated with fibrosis progression (P < 0.01). In conclusion, GFAP could represent a useful marker of early activation of HSCs in HCV-CH and seems to predict fibrosis progression in HCV-PTR.
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Affiliation(s)
- Simone Carotti
- Department of Human Anatomy, University of Rome La Sapienza, Italy, Rome
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Strnad P, Stumptner C, Zatloukal K, Denk H. Intermediate filament cytoskeleton of the liver in health and disease. Histochem Cell Biol 2008; 129:735-49. [PMID: 18443813 PMCID: PMC2386529 DOI: 10.1007/s00418-008-0431-x] [Citation(s) in RCA: 83] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 04/14/2008] [Indexed: 02/06/2023]
Abstract
Intermediate filaments (IFs) represent the largest cytoskeletal gene family comprising approximately 70 genes expressed in tissue specific manner. In addition to scaffolding function, they form complex signaling platforms and interact with various kinases, adaptor, and apoptotic proteins. IFs are established cytoprotectants and IF variants are associated with >30 human diseases. Furthermore, IF-containing inclusion bodies are characteristic features of several neurodegenerative, muscular, and other disorders. Acidic (type I) and basic keratins (type II) build obligatory type I and type II heteropolymers and are expressed in epithelial cells. Adult hepatocytes contain K8 and K18 as their only cytoplasmic IF pair, whereas cholangiocytes express K7 and K19 in addition. K8/K18-deficient animals exhibit a marked susceptibility to various toxic agents and Fas-induced apoptosis. In humans, K8/K18 variants predispose to development of end-stage liver disease and acute liver failure (ALF). K8/K18 variants also associate with development of liver fibrosis in patients with chronic hepatitis C. Mallory-Denk bodies (MDBs) are protein aggregates consisting of ubiquitinated K8/K18, chaperones and sequestosome1/p62 (p62) as their major constituents. MDBs are found in various liver diseases including alcoholic and non-alcoholic steatohepatitis and can be formed in mice by feeding hepatotoxic substances griseofulvin and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). MDBs also arise in cell culture after transfection with K8/K18, ubiquitin, and p62. Major factors that determine MDB formation in vivo are the type of stress (with oxidative stress as a major player), the extent of stress-induced protein misfolding and resulting chaperone, proteasome and autophagy overload, keratin 8 excess, transglutaminase activation with transamidation of keratin 8 and p62 upregulation.
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Affiliation(s)
- P Strnad
- Department of Internal Medicine I, University of Ulm, Robert-Koch-Strabe 8, 89081, Ulm, Germany.
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40
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Wada T, Kang HS, Angers M, Gong H, Bhatia S, Khadem S, Ren S, Ellis E, Strom SC, Jetten AM, Xie W. Identification of oxysterol 7alpha-hydroxylase (Cyp7b1) as a novel retinoid-related orphan receptor alpha (RORalpha) (NR1F1) target gene and a functional cross-talk between RORalpha and liver X receptor (NR1H3). Mol Pharmacol 2008; 73:891-9. [PMID: 18055760 DOI: 10.1124/mol.107.040741] [Citation(s) in RCA: 81] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
Abstract
The retinoid-related orphan receptors (RORs) and liver X receptors (LXRs) were postulated to have distinct functions. RORs play a role in tissue development and circadian rhythm, whereas LXRs are sterol sensors that affect lipid homeostasis. In this study, we revealed a novel function of RORalpha (NR1F1) in regulating the oxysterol 7alpha-hydroxylase (Cyp7b1), an enzyme critical for the homeostasis of cholesterol, bile acids, and oxysterols. The expression of Cyp7b1 gene was suppressed in the RORalpha null (RORalpha(sg/sg)) mice, suggesting RORalpha as a positive regulator of Cyp7b1. Promoter analysis established Cyp7b1 as a transcriptional target of RORalpha, and transfection of RORalpha induced the expression of endogenous Cyp7b1 in the liver. Interestingly, Cyp7b1 regulation seemed to be RORalpha-specific, because RORgamma had little effect. Reporter gene analysis showed that the activation of Cyp7b1 gene promoter by RORalpha was suppressed by LXRalpha (NR1H3), whereas RORalpha inhibited both the constitutive and ligand-dependent activities of LXRalpha. The mutual suppression between RORalpha and LXR was supported by the in vivo observation that loss of RORalpha increased the expression of selected LXR target genes, leading to hepatic triglyceride accumulation. Likewise, mice deficient of LXR alpha and beta isoforms showed activation of selected RORalpha target genes. Our results have revealed a novel role for RORalpha and a functional interplay between RORalpha and LXR in regulating endo- and xenobiotic genes, which may have broad implications in metabolic homeostasis.
