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Porto E, De Backer J, Thuy LTT, Kawada N, Hankeln T. Transcriptomics of a cytoglobin knockout mouse: Insights from hepatic stellate cells and brain. J Inorg Biochem 2024; 250:112405. [PMID: 37977965 DOI: 10.1016/j.jinorgbio.2023.112405] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2023] [Revised: 10/10/2023] [Accepted: 10/16/2023] [Indexed: 11/19/2023]
Abstract
The vertebrate respiratory protein cytoglobin (Cygb) is thought to exert multiple cellular functions. Here we studied the phenotypic effects of a Cygb knockout (KO) in mouse on the transcriptome level. RNA sequencing (RNA-Seq) was performed for the first time on sites of major endogenous Cygb expression, i.e. quiescent and activated hepatic stellate cells (HSCs) and two brain regions, hippocampus and hypothalamus. The data recapitulated the up-regulation of Cygb during HSC activation and its expression in the brain. Differential gene expression analyses suggested a role of Cygb in the response to inflammation in HSCs and its involvement in retinoid metabolism, retinoid X receptor (RXR) activation-induced xenobiotics metabolism, and RXR activation-induced lipid metabolism and signaling in activated cells. Unexpectedly, only minor effects of the Cygb KO were detected in the transcriptional profiles in hippocampus and hypothalamus, precluding any enrichment analyses. Furthermore, the transcriptome data pointed at a previously undescribed potential of the Cygb- knockout allele to produce cis-acting effects, necessitating future verification studies.
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Affiliation(s)
- Elena Porto
- Institute of Organismic and Molecular Evolution, Molecular Genetics & Genome Analysis Group, Johannes Gutenberg University Mainz, J. J. Becher-Weg 30A, Mainz D-55128, Germany
| | - Joey De Backer
- Research Group PPES, Department of Biomedical Sciences, University of Antwerp, Universiteitsplein 1, Wilrijk, Antwerp 1610, Belgium
| | - Le Thi Thanh Thuy
- Department of Hepatology, Graduate School of Medicine, Osaka Metropolitan University, Osaka 545-8585, Japan
| | - Norifumi Kawada
- Department of Hepatology, Graduate School of Medicine, Osaka Metropolitan University, Osaka 545-8585, Japan
| | - Thomas Hankeln
- Institute of Organismic and Molecular Evolution, Molecular Genetics & Genome Analysis Group, Johannes Gutenberg University Mainz, J. J. Becher-Weg 30A, Mainz D-55128, Germany.
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Overexpression of Tumor Necrosis Factor-Like Ligand 1 A in Myeloid Cells Aggravates Liver Fibrosis in Mice. J Immunol Res 2019; 2019:7657294. [PMID: 30906791 PMCID: PMC6393882 DOI: 10.1155/2019/7657294] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2018] [Revised: 10/26/2018] [Accepted: 11/15/2018] [Indexed: 11/18/2022] Open
Abstract
Macrophages are the master regulator of the dynamic fibrogenesis-fibrosis resolution paradigm. TNF-like ligand 1 aberrance (TL1A) was found to be able to induce intestinal inflammation and fibrosis. Furthermore, significantly increased TL1A had been detected in liver tissues and mononuclear cells of patients with primary biliary cirrhosis (PBC). This study was to investigate the effect of myeloid cells with constitutive TL1A expression on liver fibrogenesis. We found that TL1A expressions in liver tissues and macrophages were significantly increased in mice with liver fibrosis induced by injection of carbon tetrachloride (CCl4). TL1A overexpression in myeloid cells induced liver function injury, accelerated the necrosis and apoptosis of hepatocytes, recruited macrophages, and promoted activation of hepatic stellate cells (HSCs) and fibrosis. In vitro results of our study showed that TL1A overexpression in macrophages promoted secretion of platelet-derived growth factor-BB (PDGF-BB), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β). Culturing macrophages with TL1A overexpression could accelerate the activation and proliferation of primary HSCs. These results indicated that constitutive TL1A expression in myeloid cells exacerbated liver fibrosis, probably through macrophage recruitment and secretion of proinflammatory and profibrotic cytokines.
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Gao L, Zhang Z, Zhang P, Yu M, Yang T. Role of canonical Hedgehog signaling pathway in liver. Int J Biol Sci 2018; 14:1636-1644. [PMID: 30416378 PMCID: PMC6216024 DOI: 10.7150/ijbs.28089] [Citation(s) in RCA: 46] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2018] [Accepted: 08/01/2018] [Indexed: 12/19/2022] Open
Abstract
Hedgehog (Hh) signaling pathway plays an important role in embryonic development. It becomes reactivated in many types of acute and chronic liver injuries. Hh signaling is required for liver regeneration, regulates capillarisation, controls the fates of hepatic stellate cells, promotes liver fibrosis and liver cancers. In this review, we summarize the current knowledge of the role of canonical Hh signaling pathway in adult liver. This help to understand the pathogenesis of liver diseases and find out the new effective targeted therapeutic strategies for liver diseases treatments.
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Affiliation(s)
- Lili Gao
- Center for Medical Research and Innovation, Shanghai Pudong Hospital, Fudan University Pudong Medical Center, Shanghai, 201399, China
| | - Zhenya Zhang
- Department of general surgery, Hebei Medical University Fourth Hospital, Shijiazhuang, 050011, China
| | - Peng Zhang
- Center for Medical Research and Innovation, Shanghai Pudong Hospital, Fudan University Pudong Medical Center, Shanghai, 201399, China
| | - Minghua Yu
- Department of Oncology, Shanghai Pudong Hospital, Fudan University Pudong Medical Center, Shanghai 201399, China
- ✉ Corresponding authors: Dr. Minghua Yu, Department of Oncology, Shanghai Pudong Hospital, Fudan University Pudong Medical Center, Shanghai 201399, China. Phone: 86-21-68030812; E-mail: and Dr. Tao Yang, Center for Medical Research and Innovation, Shanghai Pudong Hospital, Fudan University Pudong Medical Center, 2800 Gongwei Road, Shanghai 201399, China. Phone: 86-21-68036516; E-mail:
| | - Tao Yang
- Center for Medical Research and Innovation, Shanghai Pudong Hospital, Fudan University Pudong Medical Center, Shanghai, 201399, China
- ✉ Corresponding authors: Dr. Minghua Yu, Department of Oncology, Shanghai Pudong Hospital, Fudan University Pudong Medical Center, Shanghai 201399, China. Phone: 86-21-68030812; E-mail: and Dr. Tao Yang, Center for Medical Research and Innovation, Shanghai Pudong Hospital, Fudan University Pudong Medical Center, 2800 Gongwei Road, Shanghai 201399, China. Phone: 86-21-68036516; E-mail:
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Wu HT, Chuang YW, Huang CP, Chang MH. Loss of angiotensin converting enzyme II (ACE2) accelerates the development of liver injury induced by thioacetamide. Exp Anim 2017; 67:41-49. [PMID: 28845018 PMCID: PMC5814313 DOI: 10.1538/expanim.17-0053] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
Angiotensin converting enzyme II (ACE2), an angiotensin converting enzyme (ACE) homologue
that displays antagonist effects on ACE/angiotensin II (Ang II) axis in renin-angiotensin
system (RAS), could play a protective role against liver damages. The purpose of this
study is to investigate whether inflammation-mediated liver injury could be affected by
ACE2 derived pathways in the RAS. Eight-weeks-old wild-type (WT; C57BL/6) and
Ace2 KO (hemizygous Ace2-/y) male mice were
used to induce liver fibrosis by thioacetamide (TAA) administration (0, 100, and 200 mg/kg
BW). The mice administrated with TAA could be successfully induced liver fibrosis in a
TAA-dose dependent manner. Compared to WT mice, the results show that
Ace2 KO mice have high sensitive, and developed more serious reaction
of hepatic inflammation and fibrosis by TAA administration. The physiological and
pathological examinations demonstrated higher serum aspartate aminotransferase (AST),
alanine aminotransferase (ALT) and alkaline phosphatase (ALP) levels, infiltration of
white blood cells and fibrotic lesions within liver in the Ace2 KO mice.
The severe liver damage of Ace2 KO mice were also confirmed by the
evidence of higher expression of hepatic inflammation-related genes (IL-6
and Tnf) and fibrosis-related genes (Col1a1,
Timp1 and Mmp9). Ace2 gene deficiency
could lead to a severe inflammation and collagen remodeling in the liver administrated by
TAA, and the responses lead the pathogenesis of liver fibrosis. Our studies provided the
main messages and favorable study directions of relationship of Ace2 and
liver disease.
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Affiliation(s)
- Hsi-Tien Wu
- Department of BioAgricultural Science, National Chia Yi University, 300 Syuefu Road, Chiayi 60004, Taiwan
| | - Ya-Wen Chuang
- Department of BioAgricultural Science, National Chia Yi University, 300 Syuefu Road, Chiayi 60004, Taiwan
| | - Cheng-Pu Huang
- Department of BioAgricultural Science, National Chia Yi University, 300 Syuefu Road, Chiayi 60004, Taiwan
| | - Ming-Huang Chang
- Department of Veterinary Medicine, National Chia Yi University, 580 Xinmin Road, Chiayi 60054, Taiwan
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Habuchi H, Ushida T, Habuchi O. Mice deficient in N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase exhibit enhanced liver fibrosis and delayed recovery from fibrosis in carbon tetrachloride-treated mice. Heliyon 2016; 2:e00138. [PMID: 27547834 PMCID: PMC4983273 DOI: 10.1016/j.heliyon.2016.e00138] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2016] [Accepted: 08/01/2016] [Indexed: 01/18/2023] Open
Abstract
Background Chondroitin/dermatan sulfate (CS/DS) rich in N-acetylgalactosamine 4,6-bissulfate (GalNAc(4,6SO4)) residues is present as decorin and/or biglycan in mouse liver, and GalNAc(4,6SO4) residues disappeared completely in N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) knockout (KO) mice. The aim of this study was to investigate whether CS/DS rich in GalNAc(4,6SO4) residues participate in the progression or resolution of liver fibrosis. Methods Wild type (WT) and GalNAc4S-6ST KO mice were treated with CCl4 for 5 weeks. After discontinuation of CCl4 administration, histochemical and biochemical changes and expression of genes related to matrix components were compared between WT and GalNAc4S-6ST KO mice. Results and conclusion On 2 days after cessation of CCl4 administration, higher fibrosis was observed in KO mice than in WT mice by Sirius Red staining. Serum alanine aminotransferase activity was higher in KO mice than in WT mice. Hydroxyproline contents and Sirius Red staining showed that repair of liver fibrosis in the recovery stages appeared to be delayed in KO mice. Expression of mRNA of matrix metalloproteinase (MMP)-2, MMP-13 and versican peaked at 2 days after cessation of CCl4 administration and was higher in KO mice than in WT mice. Expression of MMP-9 in the recovery stage was lower in KO mice than in WT mice. Our findings demonstrate that defect in GalNAc4S-6ST, which resulted in disappearance of CS/DS containing GalNAc(4,6SO4), appear to contribute to progression of liver fibrosis, delayed recovery from fibrosis, and various changes in the expression of proteoglycans and MMPs in carbon tetrachloride–treated mice.
