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Luangmonkong T, Parichatikanond W, Olinga P. Targeting collagen homeostasis for the treatment of liver fibrosis: Opportunities and challenges. Biochem Pharmacol 2023; 215:115740. [PMID: 37567319 DOI: 10.1016/j.bcp.2023.115740] [Citation(s) in RCA: 13] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2023] [Revised: 07/24/2023] [Accepted: 08/08/2023] [Indexed: 08/13/2023]
Abstract
Liver fibrosis is an excessive production, aberrant deposition, and deficit degradation of extracellular matrix (ECM). Patients with unresolved fibrosis ultimately undergo end-stage liver diseases. To date, the effective and safe strategy to cease fibrosis progression remains an unmet clinical need. Since collagens are the most abundant ECM protein which play an essential role in fibrogenesis, the suitable regulation of collagen homeostasis could be an effective strategy for the treatment of liver fibrosis. Therefore, this review provides a brief overview on the dysregulation of ECM homeostasis, focusing on collagens, in the pathogenesis of liver fibrosis. Most importantly, promising therapeutic mechanisms related to biosynthesis, deposition and extracellular interactions, and degradation of collagens, together with preclinical and clinical antifibrotic evidence of drugs affecting each target are orderly criticized. In addition, challenges for targeting collagen homeostasis in the treatment of liver fibrosis are discussed.
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Affiliation(s)
- Theerut Luangmonkong
- Department of Pharmacology, Faculty of Pharmacy, Mahidol University, Thailand; Centre of Biopharmaceutical Science for Healthy Ageing (BSHA), Faculty of Pharmacy, Mahidol University, Bangkok, Thailand.
| | - Warisara Parichatikanond
- Department of Pharmacology, Faculty of Pharmacy, Mahidol University, Thailand; Centre of Biopharmaceutical Science for Healthy Ageing (BSHA), Faculty of Pharmacy, Mahidol University, Bangkok, Thailand
| | - Peter Olinga
- Department of Pharmaceutical Technology and Biopharmacy, University of Groningen, The Netherlands
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Bellaye PS, Burgy O, Bonniaud P, Kolb M. HSP47: a potential target for fibrotic diseases and implications for therapy. Expert Opin Ther Targets 2021; 25:49-62. [PMID: 33287600 DOI: 10.1080/14728222.2021.1861249] [Citation(s) in RCA: 31] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
Introduction: Chronic fibrotic disorders are challenging clinical problems. The major challenge is the identification of specific targets expressed selectively in fibrotic tissues. Collagen accumulation is the hallmark fibrosis. HSP47 is a collagen-specific chaperon with critical role in collagen folding. This review discusses the anti-fibrotic potential of HSP47. Areas covered: This review compiles data retrieved from the PubMed database with keywords 'HSP47+fibrosis' from 01/2005 to 06/2020. We examined 1) collagen biology and its role in fibrotic diseases, 2) HSP47 role in fibrosis, 3) HSP47 inhibition strategies and 4) clinical investigations. The identification of the HSP47-collagen binding site led to the development of methods to screen HSP47 inhibitors with anti-fibrotic potential. Specific in vivo delivery systems of HSP47 siRNA to fibrotic tissue reduced collagen production/secretion associated with fibrosis inhibition in preclinical models. This strategy is about to be tested in clinical trials. Expert opinion: As a collagen-specific chaperon, HSP47 is a promising therapeutic target in fibrosis. Preclinical models have shown encouraging anti-fibrotic results. Anti-HSP47 strategies need to be further evaluated in clinical trials. The increase in circulating-HSP47 in lung fibrosis patients highlights the potential of HSP47 as a noninvasive biomarker and may represent an important step toward personalized medicine in fibrotic disorders.