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MESH Headings
- Adult
- Animals
- Cells, Cultured
- Cholesterol/blood
- Cytochrome P-450 Enzyme System/genetics
- Cytochrome P-450 Enzyme System/metabolism
- DNA-Binding Proteins/deficiency
- DNA-Binding Proteins/genetics
- DNA-Binding Proteins/physiology
- Fasting
- Female
- Gene Expression Regulation
- Genes, Reporter
- Hepatocytes/cytology
- Hepatocytes/metabolism
- Humans
- Liver X Receptors
- Luciferases/metabolism
- Male
- Mice
- Mice, Inbred C57BL
- Mice, Inbred Strains
- Mice, Knockout
- Nuclear Receptor Subfamily 1, Group F, Member 1
- Orphan Nuclear Receptors
- Promoter Regions, Genetic
- Receptors, Cytoplasmic and Nuclear/deficiency
- Receptors, Cytoplasmic and Nuclear/genetics
- Receptors, Cytoplasmic and Nuclear/physiology
- Steroid Hydroxylases/genetics
- Steroid Hydroxylases/metabolism
- Trans-Activators/deficiency
- Trans-Activators/genetics
- Trans-Activators/physiology
- Transfection
- Triglycerides/blood
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Affiliation(s)
- Taira Wada
- Center for Pharmacogenetics, 633 Salk Hall, University of Pittsburgh, Pittsburgh, PA 15213, USA.
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Wang B, Dolinski BM, Kikuchi N, Leone DR, Peters MG, Weinreb PH, Violette SM, Bissell DM. Role of alphavbeta6 integrin in acute biliary fibrosis. Hepatology 2007; 46:1404-12. [PMID: 17924447 PMCID: PMC4144397 DOI: 10.1002/hep.21849] [Citation(s) in RCA: 127] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
UNLABELLED Acute biliary obstruction leads to periductal myofibroblasts and fibrosis, the origin of which is uncertain. Our study provides new information on this question in mice and humans. We show that bile duct obstruction induces a striking increase in cholangiocyte alphavbeta6 integrin and that expression of this integrin is directly linked to fibrogenesis through activation of transforming growth factor beta (TGF-beta). Administration of blocking antibody to alphavbeta6 significantly reduces the extent of acute fibrosis after bile duct ligation. Moreover, in beta6-null mice subjected to the injury, fibrosis is reduced by 50% relative to that seen in wild-type mice, whereas inflammation occurs to the same extent. The data indicate that alphavbeta6, rather than inflammation, is linked to fibrogenesis. It is known that alphavbeta6 binds latent TGF-beta and that binding results in release of active TGFbeta. Consistent with this, intracellular signaling from the TGFbeta receptor is increased after bile duct ligation in wild-type mice but not in beta6(-/-) mice, and a competitive inhibitor of the TGFbeta receptor type II blocks fibrosis to the same extent as antibody to alphavbeta6. In a survey of human liver disease, expression of alphavbeta6 is increased in acute, but not chronic, biliary injury and is localized to cholangiocyte-like cells. CONCLUSION Cholangiocytes respond to acute bile duct obstruction with markedly increased expression of alphavbeta6 integrin, which is closely linked to periductal fibrogenesis. The findings provide a rationale for the use of inhibitors of alphavbeta6 integrin or TGFbeta for down-regulating fibrosis in the setting of acute or ongoing biliary injury.