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Affiliation(s)
- Hiroko Habuchi
- Advanced Medical Research Center, Aichi Medical University, 1-1 Yazakokarimata, Nagakute, Aichi 480-1195, Japan; Multidisciplinary Pain Center, Aichi Medical University, 1-1 Yazakokarimata, Nagakute, Aichi 480-1195, Japan
| | - Takahiro Ushida
- Multidisciplinary Pain Center, Aichi Medical University, 1-1 Yazakokarimata, Nagakute, Aichi 480-1195, Japan
| | - Osami Habuchi
- Advanced Medical Research Center, Aichi Medical University, 1-1 Yazakokarimata, Nagakute, Aichi 480-1195, Japan; Multidisciplinary Pain Center, Aichi Medical University, 1-1 Yazakokarimata, Nagakute, Aichi 480-1195, Japan
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Chung SI, Moon H, Ju HL, Cho KJ, Kim DY, Han KH, Eun JW, Nam SW, Ribback S, Dombrowski F, Calvisi DF, Ro SW. Hepatic expression of Sonic Hedgehog induces liver fibrosis and promotes hepatocarcinogenesis in a transgenic mouse model. J Hepatol 2016; 64:618-27. [PMID: 26471504 DOI: 10.1016/j.jhep.2015.10.007] [Citation(s) in RCA: 77] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/13/2015] [Revised: 09/15/2015] [Accepted: 10/01/2015] [Indexed: 12/13/2022]
Abstract
BACKGROUND & AIMS Liver fibrosis is an increasing health concern worldwide and a major risk factor for hepatocellular carcinoma (HCC). Although the involvement of Hedgehog signaling in hepatic fibrosis has been known for some time, the causative role of activated Hedgehog signaling in liver fibrosis has not been verified in vivo. METHODS Using hydrodynamics-based transfection, a transgenic mouse model has been developed that expresses Sonic Hedgehog (SHH), a ligand for Hedgehog signaling, in the liver. Levels of hepatic fibrosis and fibrosis-related gene expression were assessed in the model. Hepatic expression of SHH was induced in a murine model for hepatocellular adenoma (HCA) and tumor development was subsequently investigated. RESULTS The transgenic mice revealed SHH expression in 2-5% of hepatocytes. Secreted SHH activated Hedgehog signaling in numerous cells of various types in the tissues. Hepatic expression of SHH led to fibrosis, activation of hepatic stellate cells, and an upregulation of various fibrogenic genes. Liver injury and hepatocyte apoptosis were observed in SHH mice. Persistent expression of SHH for up to 13months failed to induce tumors in the liver; however, it promoted liver tumor development induced by other oncogenes. By employing a HCA model induced by P53(R172H) and KRAS(G12D), we found that the SHH expression promoted the transition from HCA to HCC. CONCLUSIONS SHH expression in the liver induces liver fibrosis with concurrent activation of hepatic stellate cells and fibrogenic genes. It can also enhance hepatocarcinogenesis induced by other oncogenes.
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Affiliation(s)
- Sook In Chung
- Institute of Gastroenterology, Yonsei University College of Medicine, Seoul, South Korea
| | - Hyuk Moon
- Institute of Gastroenterology, Yonsei University College of Medicine, Seoul, South Korea
| | - Hye-Lim Ju
- Liver Cirrhosis Clinical Research Center, Yonsei University College of Medicine, Seoul, South Korea
| | - Kyung Joo Cho
- Institute of Gastroenterology, Yonsei University College of Medicine, Seoul, South Korea
| | - Do Young Kim
- Department of Internal Medicine, Yonsei University College of Medicine, Seoul, South Korea
| | - Kwang-Hyub Han
- Department of Internal Medicine, Yonsei University College of Medicine, Seoul, South Korea
| | - Jung Woo Eun
- Department of Pathology, College of Medicine, The Catholic University of Korea, Seoul, South Korea
| | - Suk Woo Nam
- Department of Pathology, College of Medicine, The Catholic University of Korea, Seoul, South Korea
| | - Silvia Ribback
- Institute of Pathology, University Medicine Greifswald, Greifswald, Germany
| | - Frank Dombrowski
- Institute of Pathology, University Medicine Greifswald, Greifswald, Germany
| | - Diego F Calvisi
- Institute of Pathology, University Medicine Greifswald, Greifswald, Germany
| | - Simon Weonsang Ro
- Institute of Gastroenterology, Yonsei University College of Medicine, Seoul, South Korea; Liver Cirrhosis Clinical Research Center, Yonsei University College of Medicine, Seoul, South Korea.
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KURPIŃSKA AK, JAROSZ A, OŻGO M, SKRZYPCZAK WF. Analysis of protein expression changes in the blood plasma of cows during the last month before parturition and 2 months after calving. Turk J Biol 2016. [DOI: 10.3906/biy-1501-30] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/03/2022] Open
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Coombes J, Swiderska-Syn M, Dollé L, Reid D, Eksteen B, Claridge L, Briones-Orta MA, Shetty S, Oo YH, Riva A, Chokshi S, Papa S, Mi Z, Kuo PC, Williams R, Canbay A, Adams DH, Diehl AM, van Grunsven LA, Choi SS, Syn WK. Osteopontin neutralisation abrogates the liver progenitor cell response and fibrogenesis in mice. Gut 2015; 64:1120-31. [PMID: 24902765 PMCID: PMC4487727 DOI: 10.1136/gutjnl-2013-306484] [Citation(s) in RCA: 73] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/25/2013] [Accepted: 05/22/2014] [Indexed: 12/29/2022]
Abstract
BACKGROUND Chronic liver injury triggers a progenitor cell repair response, and liver fibrosis occurs when repair becomes deregulated. Previously, we reported that reactivation of the hedgehog pathway promotes fibrogenic liver repair. Osteopontin (OPN) is a hedgehog-target, and a cytokine that is highly upregulated in fibrotic tissues, and regulates stem-cell fate. Thus, we hypothesised that OPN may modulate liver progenitor cell response, and thereby, modulate fibrotic outcomes. We further evaluated the impact of OPN-neutralisation on murine liver fibrosis. METHODS Liver progenitors (603B and bipotential mouse oval liver) were treated with OPN-neutralising aptamers in the presence or absence of transforming growth factor (TGF)-β, to determine if (and how) OPN modulates liver progenitor function. Effects of OPN-neutralisation (using OPN-aptamers or OPN-neutralising antibodies) on liver progenitor cell response and fibrogenesis were assessed in three models of liver fibrosis (carbon tetrachloride, methionine-choline deficient diet, 3,5,-diethoxycarbonyl-1,4-dihydrocollidine diet) by quantitative real time (qRT) PCR, Sirius-Red staining, hydroxyproline assay, and semiquantitative double-immunohistochemistry. Finally, OPN expression and liver progenitor response were corroborated in liver tissues obtained from patients with chronic liver disease. RESULTS OPN is overexpressed by liver progenitors in humans and mice. In cultured progenitors, OPN enhances viability and wound healing by modulating TGF-β signalling. In vivo, OPN-neutralisation attenuates the liver progenitor cell response, reverses epithelial-mesenchymal-transition in Sox9+ cells, and abrogates liver fibrogenesis. CONCLUSIONS OPN upregulation during liver injury is a conserved repair response, and influences liver progenitor cell function. OPN-neutralisation abrogates the liver progenitor cell response and fibrogenesis in mouse models of liver fibrosis.
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Affiliation(s)
- J Coombes
- Regeneration and Repair Group, The Institute of Hepatology, Foundation for Liver Research, London, UK
| | - M Swiderska-Syn
- Division of Gastroenterology, Department of Medicine, Duke University, NC, USA
| | - L Dollé
- Liver Cell Biology Lab (LIVR), Department of Cell Biology (CYTO), Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Brussels, Belgium
| | - D Reid
- Snyder Institute for Chronic Diseases, Health Research and Innovation Centre (HRIC), University of Calgary, Canada
| | - B Eksteen
- Snyder Institute for Chronic Diseases, Health Research and Innovation Centre (HRIC), University of Calgary, Canada
| | - L Claridge
- Centre for Liver Research, NIHR Institute for Biomedical Research, University of Birmingham, UK
| | - MA Briones-Orta
- Regeneration and Repair Group, The Institute of Hepatology, Foundation for Liver Research, London, UK
| | - S Shetty
- Centre for Liver Research, NIHR Institute for Biomedical Research, University of Birmingham, UK
| | - YH Oo
- Centre for Liver Research, NIHR Institute for Biomedical Research, University of Birmingham, UK
| | - A Riva
- Viral Hepatitis Group, The Institute of Hepatology, Foundation for Liver Research, London, UK
| | - S Chokshi
- Viral Hepatitis Group, The Institute of Hepatology, Foundation for Liver Research, London, UK
| | - S Papa
- Cell Signaling Group, The Institute of Hepatology, Foundation for Liver Research, London, UK
| | - Z Mi
- Department of Surgery, Loyola University, Chicago, USA
| | - PC Kuo
- Department of Surgery, Loyola University, Chicago, USA
| | - R Williams
- Regeneration and Repair Group, The Institute of Hepatology, Foundation for Liver Research, London, UK
| | - A Canbay
- Department of Gastroenterology and Hepatology, Essen University Hospital, Essen, Germany
| | - DH Adams
- Centre for Liver Research, NIHR Institute for Biomedical Research, University of Birmingham, UK
| | - AM Diehl
- Division of Gastroenterology, Department of Medicine, Duke University, NC, USA
| | - LA van Grunsven
- Liver Cell Biology Lab (LIVR), Department of Cell Biology (CYTO), Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Brussels, Belgium
| | - SS Choi
- Division of Gastroenterology, Department of Medicine, Duke University, NC, USA,Section of Gastroenterology, Department of Medicine, Durham Veteran Affairs Medical Center, Durham, NC, USA
| | - WK Syn
- Regeneration and Repair Group, The Institute of Hepatology, Foundation for Liver Research, London, UK,Centre for Liver Research, NIHR Institute for Biomedical Research, University of Birmingham, UK,Department of Hepatology, Barts Health NHS Trust, London, UK,Senior and Corresponding Author: Dr Wing-Kin Syn, Head of Liver Regeneration and Repair, The Institute of Hepatology, Foundation for Liver Research, London WC1E 6HX, Tel: 44-20272559837,
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Abstract
Hepatic stellate cells are resident perisinusoidal cells distributed throughout the liver, with a remarkable range of functions in normal and injured liver. Derived embryologically from septum transversum mesenchyme, their precursors include submesothelial cells that invade the liver parenchyma from the hepatic capsule. In normal adult liver, their most characteristic feature is the presence of cytoplasmic perinuclear droplets that are laden with retinyl (vitamin A) esters. Normal stellate cells display several patterns of intermediate filaments expression (e.g., desmin, vimentin, and/or glial fibrillary acidic protein) suggesting that there are subpopulations within this parental cell type. In the normal liver, stellate cells participate in retinoid storage, vasoregulation through endothelial cell interactions, extracellular matrix homeostasis, drug detoxification, immunotolerance, and possibly the preservation of hepatocyte mass through secretion of mitogens including hepatocyte growth factor. During liver injury, stellate cells activate into alpha smooth muscle actin-expressing contractile myofibroblasts, which contribute to vascular distortion and increased vascular resistance, thereby promoting portal hypertension. Other features of stellate cell activation include mitogen-mediated proliferation, increased fibrogenesis driven by connective tissue growth factor, and transforming growth factor beta 1, amplified inflammation and immunoregulation, and altered matrix degradation. Evolving areas of interest in stellate cell biology seek to understand mechanisms of their clearance during fibrosis resolution by either apoptosis, senescence, or reversion, and their contribution to hepatic stem cell amplification, regeneration, and hepatocellular cancer.
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Affiliation(s)
- Juan E Puche
- Division of Liver Diseases, Icahn School of Medicine at Mount Sinai Hospital, New York, New York, New York
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Swiderska-Syn M, Syn WK, Xie G, Krüger L, Machado MV, Karaca G, Michelotti GA, Choi SS, Premont RT, Diehl AM. Myofibroblastic cells function as progenitors to regenerate murine livers after partial hepatectomy. Gut 2014; 63:1333-44. [PMID: 24173292 PMCID: PMC4006344 DOI: 10.1136/gutjnl-2013-305962] [Citation(s) in RCA: 91] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
OBJECTIVE Smoothened (SMO), a coreceptor of the Hedgehog (Hh) pathway, promotes fibrogenic repair of chronic liver injury. We investigated the roles of SMO+ myofibroblast (MF) in liver regeneration by conditional deletion of SMO in α smooth muscle actin (αSMA)+ cells after partial hepatectomy (PH). DESIGN αSMA-Cre-ER(T2)×SMO/flox mice were treated with vehicle (VEH) or tamoxifen (TMX), and sacrificed 24-96 h post-PH. Regenerating livers were analysed for proliferation, progenitors and fibrosis by qRT-PCR and quantitative immunohistochemistry (IHC). Results were normalised to liver segments resected at PH. For lineage-tracing studies, αSMA-Cre-ER(T2)×ROSA-Stop-flox-yellow fluorescent protein (YFP) mice were treated with VEH or TMX; livers were stained for YFP, and hepatocytes isolated 48 and 72 h post-PH were analysed for YFP by flow cytometric analysis (FACS). RESULTS Post-PH, VEH-αSMA-SMO mice increased expression of Hh-genes, transiently accumulated MF, fibrosis and liver progenitors, and ultimately exhibited proliferation of hepatocytes and cholangiocytes. In contrast, TMX-αSMA-SMO mice showed loss of whole liver SMO expression, repression of Hh-genes, enhanced accumulation of quiescent HSC but reduced accumulation of MF, fibrosis and progenitors, as well as inhibition of hepatocyte and cholangiocyte proliferation, and reduced recovery of liver weight. In TMX-αSMA-YFP mice, many progenitors, cholangiocytes and up to 25% of hepatocytes were YFP+ by 48-72 h after PH, indicating that liver epithelial cells were derived from αSMA-YFP+ cells. CONCLUSIONS Hh signalling promotes transition of quiescent hepatic stellate cells to fibrogenic MF, some of which become progenitors that regenerate the liver epithelial compartment after PH. Hence, scarring is a component of successful liver regeneration.