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Affiliation(s)
- Pierre-Simon Bellaye
- Centre George-Franrçois Leclerc, Nuclear Medicine department, Plateforme d'imagerie et de radiothérapie préclinique, 1 rue du professeur Marion, Dijon, France.,Centre de Référence Constitutif des Maladies Pulmonaires Rares de l'Adultes de Dijon, Réseau OrphaLung, Filère RespiFil, Centre Hospitalier Universitaire de Bourgogne , Dijon,France
| | - Olivier Burgy
- Centre de Référence Constitutif des Maladies Pulmonaires Rares de l'Adultes de Dijon, Réseau OrphaLung, Filère RespiFil, Centre Hospitalier Universitaire de Bourgogne , Dijon,France.,INSERM U1231 Department HSP-pathies 7 Boulevard Jeanne d'Arc ,Dijon France
| | - Philippe Bonniaud
- Centre de Référence Constitutif des Maladies Pulmonaires Rares de l'Adultes de Dijon, Réseau OrphaLung, Filère RespiFil, Centre Hospitalier Universitaire de Bourgogne , Dijon,France
| | - Martin Kolb
- McMaster University, Department of medicine, FIRH, 50 Charlton Avenue East, Hamilton , Ontario, Canada
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Smith-Cortinez N, Fagundes RR, Gomez V, Kong D, de Waart DR, Heegsma J, Sydor S, Olinga P, de Meijer VE, Taylor CT, Bank R, Paulusma CC, Faber KN. Collagen release by human hepatic stellate cells requires vitamin C and is efficiently blocked by hydroxylase inhibition. FASEB J 2020; 35:e21219. [PMID: 33236467 DOI: 10.1096/fj.202001564rr] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2020] [Revised: 11/02/2020] [Accepted: 11/10/2020] [Indexed: 12/13/2022]
Abstract
Liver fibrosis is characterized by the accumulation of extracellular matrix proteins, mainly composed of collagen. Hepatic stellate cells (HSCs) mediate liver fibrosis by secreting collagen. Vitamin C (ascorbic acid) is a cofactor of prolyl-hydroxylases that modify newly synthesized collagen on the route for secretion. Unlike most animals, humans cannot synthesize ascorbic acid and its role in liver fibrosis remains unclear. Here, we determined the effect of ascorbic acid and prolyl-hydroxylase inhibition on collagen production and secretion by human HSCs. Primary human HSCs (p-hHSCs) and the human HSCscell line LX-2 were treated with ascorbic acid, transforming growth factor-beta (TGFβ) and/or the pan-hydroxylase inhibitor dimethyloxalylglycine (DMOG). Expression of collagen-I was analyzed by RT-qPCR (COL1A1), Western blotting, and immunofluorescence microscopy. Collagen secretion was determined in the medium by Western blotting for collagen-I and by HPLC for hydroxyproline concentrations. Expression of solute carrier family 23 members 1 and 2 (SLC23A1/SLC23A2), encoding sodium-dependent vitamin C transporters 1 and 2 (SVCT1/SVCT2) was quantified in healthy and cirrhotic human tissue. In the absence of ascorbic acid, collagen-I accumulated intracellularly in p-hHSCs and LX-2 cells, which was potentiated by TGFβ. Ascorbic acid co-treatment strongly promoted collagen-I excretion and enhanced extracellular hydroxyproline concentrations, without affecting collagen-I (COL1A1) mRNA levels. DMOG inhibited collagen-I release even in the presence of ascorbic acid and suppressed COL1A1 and alpha-smooth muscle actin (αSMA/ACTA2) mRNA levels, also under hypoxic conditions. Hepatocytes express both ascorbic acid transporters, while p-hHSCs and LX-2 express the only SVCT2, which is selectively enhanced in cirrhotic livers. Human HSCs rely on ascorbic acid for the efficient secretion of collagen-I, which can be effectively blocked by hydroxylase antagonists, revealing new therapeutic targets to treat liver fibrosis.