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Affiliation(s)
- Bruce Wang
- The Liver Center, Department of Medicine, University of California, San Francisco
| | | | - Noriko Kikuchi
- The Liver Center, Department of Medicine, University of California, San Francisco
| | | | - Marion G. Peters
- The Liver Center, Department of Medicine, University of California, San Francisco
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Muhanna N, Horani A, Doron S, Safadi R. Lymphocyte-hepatic stellate cell proximity suggests a direct interaction. Clin Exp Immunol 2007; 148:338-47. [PMID: 17437422 PMCID: PMC1868876 DOI: 10.1111/j.1365-2249.2007.03353.x] [Citation(s) in RCA: 56] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Recent functional research studies suggest an anti-fibrotic role for natural killer (NK) cells coupled with a profibrotic role for CD8 cells. However, the morphological cellular interplay between the different cell types is less clear. To investigate lymphocyte/hepatic stellate cell (HSC) interactions, hepatic fibrosis was induced by administering carbon tetrachloride (CCl4) intraperitoneally (i.p.) for 4 weeks in C57Bl/6 mice. Animals were killed at 0, 1, 2, 3 and 4 weeks. Liver sections were stained for Sirius red. Confocal microscopy was used to evaluate alpha smooth-muscle actin (alphaSMA) and lymphocyte subsets in liver sections. At weeks 0 and 4, liver protein extracts were assessed for alphaSMA by Western blotting and isolated liver lymphocytes as well as HSC were analysed by fluorescence activated cell sorter (FACS). Similar to the results obtained from classical Sirius red staining and alphaSMA blotting, analysis of liver sections by confocal microscopy revealed a marked and continuous accumulation of alphaSMA staining along sequential experimental check-points after administering CCl4. Although the number of all liver lymphocyte subsets increased following fibrosis induction, FACS analysis revealed an increase in the distribution of liver CD8 subsets and a decrease of CD4 T cells. Confocal microscopy showed a significant early appearance of CD8 and NK cells, and to a lesser extent CD4 T cells, appearing only from week 2. Lymphocytes were seen in proximity only to HSC, mainly in the periportal area and along fibrotic septa, suggesting a direct interaction. Notably, lymphocyte subsets were undetectable in naive liver sections. Freshly isolated HCS show high expression of major histocompatibility complex (MHC) class II and CD11c. In the animal model of hepatic fibrosis, lymphocytes infiltrate into the liver parenchyma and it is thought that they attach directly to activated HSC. Because HSCs express CD11c/class II molecules, interactions involving them might reflect that HSCs have an antigen-presenting capacity.
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Affiliation(s)
- N Muhanna
- Liver/Gastroenterology Units, Division of Medicine, Hadassah University Hospital, Jerusalem, Israel
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43
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Chu D, Luo Q, Li C, Gao Y, Yu L, Wei W, Wu Q, Shen J. Paeoniflorin inhibits TGF-beta1-mediated collagen production by Schistosoma japonicum soluble egg antigen in vitro. Parasitology 2007; 134:1611-21. [PMID: 17524166 DOI: 10.1017/s0031182007002946] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
The main pathological characteristics of hepatic fibrosis in schistosomiasis are the proliferation of hepatic stellate cells (HSCs) and the deposition of collagen type I (Col I) and collagen type III (Col III). Transforming growth factor beta-1 (TGF-beta1) plays an important role in hepatic fibrosis. Paeoniflorin (PAE) has been reported to have immunoregulatory effects; however, the mechanism of its anti-hepatic fibrosis in S. japonicum has not been elucidated. In the present study, we found that mouse peritoneal macrophages (PMphis) stimulated by soluble egg antigen (SEA) of S. japonicum could secrete TGF-beta1, and the TGF-beta1 in the peritoneal macrophage-conditioned medium (PMCM) could induce proliferation of HSCs and secretion of Col I and III. We selected PMCM at 1 : 2 dilution as the optimum PMCM (OPMCM). Then we treated HSCs pre-incubated with OPMCM with PAE, and found that the inhibition of HSC proliferation or Col I and III production were closely correlated with the concentration of PAE. Further investigation found that PAE significantly decreased the Smad3 transcription and phosphorylation in HSCs stimulated by OPMCM. In conclusion, SEA plays a key role in hepatic fibrosis by inducing TGF-beta1 from PMphis. PAE can exert anti-fibrogenic effects by inhibiting HSCs proliferation and down-regulating Smad3 expression and phosphorylation through TGF-beta1 signalling.