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Affiliation(s)
- M Swiderska-Syn
- Division of Gastroenterology, Department of Medicine, Duke University Medical Center, Durham, NC
| | - WK Syn
- Division of Gastroenterology, Department of Medicine, Duke University Medical Center, Durham, NC,Regeneration and Repair, Institute of Hepatology, Foundation for Liver Research, London
| | - G Xie
- Division of Gastroenterology, Department of Medicine, Duke University Medical Center, Durham, NC
| | - L Krüger
- Division of Gastroenterology, Department of Medicine, Duke University Medical Center, Durham, NC
| | - MV Machado
- Division of Gastroenterology, Department of Medicine, Duke University Medical Center, Durham, NC
| | - G Karaca
- Division of Gastroenterology, Department of Medicine, Duke University Medical Center, Durham, NC
| | - GA Michelotti
- Division of Gastroenterology, Department of Medicine, Duke University Medical Center, Durham, NC
| | - SS Choi
- Division of Gastroenterology, Department of Medicine, Duke University Medical Center, Durham, NC,Section of Gastroenterology, Durham Veterans Affairs Medical Center, Durham, NC
| | - RT Premont
- Division of Gastroenterology, Department of Medicine, Duke University Medical Center, Durham, NC
| | - AM Diehl
- Division of Gastroenterology, Department of Medicine, Duke University Medical Center, Durham, NC,Corresponding author: Anna Mae Diehl, MD, Division of Gastroenterology, Duke University Medical Center, 595 LaSalle Street, Snyderman Building, Suite 1073, Durham, NC 27710, 919-684-4173,
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Chen YR, Chang KT, Tsai MJ, Lee CH, Huang KJ, Cheng H, Ho YP, Chen JC, Yang HH, Weng CF. Antrodia cinnamomea profoundly exalted the reversion of activated hepatic stellate cells by the alteration of cellular proteins. Food Chem Toxicol 2014; 69:150-62. [PMID: 24751970 DOI: 10.1016/j.fct.2014.04.006] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2013] [Revised: 03/24/2014] [Accepted: 04/04/2014] [Indexed: 01/05/2023]
Abstract
The direct modulation of Antrodia cinnamomea (AC) on the prominent role of liver fibrosis-hepatic stellate cells (HSCs) in situ remains unclear. Firstly, the administration of A. cinnamomea mycelial extract (ACME) could improve liver morphology and histological changes including collagen formation and GPT activity in the liver of thioacetamide (TAA)-injured rats. The morphology and fatty acid restore of TAA-induced HSCs (THSCs) returned to the non-chemical induced HSCs (NHSCs) type as measured by immunofluorescence and Oil Red O staining. PPARγ was upregulated associated with the lowering of α-SMA protein in NHSC-ACME. ACME inhibited the MMP-2 activity in NHSCs by gelatin Zymography. After LC-MS/MS, the cytoskeleton (tubulin, lamin A) and heat shock protein 8 in NHSC-ACME, and guanylate kinase, brain-specific kinase, SG-II and p55 proteins were downregulated in THSC-ACME. Whereas MHC class II, SMC6 protein, and phospholipase D were upregulated in NHSC-ACME. Furthermore, PKG-1 was downregulated in NHSC-ACME and upregulated in THSC-ACME. SG-II and p55 proteins were downregulated in NHSC-ACME and THSC-ACME by Western blotting. Taken together, the beneficial effect of A. cinnamomea on the induction of HSC cellular proteins is potentially applied as an alternative and complementary medicine for the prevention and amelioration of a liver injury.
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Affiliation(s)
- Yi-Ren Chen
- Institute of Biotechnology, National Dong Hwa University, Hualien 974, Taiwan
| | - Kai-Ting Chang
- Institute of Biotechnology, National Dong Hwa University, Hualien 974, Taiwan
| | - May-Jywan Tsai
- Neural Regeneration Laboratory, Neurological Institute, Taipei Veterans General Hospital, Taipei 112, Taiwan
| | - Chia-Hung Lee
- Institute of Biotechnology, National Dong Hwa University, Hualien 974, Taiwan
| | - Kao-Jean Huang
- Institute of Biotechnology, National Dong Hwa University, Hualien 974, Taiwan
| | - Henrich Cheng
- Neural Regeneration Laboratory, Neurological Institute, Taipei Veterans General Hospital, Taipei 112, Taiwan
| | - Yen-Peng Ho
- Department of Chemistry, National Dong Hwa University, Hualien 974, Taiwan
| | - Jian-Chyi Chen
- Department of Biotechnology, Southern Taiwan University, Tainan 710, Taiwan
| | - Hsueh-Hui Yang
- Department of Research, Buddhist Tzu Chi General Hospital, General Education Center, Tzu Chi College of Technology, Hualien 970, Taiwan
| | - Ching-Feng Weng
- Institute of Biotechnology, National Dong Hwa University, Hualien 974, Taiwan.
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12
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Iredale JP, Thompson A, Henderson NC. Extracellular matrix degradation in liver fibrosis: Biochemistry and regulation. Biochim Biophys Acta Mol Basis Dis 2012; 1832:876-83. [PMID: 23149387 DOI: 10.1016/j.bbadis.2012.11.002] [Citation(s) in RCA: 177] [Impact Index Per Article: 13.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2012] [Revised: 10/31/2012] [Accepted: 11/01/2012] [Indexed: 02/06/2023]
Abstract
Fibrosis is a highly conserved wound healing response and represents the final common pathway of virtually all chronic inflammatory injuries. Over the past 3 decades detailed analysis of hepatic extracellular matrix synthesis and degradation using approaches incorporating human disease, experimental animal models and cell culture have highlighted the extraordinarily dynamic nature of tissue repair and remodelling in this solid organ. Furthermore emerging studies of fibrosis in other organs demonstrate that basic common mechanisms exist, suggesting that bidirectionality of the fibrotic process may not solely be the preserve of the liver. In this review we will examine the cellular and molecular mechanisms that govern extracellular matrix degradation and fibrosis resolution, and highlight how manipulation of these processes may result in the development of effective anti-fibrotic therapies. This article is part of a Special Issue entitled: Fibrosis: Translation of basic research to human disease.
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Affiliation(s)
- John P Iredale
- MRC Centre for Inflammation Research, The Queen's Medical Research Institute, University of Edinburgh, Edinburgh, UK.
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13
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Abstract
Fibrosis is a hallmark histologic event of chronic liver diseases and is characterized by the excessive accumulation and reorganization of the extracellular matrix (ECM). The gold standard for assessment of fibrosis is liver biopsy. As this procedure has various limitations, including risk of patient injury and sampling error, a non-invasive serum marker for liver fibrosis is desirable. The increasing understanding of the pathogenesis of hepatic fibrosis has suggested several markers which could be useful indicators of hepatic fibrogenesis and fibrosis. These markers include serum markers of liver function, ECM synthesis, fibrolytic processes, ECM degradation and fibrogenesis related cytokines. Recently, neo-epitopes, which are post-translational modifications of proteins, have been successfully used in bone and cartilage diseases which are characterized by extensive ECM remodeling. Increasing numbers of studies are being undertaken to identify neo-epitopes generated during liver fibrosis, and which ultimately might be useful for diagnosing and monitoring fibrogenesis. To date, the metalloproteinases generated fragment of collagen I, III, IV and VI have been proven to be elevated in two rat models of fibrosis. This review summarizes the recent efforts that have been made to identify potentially reliable non-invasive serum markers. We used the recently proposed BIPED (Burden of disease, Investigative, Prognostic, Efficacy and Diagnostic) system to characterize potential serum markers and neo-epitope markers that have been identified to date.
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Affiliation(s)
- Tianhui Liu
- Liver Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing, China
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Bourd-Boittin K, Bonnier D, Leyme A, Mari B, Tuffery P, Samson M, Ezan F, Baffet G, Theret N. Protease profiling of liver fibrosis reveals the ADAM metallopeptidase with thrombospondin type 1 motif, 1 as a central activator of transforming growth factor beta. Hepatology 2011; 54:2173-84. [PMID: 21826695 DOI: 10.1002/hep.24598] [Citation(s) in RCA: 64] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
UNLABELLED During chronic liver disease, tissue remodeling leads to dramatic changes and accumulation of matrix components. Matrix metalloproteases and their inhibitors have been involved in the regulation of matrix degradation. However, the role of other proteases remains incompletely defined. We undertook a gene-expression screen of human liver fibrosis samples using a dedicated gene array selected for relevance to protease activities, identifying the ADAMTS1 (A Disintegrin And Metalloproteinase [ADAM] with thrombospondin type 1 motif, 1) gene as an important node of the protease network. Up-regulation of ADAMTS1 in fibrosis was found to be associated with hepatic stellate cell (HSC) activation. ADAMTS1 is synthesized as 110-kDa latent forms and is processed by HSCs to accumulate as 87-kDa mature forms in fibrotic tissues. Structural evidence has suggested that the thrombospondin motif-containing domain from ADAMTS1 may be involved in interactions with, and activation of, the major fibrogenic cytokine, transforming growth factor beta (TGF-β). Indeed, we observed direct interactions between ADAMTS1 and latency-associated peptide-TGF-β (LAP-TGF-β). ADAMTS1 induces TGF-β activation through the interaction of the ADAMTS1 KTFR peptide with the LAP-TGF-β LKSL peptide. Down-regulation of ADAMTS1 in HSCs decreases the release of TGF-β competent for transcriptional activation, and KTFR competitor peptides directed against ADAMTS1 block the HSC-mediated release of active TGF-β. Using a mouse liver fibrosis model, we show that carbon tetrachloride treatment induces ADAMTS1 expression in parallel to that of type I collagen. Importantly, concurrent injection of the KTFR peptide prevents liver damage. CONCLUSION Our results indicate that up-regulation of ADAMTS1 in HSCs constitutes a new mechanism for control of TGF-β activation in chronic liver disease.
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Affiliation(s)
- Katia Bourd-Boittin
- Institut de Recherche en Santé, Environnement et Travail EA4427 SeRAIC, Université de Rennes 1, IFR14, Rennes, France
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15
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Down regulation of a matrix degrading cysteine protease cathepsin L, by acetaldehyde: role of C/EBPα. PLoS One 2011; 6:e20768. [PMID: 21687683 PMCID: PMC3110794 DOI: 10.1371/journal.pone.0020768] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2011] [Accepted: 05/08/2011] [Indexed: 12/21/2022] Open
Abstract
Background The imbalance between extra cellular matrix (ECM) synthesis and degradation is critical aspect of various hepatic pathologies including alcohol induced liver fibrosis. This study was carried out to investigate the effect of acetaldehyde on expression of an extra cellular matrix degrading protease cathepsin L (CTSL) in HepG2 cells. Methodology and Results We measured the enzymatic activity, protein and, mRNA levels of CTSL in acetaldehyde treated and untreated cells. The binding of CAAT enhancer binding protein α (C/EBP α) to CTSL promoter and its key role in the transcription from this promoter and conferring responsiveness to acetaldehyde was established by site directed mutagenesis, electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP) assays and siRNA technology. Acetaldehyde treatment significantly decreased CTSL activity and protein levels in HepG2 cells. A similar decrease in the mRNA levels and promoter activity was also observed. This decrease by acetaldehyde was attributed to the fall in the liver enriched transcription factor C/EBP α levels and it's binding to the CTSL promoter. Mutagenesis of C/EBP α binding motifs revealed the key role of this factor in CTSL transcription as well as conferring responsiveness to acetaldehyde. The siRNA mediated silencing of the C/EBP α expression mimicked the effect of acetaldehyde on CTSL levels and its promoter activity. It also abolished the responsiveness of this promoter to acetaldehyde. Conclusion Acetaldehyde down regulates the C/EBP α mediated CTSL expression in hepatic cell lines. The decreased expression of CTSL may at least in part contribute to ECM deposition in liver which is a hallmark of alcoholic liver fibrosis.
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Pereira TN, Walsh MJ, Lewindon PJ, Ramm GA. Paediatric cholestatic liver disease: Diagnosis, assessment of disease progression and mechanisms of fibrogenesis. World J Gastrointest Pathophysiol 2010; 1:69-84. [PMID: 21607144 PMCID: PMC3097948 DOI: 10.4291/wjgp.v1.i2.69] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/09/2010] [Revised: 03/26/2010] [Accepted: 04/02/2010] [Indexed: 02/06/2023] Open
Abstract
Cholestatic liver disease causes significant morbidity and mortality in children. The diagnosis and management of these diseases can be complicated by an inability to detect early stages of fibrosis and a lack of adequate interventional therapy. There is no single gold standard test that accurately reflects the presence of liver disease, or that can be used to monitor fibrosis progression, particularly in conditions such as cystic fibrosis. This has lead to controversy over how suspected liver disease in children is detected and diagnosed. This review discusses the challenges in using commonly available methods to diagnose hepatic fibrosis and monitor disease progression in children with cholestatic liver disease. In addition, the review examines the mechanisms hypothesised to be involved in the development of hepatic fibrogenesis in paediatric cholestatic liver injury which may ultimately aid in identifying new modalities to assist in both disease detection and therapeutic intervention.
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Bourd-Boittin K, Basset L, Bonnier D, L'helgoualc'h A, Samson M, Théret N. CX3CL1/fractalkine shedding by human hepatic stellate cells: contribution to chronic inflammation in the liver. J Cell Mol Med 2009; 13:1526-35. [PMID: 19432809 PMCID: PMC3828864 DOI: 10.1111/j.1582-4934.2009.00787.x] [Citation(s) in RCA: 57] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
Abstract
Chemokines are the inflammatory mediators that modulate liver fibrosis, a common feature of chronic inflammatory liver diseases. CX3CL1/fractalkine is a membrane-associated chemokine that requires step processing for chemotactic activity and has been recently implicated in liver disease. Here, we investigated the potential shedding activities involved in the release of the soluble chemotactic peptides from CX3CL1 in the injured liver. We showed an increased expression of the sheddases ADAM10 and ADAM17 in patients with chronic liver diseases that was associated with the severity of liver fibrosis. We demonstrated that hepatic stellate cells (HSC) were an important source of ADAM10 and ADAM17 and that treatment with the inflammatory cytokine inter-feron-γ induced the expression of CX3CL1 and release of soluble peptides. This release was inhibited by the metalloproteinase inhibitor batimastat; however, ADAM10/ADAM17 inhibitor GW280264X only partially affected shedding activity. By using selective tissue metalloprotease inhibitors and overexpression analyses, we showed that CX3CL1 was mainly processed by matrix metalloproteinase (MMP)-2, a metalloprotease highly expressed by HSC. We further demonstrated that the CX3CL1 soluble peptides released from stimulated HSC induced the activation of the CX3CR1-dependent signalling pathway and promoted chemoattraction of monocytes in vitro. We conclude that ADAM10, ADAM17 and MMP-2 synthesized by activated HSC mediate CX3CL1 shedding and release of chemotactic peptides, thereby facilitating recruitment of inflammatory cells and paracrine stimulation of HSC in chronic liver diseases.
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18
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Velayudham A, Dolganiuc A, Ellis M, Petrasek J, Kodys K, Mandrekar P, Szabo G. VSL#3 probiotic treatment attenuates fibrosis without changes in steatohepatitis in a diet-induced nonalcoholic steatohepatitis model in mice. Hepatology 2009; 49:989-97. [PMID: 19115316 PMCID: PMC3756672 DOI: 10.1002/hep.22711] [Citation(s) in RCA: 195] [Impact Index Per Article: 12.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Nonalcoholic fatty liver disease (NAFLD) and its advanced stage, nonalcoholic steatohepatitis (NASH), are the most common causes of chronic liver disease in the United States. NASH features the metabolic syndrome, inflammation, and fibrosis. Probiotics exhibit immunoregulatory and anti-inflammatory activity. We tested the hypothesis that probiotic VSL#3 may ameliorate the methionine-choline-deficient (MCD) diet-induced mouse model of NASH. MCD diet resulted in NASH in C57BL/6 mice compared to methionine-choline-supplemented (MCS) diet feeding evidenced by liver steatosis, increased triglycerides, inflammatory cell accumulation, increased tumor necrosis factor alpha levels, and fibrosis. VSL#3 failed to prevent MCD-induced liver steatosis or inflammation. MCD diet, even in the presence of VSL#3, induced up-regulation of serum endotoxin and expression of the Toll-like receptor 4 signaling components, including CD14 and MD2, MyD88 adaptor, and nuclear factor kappaB activation. In contrast, VSL#3 treatment ameliorated MCD diet-induced liver fibrosis resulting in diminished accumulation of collagen and alpha-smooth muscle actin. We identified increased expression of liver peroxisome proliferator-activated receptors and decreased expression of procollagen and matrix metalloproteinases in mice fed MCD+VSL#3 compared to MCD diet alone. MCD diet triggered up-regulation of transforming growth factor beta (TGFbeta), a known profibrotic agent. In the presence of VSL#3, the MCD diet-induced expression of TGFbeta was maintained; however, the expression of Bambi, a TGFbeta pseudoreceptor with negative regulatory function, was increased. In summary, our data indicate that VSL#3 modulates liver fibrosis but does not protect from inflammation and steatosis in NASH. The mechanisms of VSL#3-mediated protection from MCD diet-induced liver fibrosis likely include modulation of collagen expression and impaired TGFbeta signaling.
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Affiliation(s)
- Arumugam Velayudham
- Department of Medicine, Liver Center, University of Massachusetts Medical School, Worcester, MA 01605-2324, USA
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19
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Lee JS, Kim JH. [The role of activated hepatic stellate cells in liver fibrosis, portal hypertension and cancer angiogenesis]. THE KOREAN JOURNAL OF HEPATOLOGY 2008; 13:309-19. [PMID: 17898548 DOI: 10.3350/kjhep.2007.13.3.309] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
Abstract
Although hepatic stellate cells, which are liver specific pericytes, have been recognized within the vasculature of the sinusoid for more than one hundred years, the biology and function of these cells is unclear. Recent studies have highlighted the key role of stellate cells in a number of fundamental processes that include wound healing/fibrosis, vasoregulation, and vascular remodeling/angiogenesis. In the liver, these processes are particularly important in the development of cirrhosis, portal hypertension and cancer. This article highlights the recent advances in our understanding of the biology of hepatic stellate cells and discusses some of the recently-ascribed functions that are relevant to liver fibrosis, portal hypertension and cancer angiogenesis.
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Affiliation(s)
- June Sung Lee
- Department of Internal Medicine, Ilsan Paik Hospital, Inje University College of Medicine, Goyang, Korea.
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20
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Liu C, Sun M, Yan X, Han L, Zhang Y, Liu C, El-Nezami H, Liu P. Inhibition of hepatic stellate cell activation following Yinchenhao decoction administration to dimethylnitrosamine-treated rats. Hepatol Res 2008; 38:919-29. [PMID: 18371158 DOI: 10.1111/j.1872-034x.2008.00346.x] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
AIM In an effort to investigate the mechanism by which Yinchenhao decoction (YCHD) acts on liver injury, we investigated the potential antifibrogenic effects of YCHD in an experimental liver fibrosis rat model, with special focus on the mechanisms inhibiting the activation and promoting apoptosis of hepatic stellate cells (HSC). METHODS The rats were initially randomized into two groups: the control (n = 10) and dimethylnitrosamine-treated (DMN; n = 30) groups. DMN (10 mg/kg body weight) was administered intraperitoneally to the DMN-treated rats for three consecutive days each week. At the end of the second week, three rats from the control and six rats from the DMN-treated groups were killed for the fibrosis development assessment. The remaining DMN rats were further randomized into two groups: the DMN-water group (n = 12) and the DMN-YCHD group (n = 12). Both groups continued to receive weekly DMN treatment for another 2 weeks in addition to daily administration of either water or YCHD, which were given intragastrically at a dose of 0.418 g/100 g body weight. RESULTS Hepatic hydroxyproline content decreased and had improved histopathology in the DMN-YCHD rats. Compared to the DMN group, alpha-smooth muscle actin (SMA) and CD68 expression in the DMN-YCHD group was reduced significantly; however, alpha-SMA-positive HSC apoptosis was not observed by confocal microscopy; Fibrogenic proteins (tissue inhibitor matrix proteinases-1 and 2 and matrix metalloproteinase [MMP]-2/14) and cytokines (tumor necrosis factor-alpha and transforming growth factor-beta(1)) were decreased; MMP-9 was significantly upregulated. CONCLUSION Yinchenhao administration attenuates liver fibrosis at least in part by inhibiting HSC activation directly, rather than promoting cell apoptosis of activated HSC, and the suppressive activation of Kupffer cells.
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Affiliation(s)
- Cheng Liu
- Institute of Liver Diseases, Shuguang Hospital, Shanghai, China
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21
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Friedman SL. Hepatic fibrosis -- overview. Toxicology 2008; 254:120-9. [PMID: 18662740 DOI: 10.1016/j.tox.2008.06.013] [Citation(s) in RCA: 261] [Impact Index Per Article: 15.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2008] [Revised: 06/27/2008] [Accepted: 06/30/2008] [Indexed: 02/08/2023]
Abstract
The study of hepatic fibrosis, or scarring in response to chronic liver injury, has witnessed tremendous progress in the past two decades. Clarification of the cellular sources of scar, and emergence of hepatic stellate cells not only as a fibrogenic cell type, but also as a critical immunomodulatory and homeostatic regulator are among the most salient advances. Activation of hepatic stellate cells remains a central event in fibrosis, complemented by evidence of additional sources of matrix-producing cells including bone marrow, portal fibroblasts, and epithelial-mesenchymal transition from both hepatocytes and cholangiocytes. A growing range of cytokines and their receptors and inflammatory cell subsets have further expanded our knowledge about this dynamic process. Collectively, these findings have laid the foundation for continued elucidation of underlying mechanisms, and more importantly for the implementation of rationally based approaches to limit fibrosis, accelerate repair and enhance liver regeneration in patients with chronic liver disease.
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Affiliation(s)
- Scott L Friedman
- Division of Liver Diseases, Box 1123, Mount Sinai School of Medicine, 1425 Madison Avenue, Room 11-70C, New York, NY 10029-6574, United States.
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Abstract
Substantial improvements in the treatment of chronic liver disease have accelerated interest in uncovering the mechanisms underlying hepatic fibrosis and its resolution. Activation of resident hepatic stellate cells into proliferative, contractile, and fibrogenic cells in liver injury remains a dominant theme driving the field. However, several new areas of rapid progress in the past 5-10 years also have taken root, including: (1) identification of different fibrogenic populations apart from resident stellate cells, for example, portal fibroblasts, fibrocytes, and bone-marrow-derived cells, as well as cells derived from epithelial mesenchymal transition; (2) emergence of stellate cells as finely regulated determinants of hepatic inflammation and immunity; (3) elucidation of multiple pathways controlling gene expression during stellate cell activation including transcriptional, post-transcriptional, and epigenetic mechanisms; (4) recognition of disease-specific pathways of fibrogenesis; (5) re-emergence of hepatic macrophages as determinants of matrix degradation in fibrosis resolution and the importance of matrix cross-linking and scar maturation in determining reversibility; and (6) hints that hepatic stellate cells may contribute to hepatic stem cell behavior, cancer, and regeneration. Clinical and translational implications of these advances have become clear, and have begun to impact significantly on the management and outlook of patients with chronic liver disease.
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Neaud V, Rosenbaum J. A red wine polyphenolic extract reduces the activation phenotype of cultured human liver myofibroblasts. World J Gastroenterol 2008; 14:2194-9. [PMID: 18407593 PMCID: PMC2703844 DOI: 10.3748/wjg.14.2194] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To test the effect of a standardized red wine polyphenolic extract (RWPE) on the phenotype of human liver myofibroblasts in culture.