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Affiliation(s)
- D Chu
- Institute of Clinical Pharmacology, Anhui Medical University and the Key Laboratory of Antiinflammatory-immunopharmacology, Anhui, China
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44
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Horani A, Muhanna N, Pappo O, Melhem A, Alvarez CE, Doron S, Wehbi W, Dimitrios K, Friedman SL, Safadi R. Beneficial effect of glatiramer acetate (Copaxone) on immune modulation of experimental hepatic fibrosis. Am J Physiol Gastrointest Liver Physiol 2007; 292:G628-38. [PMID: 17038628 DOI: 10.1152/ajpgi.00137.2006] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
While CD8 subsets activate hepatic fibrosis, natural killer (NK) cells exhibit antifibrotic activity. Glatiramer acetate (GA) is an immune modulator for multiple sclerosis. We assessed the potential impact of GA on mouse hepatic fibrogenesis. Hepatic fibrosis was induced in C57BL/6 mice by intraperitoneal administration of carbon tetrachloride (CCl(4)) for 6 wk. During the last 2 wk, animals were also treated with either GA (200 mu/day ip) or medium and compared with naive and fibrotic mice (8 animals/group). GA markedly attenuated fibrosis without altering reactive oxygen species production. By morphometric measurement of Sirius red-stained tissue sections, the relative fibrosis area decreased from 5.28 +/- 0.32% (mean +/- SE) in the untreated CCl(4) group to 2.01 +/- 0.28% in CCl(4)+GA-treated animals, compared with 0.38 +/- 0.07% in naive mice. alpha-Smooth muscle actin immunoblotting and mRNA expression revealed a similar pattern. Serum aminotransferase and Ishak-Knodell necroinflammatory score were markedly elevated, to the same extent, in both CCl(4)-treated groups. Fibrosis induction was associated with significant increase in CD8 subsets and decrease in CD4 T cells. After GA treatment, however, NK content, CD4(+)CD25(+)FoxP3(+) cells, hepatic expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and apoptosis of hepatic stellate cells were all increased. Serum interleukin (IL)-10 levels markedly rose, whereas IL-4 fell. In vitro activation of human hepatic stellate cells cocultured with hepatitis C virus-derived peripheral blood lymphocytes decreased when lymphocytes were preincubated with GA before coculture. In an animal model of hepatic fibrosis, GA has an antifibrotic effect associated with decreased CD8 cells and reduced serum IL-4 levels and increased NK cells, CD4(+)CD25(+)FoxP3(+) cells, TRAIL, and elevated serum IL-10 levels.
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Affiliation(s)
- Amjad Horani
- Liver and Gastroenterology Units, Div of Medicine, Hadassah University Hospital, 91120 Jerusalem, Israel
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45
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Jiang F, Parsons CJ, Stefanovic B. Gene expression profile of quiescent and activated rat hepatic stellate cells implicates Wnt signaling pathway in activation. J Hepatol 2006; 45:401-9. [PMID: 16780995 DOI: 10.1016/j.jhep.2006.03.016] [Citation(s) in RCA: 178] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/29/2005] [Revised: 03/24/2006] [Accepted: 03/31/2006] [Indexed: 12/19/2022]
Abstract
BACKGROUND/AIMS Liver fibrosis is characterized by accumulation of extracellular matrix proteins synthesized by activated hepatic stellate cells (HSCs). To understand molecular mechanisms of HSCs activation a comprehensive comparison of gene expression between quiescent and activated HSCs is needed. METHODS Using DNA microarrays we compared expression of 31,100 genes between quiescent rat HSCs and culture activated rat HSCs. Expression of the components of Wnt signaling was analyzed in HSCs and fibrotic livers by RT-PCR. Activation of beta-catenin was analyzed by Western blot. RESULTS Nine hundred genes were upregulated more than 4.6-fold and 500 genes were downregulated more than 5.7-fold in activated HSCs. The upregulated genes included Wnt receptor frizzled 2, ligands Wnt4 and Wnt5, which was confirmed in fibrotic livers. Expression of the target genes of Wnt signaling was increased from 5- to 70-fold. Phosphorylation and nuclear translocation of beta-catenin were unchanged, indicating activation of the noncanonical Wnt pathway. CONCLUSIONS Highly upregulated expression of Wnt5a and its receptor frizzled 2 implicates this pathway in differentiation of quiescent HSCs into myofibroblasts. Activation of Wnt signaling pathway in HSCs and in animal models of liver fibrosis has not been described previously, suggesting an important role of Wnt signaling in development of liver fibrosis.