METHODS: Human myofibroblasts grown from liver explants were used in this study. Cell proliferation was measured with the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay. Signaling events were analyzed by western blot with phospho-specific antibodies. Matrix-metalloproteinase activity was measured with gel zymography.
RESULTS: We found that cell proliferation was dose-dependently decreased by up to 90% by RWPE while cell viability was not affected. Exposure to RWPE also greatly decreased the phosphorylation of ERK1/ERK2 and Akt in response to stimulation by the mitogenic factor platelet-derived growth factor BB (PDGF-BB). Finally, RWPE affected extracellular matrix remodeling by decreasing the secretion by myofibroblasts of matrix-metalloproteinase-2 and of tissue inhibitor of matrix-metalloproteinases-1.
CONCLUSION: Altogether, RWPE decreases the activation state of liver myofibroblasts. The identification of the active compounds in RWPE could offer new therapeutic strategies against liver fibrosis.
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Bellacchio E, Paggi MG. Protease-mediated arsenic prodrug strategy in cancer and infectious diseases: a hypothesis for targeted activation. J Cell Physiol 2008; 214:681-6. [PMID: 17894417 DOI: 10.1002/jcp.21261] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
A strategy for the selective in vivo activation of prodrugs by proteases is presented. The approach is based on the design of polythiol peptides able to neutralize the toxicity of As(III) through chelation, and contemporarily to be recognized as substrates of a disease-linked specific protease. Enzyme digestion implies conversion of such polythiol peptides into monothiol fragments with irreversible loss of the ability to chelate the metalloid, thus triggering the release in its free and pharmacologically effective form. The proteases whose activity appears dramatically up-regulated in various pathologies, ranging from cancer to infectious diseases, can be conveniently employed as prodrug activators in the disease microenvironment. The design of the representative peptide shown here has been assisted by molecular modeling in order to fulfill the dual characteristic to be an efficient As(III) chelator and simultaneously a substrate of the matrix metalloproteinase-9 (MMP-9) whose activity results dramatically increased at the surface of cells affected by several pathologies.
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Abstract
The hepatic stellate cell has surprised and engaged physiologists, pathologists, and hepatologists for over 130 years, yet clear evidence of its role in hepatic injury and fibrosis only emerged following the refinement of methods for its isolation and characterization. The paradigm in liver injury of activation of quiescent vitamin A-rich stellate cells into proliferative, contractile, and fibrogenic myofibroblasts has launched an era of astonishing progress in understanding the mechanistic basis of hepatic fibrosis progression and regression. But this simple paradigm has now yielded to a remarkably broad appreciation of the cell's functions not only in liver injury, but also in hepatic development, regeneration, xenobiotic responses, intermediary metabolism, and immunoregulation. Among the most exciting prospects is that stellate cells are essential for hepatic progenitor cell amplification and differentiation. Equally intriguing is the remarkable plasticity of stellate cells, not only in their variable intermediate filament phenotype, but also in their functions. Stellate cells can be viewed as the nexus in a complex sinusoidal milieu that requires tightly regulated autocrine and paracrine cross-talk, rapid responses to evolving extracellular matrix content, and exquisite responsiveness to the metabolic needs imposed by liver growth and repair. Moreover, roles vital to systemic homeostasis include their storage and mobilization of retinoids, their emerging capacity for antigen presentation and induction of tolerance, as well as their emerging relationship to bone marrow-derived cells. As interest in this cell type intensifies, more surprises and mysteries are sure to unfold that will ultimately benefit our understanding of liver physiology and the diagnosis and treatment of liver disease.
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Affiliation(s)
- Scott L Friedman
- Division of Liver Diseases, Mount Sinai School of Medicine, New York, New York 10029-6574, USA.
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26
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Rullier A, Gillibert-Duplantier J, Costet P, Cubel G, Haurie V, Petibois C, Taras D, Dugot-Senant N, Deleris G, Bioulac-Sage P, Rosenbaum J. Protease-activated receptor 1 knockout reduces experimentally induced liver fibrosis. Am J Physiol Gastrointest Liver Physiol 2008; 294:G226-35. [PMID: 17962354 DOI: 10.1152/ajpgi.00444.2007] [Citation(s) in RCA: 53] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Thrombin inhibition protects against liver fibrosis. However, it is not known whether the thrombin profibrogenic effect is due to effects on blood coagulation or to signaling via protease-activated receptors (PARs). We took advantage of the lack of blood coagulation defects in PAR-1-knockout mice. Acute carbon tetrachloride (CCl(4)) toxicity was similar in wild-type (WT), PAR-1(-/-), and PAR-1(+/-) mice as judged by aminotransferase levels, area of liver necrosis, and liver peroxidation measured by Fourier-transformed infrared spectroscopy. Fifteen mice/group received CCl(4) or its solvent for 6 wk (300 microl/kg, 3 times a week). Fibrosis area was increased 10-fold by CCl(4) treatment in WT mice. PAR-1 deficiency protected against fibrosis, with 36% and 56% decrease in PAR-1(+/-) and PAR-1(-/-) mice, respectively (P < 0.001). Similar results were obtained for area of activated fibrogenic cells (64% and 79% decrease in PAR-1(+/-) and PAR-1(-/-) mice, respectively, P < 0.001). These findings were corroborated by measurements of type I collagen, matrix metalloproteinase-2, and PDGF-beta receptor mRNA levels. There was also a significant decrease in T lymphocyte infiltration in PAR-1-deficient mice. Altogether, these results suggest that thrombin profibrogenic effects are independent of effects on blood coagulation and are instead due to direct effects on fibrogenic cells and possibly on T lymphocytes.
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Affiliation(s)
- Anne Rullier
- INSERM U889, Université Victor Segalen Bordeaux 2, 146, Rue Léo Saignat, 33076 Bordeaux, France
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27
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Hu YB, Li DG, Lu HM. Modified synthetic siRNA targeting tissue inhibitor of metalloproteinase-2 inhibits hepatic fibrogenesis in rats. J Gene Med 2007; 9:217-29. [PMID: 17351970 DOI: 10.1002/jgm.1009] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
Abstract
BACKGROUND/AIMS Fibrosis occurs in most chronic liver injuries and results from changes in the balance between synthesis and degradation of extracellular matrix (ECM) components. Matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs) are known to regulate the ECM turnover. We investigate the effect of modified synthetic small interfering RNA (siRNA) targeting TIMP-2 in rat model of liver fibrosis. METHODS Rat hepatic fibrosis was induced by CCl4 for 8 weeks. After the 2-week CCl4 injection period, rats in the three siRNA groups simultaneously received a different dosage (0.05, 0.1 and 0.2 mg.kg(-1), respectively) of modified synthetic siRNA targeting TIMP-2 via the tail vein every 3 days for 6 weeks. The pathological changes in liver tissues were observed by light microscopy and transmission electron microscopy. Portal vein pressure and proliferating cell nuclear antigen were measured. Expression of TIMP-2, MMP-2, MT1-MMP, MMP-13, hepatocyte growth factor, collagen type I, collagen type III and alpha-SMA were evaluated by quantitative real-time polymerase chain reaction or Western blotting or gelatin zymography. RESULTS Modified synthetic siRNA targeting TIMP-2 induced a dose-dependent inhibition of the TIMP-2 expression in the rat model of liver fibrosis with a similar trend in MMP-2 and MT1-MMP, but an increase in MMP-13. Rats administered siRNA targeting TIMP-2 showed promotion of ECM degradation, reduction in activated hepatic stellate cells and enhancement of hepatocyte regeneration. Furthermore, portal hypertension was also ameliorated after treatment with siRNA targeting TIMP-2. CONCLUSIONS Knock-down of TIMP-2 expression attenuates CCl4-induced liver fibrosis and is a potential pharmacological target for gene therapy in liver fibrosis.
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Affiliation(s)
- Ying-Bin Hu
- Department of Digestive Diseases, Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, 1665 Kongjiang Road, Shanghai, 200092, P. R. China.
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Iredale JP. Models of liver fibrosis: exploring the dynamic nature of inflammation and repair in a solid organ. J Clin Invest 2007; 117:539-48. [PMID: 17332881 PMCID: PMC1804370 DOI: 10.1172/jci30542] [Citation(s) in RCA: 680] [Impact Index Per Article: 37.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Models of liver fibrosis, which include cell culture models, explanted and biopsied human material, and experimental animal models, have demonstrated that liver fibrosis is a highly dynamic example of solid organ wound healing. Recent work in human and animal models has shown that liver fibrosis is potentially reversible and, in specific circumstances, demonstrates resolution with a restoration of near normal architecture. This Review highlights the manner in which studies of models of liver fibrosis have contributed to the paradigm of dynamic wound healing in this solid organ.
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Affiliation(s)
- John P Iredale
- Medical Research Council/University of Edinburgh Centre for Inflammation Research, Queen's Medical Research Institute, Edinburgh, United Kingdom.
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Jiroutova A, Slavkovsky R, Cermakova M, Majdiakova L, Hanovcova I, Bolehovska R, Hajzlerova M, Radilova H, Ruszova E, Kanta J. Expression of mRNAs related to connective tissue metabolism in rat hepatic stellate cells and myofibroblasts. ACTA ACUST UNITED AC 2007; 58:263-73. [PMID: 17056239 DOI: 10.1016/j.etp.2006.06.005] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2005] [Accepted: 06/07/2006] [Indexed: 01/18/2023]
Abstract
Hepatic stellate cells (HSC) and liver myofibroblasts (MFB) are two cell populations most likely responsible for the synthesis of most connective tissue components in fibrotic liver. They differ in their origin and location, and possibly in patterns of gene expression. Normal and carbon tetrachloride-cirrhotic livers from rats were used to isolate HSC. Liver was perfused with pronase and collagenase solutions, followed by centrifugation of the cell suspension on a density gradient. HSC were quiescent 2 days after plating on plastic but they became activated after another 5 days in culture. When the culture was passaged 5 times, its character changed profoundly as HSC were replaced by MFB. Microarray analysis was used to determine gene expression in quiescent HSC, activated HSC and MFB. The expression of 49 genes coding for connective tissue proteins, proteoglycans, metalloproteinases and their inhibitors, growth factors and cellular markers was determined. The pattern of gene expression changed during HSC activation and there were distinct differences between HSC and MFB. Little difference between normal cells and cells isolated from cirrhotic liver was found.
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Affiliation(s)
- Alena Jiroutova
- Charles University in Prague, Faculty of Medicine in Hradec Kralove, Department of Medical Biochemistry, Simkova 870, 500 38 Hradec Kralove, Czech Republic
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Abstract
Fibrotic liver disease occurs after any of the various forms of injury to the liver. Fibrosis is a critical factor leading to hepatic dysfunction and portal hypertension and its complications. The fibrogenic cascade is complex but leads to accumulation of extracellular matrix proteins, followed by nodular fibrosis, tissue contraction, and alteration in blood flow. A critical concept emerging is that activation of effector cells, which produce extracellular matrix, underlies the fibrogenic process. The aggregate data has not only helped lead to an understanding of the pathophysiologic basis of hepatic fibrogenesis, but it has also provided an important context with which to base novel antifibrotic therapy.
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Affiliation(s)
- Don C Rockey
- Division of Digestive and Liver Diseases, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-8887, USA.
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Zhou X, Jamil A, Nash A, Chan J, Trim N, Iredale JP, Benyon RC. Impaired proteolysis of collagen I inhibits proliferation of hepatic stellate cells: implications for regulation of liver fibrosis. J Biol Chem 2006; 281:39757-65. [PMID: 17060319 DOI: 10.1074/jbc.m605621200] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
Abstract
Myofibroblastic-activated hepatic stellate cells are the major source of the collagen I-rich extracellular matrix in liver fibrosis but also produce matrix metalloproteinases, which remodel this protein. We have investigated the role of collagen I proteolysis in both regulating proliferation and maintaining the activated myofibroblastic phenotype of stellate cells in vitro. Compared with stellate cells plated on normal collagen I, those plated on a collagenase-resistant form of collagen I (r/r collagen) had reduced thymidine incorporation and proliferating cell nuclear antigen expression but increased p21 expression. Collagen I was shown to be rendered resistant to matrix metalloproteinases by artificial cross-linking in vitro using tissue transglutaminase exerted similar antiproliferative effects on stellate cells to r/r collagen. Of the stellate cell activation markers examined (tissue inhibitor of metalloproteinases-1, alpha-smooth muscle actin, matrix metalloproteinases-2 and -9, and procollagen I) only the last was decreased by culture on r/r collagen relative to normal collagen I. Antagonists of integrin alphavbeta3, an integrin reported to stimulate stellate cell proliferation, significantly inhibited adhesion, proliferation, and procollagen I synthesis of stellate cells plated on normal collagen I but had reduced effectiveness on these parameters in cells on r/r collagen. We conclude that proliferation of stellate cells is promoted by pericellular collagen I proteolysis acting via alphavbeta3 integrin. Cross-linking of collagen I by tissue transglutaminase, a process known to occur in chronic liver fibrosis, might not only increase its resistance to matrix metalloproteinases thereby inhibiting resolution of fibrosis but also functions to constrain the fibroproliferative process.