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Affiliation(s)
- Feng Jiang
- Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, FL 32306, USA
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Melhem A, Muhanna N, Bishara A, Alvarez CE, Ilan Y, Bishara T, Horani A, Nassar M, Friedman SL, Safadi R. Anti-fibrotic activity of NK cells in experimental liver injury through killing of activated HSC. J Hepatol 2006; 45:60-71. [PMID: 16515819 DOI: 10.1016/j.jhep.2005.12.025] [Citation(s) in RCA: 206] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/24/2005] [Revised: 11/30/2005] [Accepted: 12/15/2005] [Indexed: 02/06/2023]
Abstract
BACKGROUND/AIMS We have investigated the role of natural killer (NK) cells in hepatic fibrogenesis. Mouse NK cells express both inhibitory/activating-killing-immunoglobulin-related-receptors (iKIR/aKIR) specific for Class-I-molecules. METHODS Hepatic fibrosis induced by carbon-tetrachloride (CCl4) was compared between wild-type (WT) male-BALBc; combined-immunodeficiency (SCID, lacking B/T-cells); and SCID-BEIGE-mice (lacking B/T/NK cells), and naive mice. RESULTS Hepatic fibrosis significantly increased in all CCl4-treated groups. SCID-BEIGE mice had more fibrosis than SCID-mice (P<0.0001) as assessed by morphometry of sirius-red stained tissue sections. Following fibrosis, hepatic NK cells significantly decreased, the aKIR:iKIR-ratio significantly increased while Class-I expression on HSC decreased (P<0.001). Both freshly isolated and in situ HSC displayed a significant increase in cellular apoptosis following fibrosis induction. Confocal microscopy demonstrated the direct adhesion of NK cells to HSC in mouse liver sections and in vitro human NK/HSC co-culture. In human HSC there was decreased Class-I expression and increased apoptosis as well, which was further increased following blocking of either HSC-related Class-I or NK-related killer inhibitory receptors. Apoptosis was inhibited by pre-incubation of NK cells with the granzyme inhibitor 3,4-dichloroisocoumarin. CONCLUSIONS During liver injury, NK cells have an anti-fibrotic activity at least in part through stimulation of HSC killing.
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Affiliation(s)
- Alaa Melhem
- Division of Medicine, Liver and Gastroenterology Units, Hadassah University Hospital, Pob 12000, 91120 Jerusalem, Israel
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Maubach G, Lim MCC, Zhang CY, Zhuo L. GFAP promoter directs lacZ expression specifically in a rat hepatic stellate cell line. World J Gastroenterol 2006; 12:723-30. [PMID: 16521185 PMCID: PMC4066122 DOI: 10.3748/wjg.v12.i5.723] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: The GFAP was traditionally considered to be a biomarker for neural glia (mainly astrocytes and non-myelinating Schwann cells). Genetically, a 2.2-kb human GFAP promoter has been successfully used to target astrocytes in vitro and in vivo. More recently, GFAP was also established as one of the several makers for identifying hepatic stellate cells (HSC). In this project, possible application of the same 2.2-kb human GFAP promoter for targeting HSC was investigated.
METHODS: The GFAP-lacZ transgene was transfected into various cell lines (HSC, hepatocyte, and other non-HSC cell types). The transgene expression specificity was determined by X-gal staining of the β-galactosidase activity. And the responsiveness of the transgene was tested with a typical pro-fibrotic cytokine TGF-β1. The expression of endogenous GFAP gene was assessed by real-time RT-PCR, providing a reference for the transgene expression.
RESULTS: The results demonstrated for the first time that the 2.2 kb hGFAP promoter was not only capable of directing HSC-specific expression, but also responding to a known pro-fibrogenic cytokine TGF-β1 by upregulation in a dose- and time-dependent manner, similar to the endogenous GFAP.
CONCLUSION: In conclusion, these findings suggested novel utilities for using the GFAP promoter to specifically manipulate HSC for therapeutic purpose.
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Affiliation(s)
- Gunter Maubach
- Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, #04-01, 138669, Singapore
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Bandapalli OR, Geheeb M, Kobelt D, Kuehnle K, Elezkurtaj S, Herrmann J, Gressner AM, Weiskirchen R, Beule D, Blüthgen N, Herzel H, Franke C, Brand K. Global analysis of host tissue gene expression in the invasive front of colorectal liver metastases. Int J Cancer 2005; 118:74-89. [PMID: 16080196 DOI: 10.1002/ijc.21307] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Host cell reactions are a crucial determinant for tumor invasion. We analyzed on a genomewide scale gene expression differences between microdissected tissues taken from unaffected liver tissue of a human colorectal tumor (LS174) growing in the livers of nude mice and tissue from the host part of the invasive front. Due to the low degree of interspecies cross-hybridization of 15% as determined on Affymetrix microarrays, our xenograft model allowed for the distinction of genes of murine versus human origin even if the respective tissues could not be isolated separately. Using the gene ontology (GO) classification, we were able to determine patterns of up- and downregulated genes in the liver part of the invasive front. We observed a pronounced overrepresentation, e.g., of the GO terms "extracellular matrix," "cell communication," "response to biotic stimulus," "structural molecule activity" and "cell growth," indicating a very pronounced host cell response to tumor invasion. On the single gene level, hepatic stellate cell (HSC) activation markers were overrepresented in the liver part of the invasion front. Immunohistochemistry and qPCR confirmed an activation of HSC as well as an increased number of HSC in the invasive front as compared to the noninvaded liver tissue. In summary, our data demonstrate the feasibility of an interspecies differential gene expression approach on a genomewide scale.