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Affiliation(s)
- Xiaoying Zhou
- Liver Group, University Division of Infection, Inflammation and Repair, Southampton General Hospital, Southampton, Hants, SO16 6YD, United Kingdom
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Czochra P, Klopcic B, Meyer E, Herkel J, Garcia-Lazaro JF, Thieringer F, Schirmacher P, Biesterfeld S, Galle PR, Lohse AW, Kanzler S. Liver fibrosis induced by hepatic overexpression of PDGF-B in transgenic mice. J Hepatol 2006; 45:419-28. [PMID: 16842882 DOI: 10.1016/j.jhep.2006.04.010] [Citation(s) in RCA: 192] [Impact Index Per Article: 10.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/04/2005] [Revised: 04/04/2006] [Accepted: 04/18/2006] [Indexed: 02/06/2023]
Abstract
BACKGROUND/AIMS In hepatic fibrogenesis, stellate cells are activated leading to production and deposition of extracellular matrix. To clarify the role of PDGF-B in liver fibrogenesis, we overexpressed PDGF-B in the liver of transgenic mice. METHODS Transgenic mice for the conditional overexpression of PDGF-B in the liver under control of an albumin promoter were generated utilising the Cre/loxP system. Constitutive PDGF-B expression was achieved after breeding with mice expressing Cre-recombinase under actin promoter control. Tamoxifen inducible expression was achieved after breeding with mice expressing Cre under transthyretin receptor promoter control. Levels of fibrosis were assessed and the expression of regulators of matrix remodelling was measured. RESULTS PDGF-B expression caused hepatic stellate cell and myofibroblast activation marked by alpha-smooth muscle actin and PDGFR-beta expression. Liver fibrosis was verified macroscopically, histologically and by collagen I mRNA quantification in 4-6 week-old animals. MMP-2, MMP-9 and TIMP-1 were upregulated whereas TGF-beta expression was unchanged. CONCLUSIONS We identified PDGF-B as a proliferative and profibrogenic stimulus and potential inducer of stellate cell transdifferentiation in vivo. PDGF-B overexpression causes liver fibrosis without significantly upregulating TGF-beta1, suggesting a TGF-beta-independent mechanism. The established model provides a tool for testing anti-PDGF-B therapeutic strategies in liver fibrosis in vivo.
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Affiliation(s)
- Piotr Czochra
- Department of Medicine, Johannes Gutenberg-University, Mainz, Germany
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33
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Abstract
Hepatic fibrogenesis is the common result of injury to the liver. It is believed to be a critical factor that leads to hepatic dysfunction and may be important in portal hypertension. The fibrogenic response is a complex process in which accumulation of extracellular matrix proteins, tissue contraction, and alteration in blood flow are prominent. A critical event in fibrogenesis is activation of resident perisinusoidal cells that are termed "hepatic stellate cells". Stellate cell activation is characterized by many important phenotypes, including enhanced extracellular matrix synthesis and prominent contractility. Given the central role of stellate cell activation in hepatic fibrogenesis (and portal hypertension), effective therapy for hepatic fibrogenesis is most likely will be directed at this event.
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Affiliation(s)
- Don C Rockey
- Division of Digestive and Liver Diseases, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-8887, USA.
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Lee SH, Seo GS, Park YN, Yoo TM, Sohn DH. Effects and regulation of osteopontin in rat hepatic stellate cells. Biochem Pharmacol 2005; 68:2367-78. [PMID: 15548383 DOI: 10.1016/j.bcp.2004.08.022] [Citation(s) in RCA: 75] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2004] [Accepted: 08/17/2004] [Indexed: 02/07/2023]
Abstract
Using a cDNA microarray, we identified osteopontin (OPN) as one of the genes upregulated in cultured activated hepatic stellate cells (HSCs). Northern and western blot analyses showed that OPN was increasingly expressed during the progressive activation of cultured rat HSCs, and a significant increase in OPN was observed in carbon tetrachloride-induced rat liver fibrosis. In biliary atresia, OPN protein was predominantly expressed in Kupffer cells and HSCs in the necrotic areas. Incubation of HSCs with recombinant OPN-induced significant proliferative and migratory effects, and induced matrix metalloproteinase 2 production and activation. Moreover, OPN increased type I collagen production and type II transforming growth factor-beta receptor mRNA and protein. In conclusion, this study shows that OPN is expressed in activated HSCs and suggests that the upregulation of OPN might be a central pathway of HSC activation.
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Affiliation(s)
- Sung Hee Lee
- Medicinal Resources Research Center, College of Pharmacy, Wonkwang University, Iksan, Jeonbuk 570-749, Republic of Korea
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Mazzocca A, Sciammetta SC, Carloni V, Cosmi L, Annunziato F, Harada T, Abrignani S, Pinzani M. Binding of hepatitis C virus envelope protein E2 to CD81 up-regulates matrix metalloproteinase-2 in human hepatic stellate cells. J Biol Chem 2004; 280:11329-39. [PMID: 15611113 DOI: 10.1074/jbc.m410161200] [Citation(s) in RCA: 96] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
Abstract
The hepatitis C virus (HCV) envelope E2 glycoprotein is a key molecule regulating the interaction of HCV with cell surface proteins. E2 binds the major extracellular loop of human CD81, a tetraspanin expressed on various cell types including hepatocytes and B lymphocytes. Regardless, information on the biological functions originating from this interaction are largely unknown. Since human hepatic stellate cells (HSC) express high levels of CD81 at the cell surface, we investigated the E2/CD81 interaction in human HSC and the possible effects arising from this interaction. Matrix metalloproteinase-2 (MMP-2; gelatinase A), a major enzyme involved in the degradation of normal hepatic extracellular matrix, was up-regulated following the interaction between E2 and CD81. In particular, by employing zymography and Western blot, we observed that E2 binding to CD81 induces a time-dependent increase in the synthesis and activity of MMP-2. This effect was abolished by preincubating HSC with an anti-CD81 neutralizing antibody. Similar effects were detected in NIH3T3 mouse fibroblasts transfected with human CD81 with identical time course features. In addition, E2/CD81 interaction in human HSC induced the up-regulation of MMP-2 by increasing activator protein-2/DNA binding activity via ERK/MAPK phosphorylation. Finally, suppression of CD81 by RNA interference in human HSC abolished the described effects of E2 on these cells, indicating that CD81 is essential for the activation of the signaling pathway leading to the up-regulation of MMP-2. These results suggest that HSC may represent a potential target for HCV. The interaction of HCV envelope with CD81 on the surface of human HSC induces an increased expression of MMP-2. Increased degradation of the normal hepatic extracellular matrix in areas where HCV is concentrated may favor inflammatory infiltration and further parenchymal damage.
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Affiliation(s)
- Antonio Mazzocca
- Dipartimento di Medicina Interna, Centro di Ricerca, Trasferimento e Alta Formazione MCIDNENT Università di Firenze, I-50134 Firenze, Italy.
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36
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Di Sario A, Bendia E, Macarri G, Candelaresi C, Taffetani S, Marzioni M, Omenetti A, De Minicis S, Trozzi L, Benedetti A. The anti-fibrotic effect of pirfenidone in rat liver fibrosis is mediated by downregulation of procollagen alpha1(I), TIMP-1 and MMP-2. Dig Liver Dis 2004; 36:744-51. [PMID: 15571005 DOI: 10.1016/j.dld.2004.05.012] [Citation(s) in RCA: 59] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
BACKGROUND Pirfenidone (5 methyl-1-phenyl-2(1H)-pyridone) is a novel anti-fibrotic agent, which has been shown to decrease collagen deposition in a variety of animal models in vivo, and recently in hepatic fibrosis also. At cellular level, we have recently demonstrated that pirfenidone is able to inhibit proliferation of hepatic stellate cells induced by platelet-derived growth factor, as well as collagen type I accumulation and alpha1(I) procollagen mRNA expression. AIMS To evaluate if pirfenidone maintains its anti-fibrotic properties also when administered after the induction of hepatic damage and to further investigate the molecular mechanisms leading to the anti-fibrotic effect of pirfenidone. METHODS AND RESULTS Rats treated with dimethylnitrosamine (10 mg/kg) for 5 weeks received a liquid diet containing 0.5% pirfenidone starting from the third week. Pirfenidone treatment reduced the degree of liver injury, as determined by alanine aminotransferase values and necro-inflammatory score, which was associated with reduced hepatic stellate cells proliferation and collagen deposition. Treatment with dimethylnitrosamine increased transcripts levels for transforming growth factorbeta1, procollagen alpha1(I), tissue inhibitors of metalloproteinase-1 and matrix metalloproteinase-2 by 7-, 7-, 4- and 15-fold, respectively. Pirfenidone administration downregulated elevated levels of those transcripts by 50-60%, and this was associated with a 70% reduction in collagen deposition. CONCLUSIONS (1) Pirfenidone is effective also if administered after the induction of the hepatic damage; (2) the anti-fibrotic effect of pirfenidone is mainly due to the reduced expression of profibrogenic procollagen alpha1(I) and TIMP-1, most likely through the downregulation of transforming growth factorbeta1 mRNA, and of matrix metalloproteinase-2, which is mainly implicated in the degradation of the normal extracellular matrix.
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Affiliation(s)
- A Di Sario
- Gastrointestinal Unit, University of Ancona, Polo Didattico, III Piano, Via Tronto 10, 60020 Torrette, Ancona, Italy.
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Zhou X, Hovell CJ, Pawley S, Hutchings MI, Arthur MJP, Iredale JP, Benyon RC. Expression of matrix metalloproteinase-2 and -14 persists during early resolution of experimental liver fibrosis and might contribute to fibrolysis. Liver Int 2004; 24:492-501. [PMID: 15482348 DOI: 10.1111/j.1478-3231.2004.0946.x] [Citation(s) in RCA: 90] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/18/2023]
Abstract
BACKGROUND/AIMS Resolution of liver fibrosis is possible but the identity of the matrix metalloproteinases (MMPs) which degrade the accumulated collagens is uncertain. We examined MMP-2 and MMP-14 expression in established and resolving fibrosis to assess their role in resolution of liver fibrosis. METHODS MMP and tissue inhibitor of metalloproteinase (TIMP)-2 expression in liver extracts was examined by ribonuclease protection assay, Western blotting and gelatin zymography. MMP activity was examined by (14)C gelatin degradation. RESULTS In human cirrhotic liver, MMP-14 mRNA was increased to 230-330% of normal liver expression. Both 63 kDa proenzyme and 60 kDa activated form were present. Cirrhotic livers had 270-320% of normal liver expression of MMP-2 protein with 20-25% being the 62 Da activated form. Protein and mRNA for MMP-2 and MMP-14 progressively increased during 8 weeks of CCl(4) treatment in rats. Between 3 and 7 days of resolution from CCl(4) liver fibrosis, MMP-2 and MMP-14 persisted at elevated levels. Gelatinolytic activity in liver homogenates peaked at 7 days of recovery, being 140% above that in livers at peak fibrosis. CONCLUSIONS Increased expression and activation of MMP-2 and -14 occurs even under conditions of elevated TIMPs during liver fibrogenesis. During liver fibrosis resolution, as TIMP expression decays, the persistence of MMP-2 and MMP-14 may permit collagen degradation.
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Affiliation(s)
- Xiaoying Zhou
- Liver Research Group, University Division of Infection, Inflammation and Repair, Southampton General Hospital, Southampton, Hampshire SO16 6YD, UK
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38
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Zhang LJ, Chen YX, Chen ZX, Huang YH, Yu JP, Wang XZ. Effect of interleukin-10 and platelet-derived growth factor on expressions of matrix metalloproteinases-2 and tissue inhibitor of metalloproteinases-1 in rat fibrotic liver and cultured hepatic stellate cells. World J Gastroenterol 2004; 10:2574-9. [PMID: 15300910 PMCID: PMC4572167 DOI: 10.3748/wjg.v10.i17.2574] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/02/2004] [Revised: 01/04/2004] [Accepted: 01/12/2004] [Indexed: 12/15/2022] Open
Abstract
AIM To examine the expressions of matrix metalloproteinases-2 (MMP-2) and tissue inhibitor of metalloproteinases-1 (TIMP-1) in rat fibrotic liver and in normal rat hepatic stellate cells, and to investigate the changes in their expressions in response to treatment with interleukin-10 (IL-10) and platelet-derived growth factor (PDGF). METHODS Rat models of CCl4-induced hepatic fibrosis were established and the liver tissues were sampled from the rats with or without IL-10 treatment, and also from the control rats. The expressions of MMP-2 and TIMP-1 in liver tissues were detected by S-P immunohistochemistry, and their expression intensities were evaluated in different groups. Hepatic stellate cells (HSCs) were isolated from normal rat and cultured in vitro prior to exposure to PDGF treatment or co-treatment with IL-10 and PDGF. MMP-2 and TIMP-1 levels were measured by semi-quantitative reverse transcriptional polymerase chain reaction (RT-PCR). RESULTS CCl4- induced rat hepatic fibrosis models were successfully established. The positive expressions of MMP-2 and TIMP-1 increased obviously with the development of hepatic fibrosis, especially in untreated model group (84.0% and 92.0%, P<0.01). The positive signals decreased significantly following IL-10 treatment (39.3% and 71.4%, P<0.01 and P<0.05) in a time-dependent manner. TIMP-1 mRNA in PDGF-treated group was significantly increased time-dependently in comparison with that of the control group, but PDGF did not obviously affect MMP-2 expression. No difference was noted in TIMP-1 and MMP-2 expressions in HSCs after IL-10 and PDGF treatment (P>0.05). CONCLUSION MMP-2 and TIMP-1 expressions increase in liver tissues with the development of fibrosis, which can be inhibited by exogenous IL-10 inhibitor. PDGF induces the up-regulation of TIMP-1 but not MMP-2 in the HSCs. IL-10 inhibits TIMP-1 and MMP-2 expressions in HSCs induced by PDGF.
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Affiliation(s)
- Li-Juan Zhang
- Department of Gastroenterology, People's Hospital, Medical School of Wuhan University, Hubei Province, China
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Li YL, Sato M, Kojima N, Miura M, Senoo H. Regulatory role of extracellular matrix components in expression of matrix metalloproteinases in cultured hepatic stellate cells. Cell Struct Funct 2004; 24:255-61. [PMID: 15216880 DOI: 10.1247/csf.24.255] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023] Open
Abstract
Hepatic stellate cells (HSCs) were changed in their morphology, proliferative activity, and functions by culturing on type I collagen gel, as compared to the culture on polystyrene surface. HSCs have been found to produce extracellular matrix components and matrix metalloproteinases (MMPs). In this study, we have assessed the effects of several types of substrata on the expression of MMPs in HSC culture. MMP-1 expression was detectable in HSC culture on polystyrene surface and on type I collagen gel by immunofluorescence staining and reverse transcriptase-polymerase chain reaction (RT-PCR). The results from in situ zymography revealed the presence of interstitial collagenase activity around HSCs and along their cellular processes. Although proMMP-2 and proMMP-9 were detectable by gelatin zymography in the conditioned medium from both cultures using type I collagen gel and Matrigel as substratum, an active form of MMP-2 but not of MMP-9 was detected only in the culture using type I collagen as a substratum. Tissue inhibitor of metalloproteinase-2 expression was observed by RT-PCR in HSCs cultured on or in type I collagen gel, suggesting the suppression of MMP-2 activity detected in HSC culture using type I collagen. These results indicate a differential expression of MMP activity, hence the remodeling of extracellular matrix components is dependent on the substratum used for HSC culture. The HSC culture using several types of substrata appears to be a useful in vitro model to study the mechanism of extracellular matrix remodeling.
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Affiliation(s)
- Y L Li
- Department of Anatomy, Akita University School of Medicine, Akita 010-8543, Japan
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Wang DR, Sato M, Sato T, Kojima N, Higashi N, Senoo H. Regulation of matrix metallo-proteinase expression by extracellular matrix components in cultured hepatic stellate cells. COMPARATIVE HEPATOLOGY 2004; 3 Suppl 1:S20. [PMID: 14960172 PMCID: PMC2410240 DOI: 10.1186/1476-5926-2-s1-s20] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
Abstract
Hepatic stellate cells (HSC) changed their morphology and function including production of matrix metalloproteinases (MMPs) in response to extracellular matrix (ECM) component used as a substratum in culture. We examined in this study the regulatory role of ECM component on expression of MMPs and tissue inhibitor of metalloproteinase (TIMP) in rat HSCs cultured on polystyrene, type I collagen-coated surface, type I collagen gel, or Matrigel, respectively. When cultured on type I collagen gel, HSCs showed the asteroid cell shape and MMP-1 activity, as detected by in situ zymography. Expression of MMP-1 protein and mRNA were examined by using immunofluorescence staining and RT-PCR analysis in HSCs cultured on type I collagen gel. Active form of MMP-2 was detected by gelatin zymography in the conditioned medium of HSCs cultured on type I collagen gel, whereas it was not detected when HSCs were cultured on polystyrene, type I collagen-coated surface, or Matrigel. Increased MMP-2 mRNA was detected by RT-PCR in HSCs cultured on type I collagen gel. Increased MT1-MMP proteins were shown to localize on the cell membrane by using immunofluorescence staining in HSCs cultured on type I collagen gel. Elevated expression of membrane-type matrix metallproteinase-1 (MT1-MMP) mRNA and tissue inhibitor of metalloproteinase-2 (TIMP-2) mRNA was detected by RT-PCR in HSCs cultured on type I collagen-coated surface or type I collagen gel. These results indicate that expression of MMPs and TIMP-2 is regulated by ECM components in cultured HSCs, suggesting an important role of HSCs in the remodeling of liver tissue.
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Affiliation(s)
- Da-Ren Wang
- Department of Cell Biology and Histology, Akita University School of Medicine, Akita 010-8543, Japan
| | - Mitsuru Sato
- Department of Cell Biology and Histology, Akita University School of Medicine, Akita 010-8543, Japan
| | - Takeya Sato
- Department of Cell Biology and Histology, Akita University School of Medicine, Akita 010-8543, Japan
| | - Naosuke Kojima
- Department of Cell Biology and Histology, Akita University School of Medicine, Akita 010-8543, Japan
| | - Nobuyo Higashi
- Department of Cell Biology and Histology, Akita University School of Medicine, Akita 010-8543, Japan
| | - Haruki Senoo
- Department of Cell Biology and Histology, Akita University School of Medicine, Akita 010-8543, Japan
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Abstract
Liver fibrosis is the hallmark of every chronic liver disease. It is also the major factor of morbidity and mortality due to the development of cirrhosis and its complications including hepatocellular carcinoma. But even at the beginning of the process of liver fibrosis and due to the strategic position of the extracellular matrix at the interface between blood flow and epithelial compartment, any quantitative or qualitative modification of extracellular matrix will rapidly affect structure and function of the liver. The development of several animal models of liver fibrosis as well as isolation and cultivation of hepatic stellate cells, the major fibrogenic cell type in the liver, led to the gathering of recent knowledge on the mechanism of liver fibrosis. Activation of hepatic stellate cells is a key event in this process and many details on this finely tuned mechanism are now available. In addition to these experimental data, experience from chronic hepatitis C now allows the development of new concepts and perspectives such as liver fibrosis regression and antifibrotic therapies.
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Affiliation(s)
- Pierre Bedossa
- Service d'Anatomie Pathologique, CNRS FRE2443, Hôpital de Bicêtre, 78 Avenue Géneral Leclerc, 94275 Le Kremlin-Bicêtre, France.
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42
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Gaça MDA, Zhou X, Issa R, Kiriella K, Iredale JP, Benyon RC. Basement membrane-like matrix inhibits proliferation and collagen synthesis by activated rat hepatic stellate cells: evidence for matrix-dependent deactivation of stellate cells. Matrix Biol 2003; 22:229-39. [PMID: 12853033 DOI: 10.1016/s0945-053x(03)00017-9] [Citation(s) in RCA: 114] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023]
Abstract
During liver fibrosis hepatic stellate cells become activated, transforming into proliferative myofibroblastic cells expressing type I collagen and alpha-smooth muscle actin. They become the major producers of the fibrotic neomatrix in injured liver. This study examines if activated stellate cells are a committed phenotype, or whether they can become deactivated by extracellular matrix. Stellate cells isolated from normal rat liver proliferated and expressed mRNA for activation markers, alpha-smooth muscle actin, type I procollagen and tissue inhibitor of metalloproteinases-1 following 5-7 day culture on plastic, but culture on Matrigel suppressed proliferation and mRNA expression. Activated stellate cells were recovered from plastic by trypsinisation and replated onto plastic, type I collagen films or Matrigel. Cells replated on plastic and type I collagen films proliferated and remained morphologically myofibroblastic, expressing alpha-smooth muscle actin and type I procollagen. However, activated cells replated on Matrigel showed <30% of the proliferative rate of these cells, and this was associated with reduced cellular expression of proliferating cell nuclear antigen and phosphorylation of mitogen-activated protein kinase in response to serum. Activated HSC replated on Matrigel for 3-7 days progressively reduced their expression of mRNA for type I procollagen and alpha-smooth muscle actin and both became undetectable after 7 days. We conclude that basement membrane-like matrix induces deactivation of stellate cells. Deactivation represents an important potential mechanism mediating recovery from liver fibrosis in vivo where type I collagen is removed from the liver and stellate cells might re-acquire contact with their normal basement membrane-like pericellular matrix.
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Affiliation(s)
- Marianna D A Gaça
- Liver Research Group, University of Southampton Division of Infection, Inflammation and Repair, Southampton General Hospital, SO16 6YD, Southampton, UK
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Le Pabic H, Bonnier D, Wewer UM, Coutand A, Musso O, Baffet G, Clément B, Théret N. ADAM12 in human liver cancers: TGF-beta-regulated expression in stellate cells is associated with matrix remodeling. Hepatology 2003; 37:1056-66. [PMID: 12717386 DOI: 10.1053/jhep.2003.50205] [Citation(s) in RCA: 167] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
"A disintegrin and metalloproteinases" (ADAMs) form a family of cell-surface glycoproteins with potential protease and cell-adhesion activities. We have investigated ADAM expression in human liver cancers and their regulation by several cytokines involved in liver injury. Using degenerative RT-PCR, cDNA encoding sequences for ADAM9 and ADAM12 were identified in human activated hepatic stellate cells (HSCs). Northern blot analyses showed that HSCs, but not hepatocytes, expressed transcripts for ADAM9 messenger RNA (mRNA) and both the long and short forms of ADAM12. This expression was associated with the transition from quiescent to activated state of rat HSCs and markedly increased in human livers with cirrhosis. ADAM12 but not ADAM9 expression was up-regulated by transforming growth factor beta (TGF-beta) in human activated HSCs. The PI3K inhibitor LY294002 and the mitogen-activated protein kinase kinase (MEK) inhibitor UO126 prevented ADAM12 induction by TGF-beta, suggesting the involvement of PI3K and MEK activities. In vivo, the steady-state of both ADAM9 and ADAM12 mRNA levels was nearly undetectable in both normal livers and benign tumors and increased in hepatocellular carcinomas (up to 3- and 6-fold, respectively) and liver metastases from colonic carcinomas (up to 40- and 60-fold, respectively). The up-regulation of both ADAM9 and ADAM12 was correlated with an increase in matrix metalloproteinase 2 expression and activity. In conclusion, in liver cancers ADAM9 and ADAM12 expression is associated with tumor aggressiveness and progression.