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Affiliation(s)
- Obul Reddy Bandapalli
- Institute of Biology, Humboldt University Berlin, Max Delbrück Center for Molecular Medicine, Berlin, Germany
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Kolterud A, Wandzioch E, Carlsson L. Lhx2 is expressed in the septum transversum mesenchyme that becomes an integral part of the liver and the formation of these cells is independent of functional Lhx2. Gene Expr Patterns 2005; 4:521-8. [PMID: 15261829 DOI: 10.1016/j.modgep.2004.03.001] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2003] [Revised: 03/01/2004] [Accepted: 03/01/2004] [Indexed: 01/03/2023]
Abstract
Liver development is based on reciprocal interactions between ventral foregut endoderm and adjacent mesenchymal tissues. Targeted disruption of the LIM-homeobox gene Lhx2 has revealed that it is important for the expansion of the liver during embryonic development, whereas it appears not to be involved in the induction of hepatic fate. It is not known whether Lhx2 is expressed in the endodermal or mesenchymal portion of the liver, or if the cells normally expressing Lhx2 are absent or present in the liver of Lhx2(-/-) embryos. To address this we have analyzed gene expression from the Lhx2 locus during hepatic development in wild type and Lhx2(-/-) mice. Lhx2 is expressed in cells of the septum transversum mesenchyme adjacent to the liver bud from embryonic day 9. The hepatic cords subsequently migrate into and intermingle with the Lhx2+ cells of the septum transversum mesenchyme. Lhx2 expression is thereafter maintained in a subpopulation of mesenchymal cells in the liver until adult life. In adult liver the Lhx2+ mesenchymal cells co-express desmin, a marker associated with stellate cells. At embryonic day 10.5, cells expressing the mutant Lhx2 allel are present in Lhx2(-/-) livers, and expression of Hlx, hepatocyte growth factor, Hex and Prox1, genes known to be important in liver development, is independent of functional Lhx2 expression. Thus, Lhx2 is specifically expressed in the liver-associated septum transversum mesenchyme that subsequently becomes an integral part of the liver and the formation of these mesenchymal cells does not require functional Lhx2.
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Affiliation(s)
- Asa Kolterud
- Umeå Centre for Molecular Medicine, Umeå University, 901 87 Umeå, Sweden
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Baba S, Fujii H, Hirose T, Yasuchika K, Azuma H, Hoppo T, Naito M, Machimoto T, Ikai I. Commitment of bone marrow cells to hepatic stellate cells in mouse. J Hepatol 2004; 40:255-60. [PMID: 14739096 DOI: 10.1016/j.jhep.2003.10.012] [Citation(s) in RCA: 126] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
BACKGROUND/AIMS Recently, several cells found within the liver have been reported to derive from bone marrow (BM). This study sought to examine the commitment of BM cells to hepatic stellate cell (HSC) lineage in mouse liver. METHODS We transplanted BM cells from green fluorescent protein (GFP) transgenic mice into age-matched C57BL/J mice. Hepatic nonparenchymal cells were isolated from the livers of BM-transplanted mice using density gradient centrifugation with Nycodenz. The expression of lineage markers by the isolated cells was evaluated by RT-PCR and immunostaining. We then examined the histology of liver tissues obtained from BM-transplanted mice with and without carbon tetrachloride-induced injury. RESULTS GFP-expressing cells with intracytoplasmic lipid droplets comprised 33.4 +/- 2.3% of the cells isolated by density gradient centrifugation. These cells expressed the HSC lineage markers, such as desmin and glial fibrillary acidic protein (GFAP), by both RT-PCR and immunostaining. During a 7-day culture, GFP-positive cells began to express alpha-smooth muscle actin, a marker of activated HSC. In the liver of BM-transplanted mice, GFP-positive nonparenchymal cells expressed GFAP and extended their process around hepatocytes. Upon liver injury, these cells also co-expressed desmin and alpha-smooth muscle actin. CONCLUSIONS Nonparenchymal cells, derived from transplanted BM, acquired HSC characteristics in both quiescent and activated states.
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Affiliation(s)
- Shinji Baba
- Department of Gastroenterological Surgery, Kyoto University Graduate School of Medicine, 54 Kawara-cho, Shogoin, Sakyo-ku, Kyoto City 606-8507, Japan.
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