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Affiliation(s)
- Hélène Le Pabic
- INSERM U456, Detoxication et Réparation Tissulaire, Université de Rennes I, France
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Abstract
In the past 20 years, the elucidation of the mechanisms responsible for liver fibrogenesis has provided many potential targets for antifibrotic treatments. Difficulty has arisen, however, from the fact that fibrogenesis is part of a general beneficial wound healing process. To be successful, an antifibrotic treatment of HCV might need to be delivered selectively to the hepatic site of fibrogenesis or targeted precisely at an HCV-specific regulatory mechanism. It is likely that in the future, besides viral eradication, another treatment goal in chronic HCV infection will be to reverse existing fibrosis, but considerable work is necessary before making this a reality.
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Affiliation(s)
- Pierre Bedossa
- Department of Pathology, Service d'anatomie pathologique, Hôpital Bicêtre, Université Paris XI, 78 rue du general Leclerc, 94725 Le Kremlin-Bicêtre, France.
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45
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Yang C, Zeisberg M, Mosterman B, Sudhakar A, Yerramalla U, Holthaus K, Xu L, Eng F, Afdhal N, Kalluri R. Liver fibrosis: insights into migration of hepatic stellate cells in response to extracellular matrix and growth factors. Gastroenterology 2003; 124:147-59. [PMID: 12512039 DOI: 10.1053/gast.2003.50012] [Citation(s) in RCA: 215] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
BACKGROUND & AIMS In liver fibrosis, alterations within the space of Disse microenvironment occur and facilitate further progression of chronic liver disease. The normal basement membrane-like matrix present within the space of Disse converts to a matrix rich in fibril-forming collagens during fibrosis. METHODS To further understand the pathogenesis of liver fibrosis, we modified an in vitro Boyden chamber system to partially mimic in vivo conditions of hepatic stellate cells (HSCs) during health and disease. RESULTS Stimulation of HSCs with platelet-derived growth factor (PDGF)-BB, transforming growth factor (TGF)-beta1, and/or epithelial growth factor (EGF) resulted in an increase in their migratory capacity and up-regulated matrix metalloproteinase (MMP)-2 activity. Migration induced by PDGF-BB was associated with increased proliferation, whereas TGF-beta1/EGF-induced migration was proliferation independent. COL-3, an inhibitor of MMP-2 and MMP-9, inhibited migration of HSCs induced by direct activation of PDGF-BB or TGF-beta1 but had no effect on migration induced by chemotactic stimuli without direct contact, suggesting 2 distinct MMP-dependent and MMP-independent mechanisms of PDGF-BB- or TGF-beta1-induced migration. Additionally, we show that type I collagen by itself induced migration of HSCs. Migration induced by PDGF-BB, TGF-beta1, and collagen I could be inhibited by alpha(1)- and/or alpha(2)-integrin blocking antibodies, collectively suggesting an integrin-dependent, MMP-2-mediated migration of HSCs. CONCLUSIONS Basement membrane matrix integrity, composition, and cell-matrix interactions play an important role in anchoring HSCs and preventing them from spreading within the space of Disse and potentially elsewhere in the liver. Additionally, our data provide strong evidence for MMPs in regulation of HSCs migration.
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Affiliation(s)
- Changqing Yang
- Program in Matrix Biology, Gastroenterology and Renal Divisions, Department of Medicine, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215, USA
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46
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Issa R, Zhou X, Trim N, Millward-Sadler H, Krane S, Benyon C, Iredale J. Mutation in collagen-1 that confers resistance to the action of collagenase results in failure of recovery from CCl4-induced liver fibrosis, persistence of activated hepatic stellate cells, and diminished hepatocyte regeneration. FASEB J 2003; 17:47-9. [PMID: 12475903 DOI: 10.1096/fj.02-0494fje] [Citation(s) in RCA: 171] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
Collagen-I, which predominates in the neomatrix of fibrotic liver, regulates hepatocyte and hepatic stellate cell (HSC) phenotypes. Recovery from liver fibrosis is accompanied by hepatocyte regeneration, matrix degradation, and HSC apoptosis. Using mice bearing a mutated collagen-I gene (r/r mice), which confers resistance to collagenase degradation, we have investigated the hypothesis that collagen-I degradation is critical to HSC apoptosis and hepatocyte regeneration during recovery from liver fibrosis. During a 28-day recovery period after 8 wk of CCl4 treatment, wild-type (WT) livers had significantly (43%) decreased hydroxyproline (OHP) content. In r/r livers, however, OHP content remained elevated at peak fibrosis levels. Expressed markers of activated HSC (alpha-smooth muscle actin, collagen-I), elevated at peak fibrosis, dropped to control levels in WT livers after 28 days but remained raised in the r/r livers. Moreover, relative to WT livers, r/r livers had significantly reduced stellate cell apoptosis and hepatocyte regeneration during the recovery period. Using extracted collagen-I from each genotype as culture substrata, relative to r/r, we show that WT collagen-I promotes hepatocyte proliferation via stimulation of integrin alpha(v)beta3. Failure to degrade collagen-I critically impairs HSC apoptosis and may prevent the effective restoration of hepatocyte mass in liver fibrosis.
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Affiliation(s)
- Razao Issa
- Liver Research Group and Department of Histopathology, Infection, Inflammation and Repair, South Lab and Path Block, University of Southampton, Southampton General Hospital, Southampton SO16 6YD, UK
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47
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Gardi C, Arezzini B, Fortino V, Comporti M. Effect of free iron on collagen synthesis, cell proliferation and MMP-2 expression in rat hepatic stellate cells. Biochem Pharmacol 2002; 64:1139-45. [PMID: 12234617 DOI: 10.1016/s0006-2952(02)01257-1] [Citation(s) in RCA: 64] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Various studies on hepatic fibrosis occurring in iron overload suggest that excess of tissue iron may be involved in the stimulation of collagen synthesis. Anyway, up to date, direct evidence on the role of iron in hepatic fibrosis is lacking. Moreover, it is not clear whether iron acts as direct initiator of fibrogenesis or as mediator of hepatocellular necrosis. In the present study, we investigated the effect of nontoxic doses of iron on collagen metabolism and proliferation, key features of liver fibrosis, by means of cultures of hepatic stellate cells, the liver cells responsible for collagen production. Iron treatment increased collagen synthesis without affecting noncollagen proteins. The maximum effect was observed at 5 microM iron (+132%). At this dose, no cell damage or proliferation was detected. Conversely, higher doses of iron (10 and 25 microM) induced cell proliferation and a lower increase in collagen synthesis, suggesting the prevalence of proliferative effect on the synthetic one. These effects occurred without the intervention of serum factors and were not mediated by lipid peroxidation. Our results strongly support the hypothesis that iron "per sé" may act as a profibrogenic agent. Finally, we provide evidence that iron plays a role also in matrix degradation, by stimulating some metalloprotease activities. Iron treatment increased metalloprotease-2 activity in hepatic stellate cells, while no changes were observed for interstitial collagenase activity suggesting that, in these conditions, a pathological accumulation of hepatic extracellular matrix may occur.
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Affiliation(s)
- Concetta Gardi
- Department of Pathophysiology and Experimental Medicine, University of Siena, via Aldo Moro, I-53100 Siena, Italy.
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48
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Shek FWT, Benyon RC, Walker FM, McCrudden PR, Pender SLF, Williams EJ, Johnson PA, Johnson CD, Bateman AC, Fine DR, Iredale JP. Expression of transforming growth factor-beta 1 by pancreatic stellate cells and its implications for matrix secretion and turnover in chronic pancreatitis. THE AMERICAN JOURNAL OF PATHOLOGY 2002; 160:1787-98. [PMID: 12000730 PMCID: PMC1850856 DOI: 10.1016/s0002-9440(10)61125-x] [Citation(s) in RCA: 240] [Impact Index Per Article: 10.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Pancreatic stellate cells mediate fibrosis in chronic pancreatitis. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs)-1 and -2 are crucial modulators of fibrosis. Transforming growth factor-beta (TGF-beta) is a key regulator of extracellular matrix production and myofibroblast proliferation. We have examined MMP and TIMP synthesis by transformed cultured pancreatic stellate cells and their regulation by TGF-beta 1. By Northern analysis they expressed mRNAs for procollagen 1, TIMP-1, TIMP-2, and MMP-2. Expression of membrane type-1 MMP was confirmed by Western blotting. By immunohistochemistry these enzymes localized to fibrotic areas in human chronic pancreatitis. Active TGF-beta 1 constitutes 2 to 5% of total TGF-beta 1 secreted by pancreatic stellate cells; they express TGF-beta receptors I and II. Exogenous TGF-beta 1 (10 ng/ml) significantly increased procollagen-1 mRNA by 69% and collagen protein synthesis by 34%. Similarly TGF-beta 1 at 0.1, 1, and 10 ng/ml significantly reduced cellular proliferation rate by 37%, 44%, and 44%, respectively, whereas pan-TGF-beta-neutralizing antibody increased proliferation by 40%. TGF-beta1 (10 ng/ml) down-regulated MMP-9 by 54% and MMP-3 by 34% whereas TGF-beta 1-neutralizing antibody increased MMP-9 expression by 39%. Pancreatic stellate cells express both mediators of matrix remodeling and the regulatory cytokine TGF-beta 1 that, by autocrine inhibition of MMP-3 and MMP-9, may enhance fibrogenesis by reducing collagen degradation.
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Affiliation(s)
- Fanny Wai-Tsing Shek
- Divisions of Infection, Inflammation, and Repair, Cellular Pathology, and Cancer Sciences, University of Southampton, Southampton General Hospital, Southampton, United Kingdom
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49
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Abstract
Hepatic fibrosis is a scaring process leading to cirrhosis, a major complication of numerous chronic liver diseases. Hepatic stellate cells play a central role in the fibrotic process. After parenchymal or biliary injury, cytokines and growth factors allow the recruitment, proliferation, and activation, of stellate cells toward myofibroblasts, which secrete the extracellular matrix. Fibrosis, resulting from the failure of the balance between synthesis and degradation of extracellular matrix, is an evolutive and potentially reversible process. Histological examination is the main investigation to quantify fibrosis. Serological tests are warranted to allow a non invasive follow up of patients. Development of antifibrotic therapies should soon permit to slow down the evolution toward cirrhosis, limiting the needs for hepatic transplantation.
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Affiliation(s)
- T Lamireau
- Groupe de recherches pour l'étude du foie, Inserm E9917, université Victor-Segalen, département de pédiatrie, hôpital Pellegrin, Bordeaux, France.
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Kharbanda KK, Shubert KA, Wyatt TA, Sorrell MF, Tuma DJ. Effect of malondialdehyde-acetaldehyde-protein adducts on the protein kinase C-dependent secretion of urokinase-type plasminogen activator in hepatic stellate cells. Biochem Pharmacol 2002; 63:553-62. [PMID: 11853706 DOI: 10.1016/s0006-2952(01)00883-8] [Citation(s) in RCA: 209] [Impact Index Per Article: 9.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Previous studies from our laboratory have shown that malondialdehyde-acetaldehyde-protein adducts (MAA adducts) are formed in hepatocytes of ethanol-fed rats and directly influence the hepatic stellate cells (HSCs) to induce their secretion of chemokines and to up-regulate their expression of adhesion molecules. Since protein kinase C (PKC) is known to play a major role in many diverse intracellular signal transduction processes, we investigated whether MAA adducts influence the function of HSCs via a PKC-dependent pathway. HSCs in culture were exposed to MAA adducts, and PKC activity was determined. We observed a time- and concentration-dependent activation of PKC when cultures were exposed to BSA-MAA as compared with unmodified BSA. Using PKC isoform-specific inhibitors, we also showed that BSA-MAA induces the activation of a specific isoform of PKC, PKC-alpha, in HSCs. No activation of PKC was observed when HSCs were exposed to other aldehyde adducts such as BSA-acetaldehyde or BSA-malondialdehyde, indicating that the effects of MAA adducts on HSCs were somewhat specific. We further examined whether the observed increase in PKC activation induced by MAA adducts in HSCs, in turn, causes a functional effect. We observed that BSA-MAA induces the increased secretion of urokinase-type plasminogen activator, a key component of the plasmin-generating system, and that PKC activation is necessary for this enhanced urokinase-type plasminogen activator secretion. These results indicate that MAA adducts via a PKC-mediated pathway may regulate plasmin-mediated matrix degradation in the liver, thereby contributing to the progression of hepatic fibrosis.
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Affiliation(s)
- Kusum K Kharbanda
- Department of Veterans Affairs Medical Center, VA Alcohol Research Center, 4101 Woolworth Avenue, Omaha, NE 68105, USA.
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