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Affiliation(s)
- Natalia Smith-Cortinez
- Department of Hepatology and Gastroenterology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands
| | - Raphael R Fagundes
- Department of Hepatology and Gastroenterology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands
| | - Valentina Gomez
- Tytgat Institute for Liver and Intestinal Research, Amsterdam Gastroenterology and Metabolism, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands
| | - Defu Kong
- Department of Hepatology and Gastroenterology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands
| | - Dirk R de Waart
- Tytgat Institute for Liver and Intestinal Research, Amsterdam Gastroenterology and Metabolism, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands
| | - Janette Heegsma
- Department of Hepatology and Gastroenterology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands
| | - Svenja Sydor
- Department of Internal Medicine, University Hospital Knappschaftskrankenhaus, Ruhr-University Bochum, Bochum, Germany
| | - Peter Olinga
- Department of Pharmaceutical Technology and Biopharmacy, University of Groningen, Groningen, the Netherlands
| | - Vincent E de Meijer
- Department of Surgery, Division of Hepato-Pancreato-Biliary Surgery and Liver Transplantation, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands
| | - Cormac T Taylor
- School of Medicine and Medical Science and the Conway Institute, University College Dublin, Dublin, Ireland
| | - Ruud Bank
- Department of Pathology and Medical Biology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands
| | - Coen C Paulusma
- Tytgat Institute for Liver and Intestinal Research, Amsterdam Gastroenterology and Metabolism, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands
| | - Klaas Nico Faber
- Department of Hepatology and Gastroenterology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands
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Strowitzki MJ, Ritter AS, Kimmer G, Schneider M. Hypoxia-adaptive pathways: A pharmacological target in fibrotic disease? Pharmacol Res 2019; 147:104364. [PMID: 31376431 DOI: 10.1016/j.phrs.2019.104364] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/01/2019] [Revised: 07/19/2019] [Accepted: 07/19/2019] [Indexed: 02/07/2023]
Abstract
Wound healing responses are physiological reactions to injuries and share common characteristics and phases independently of the injured organ or tissue. A major hallmark of wound healing responses is the formation of extra-cellular matrix (ECM), mainly consisting of collagen fibers, to restore the initial organ architecture and function. Overshooting wound healing responses result in unphysiological accumulation of ECM and collagen deposition, a process called fibrosis. Importantly, hypoxia (oxygen demand exceeds supply) plays a significant role during wound healing responses and fibrotic diseases. Under hypoxic conditions, cells activate a gene program, including the stabilization of hypoxia-inducible factors (HIFs), which induces the expression of HIF target genes counteracting hypoxia. In contrast, in normoxia, so-called HIF-prolyl hydroxylases (PHDs) oxygen-dependently hydroxylate HIF-α, which marks it for proteasomal degradation. Importantly, PHDs can be pharmacologically inhibited (PHI) by so-called PHD inhibitors. There is mounting evidence that the HIF-pathway is continuously up-regulated during the development of tissue fibrosis, and that pharmacological (HIFI) or genetic inhibition of HIF can prevent organ fibrosis. By contrast, initial (short-term) activation of the HIF pathway via PHI during wound healing seems to be beneficial in several models of inflammation or acute organ injury. Thus, timing and duration of PHI and HIFI treatment seem to be crucial. In this review, we will highlight the role of hypoxia-adaptive pathways during wound healing responses and development of fibrotic disease. Moreover, we will discuss whether PHI and HIFI might be a promising treatment option in fibrotic disease, and consider putative pitfalls that might result from this approach.
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Affiliation(s)
- Moritz J Strowitzki
- Department of General, Visceral and Transplantation Surgery, University of Heidelberg, Heidelberg, Germany
| | - Alina S Ritter
- Department of General, Visceral and Transplantation Surgery, University of Heidelberg, Heidelberg, Germany
| | - Gwendolyn Kimmer
- Department of General, Visceral and Transplantation Surgery, University of Heidelberg, Heidelberg, Germany
| | - Martin Schneider
- Department of General, Visceral and Transplantation Surgery, University of Heidelberg, Heidelberg, Germany.
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Sivakumar P, Kitson C, Jarai G. Modeling and measuring extracellular matrix alterations in fibrosis: challenges and perspectives for antifibrotic drug discovery. Connect Tissue Res 2019; 60:62-70. [PMID: 30071759 DOI: 10.1080/03008207.2018.1500557] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Abstract
An imbalance of extracellular matrix (ECM) deposition and turnover is a hallmark of fibrotic pathologies as opposed to normal repair response to injury across several organs. Antifibrotic approaches to date have targeted multiple mechanisms and pathways involved in inflammation, angiogenesis, injury, wound repair, ECM biosynthesis, assembly, crosslinking and degradation. Many of these approaches have been unsuccessful which may in part be due to suboptimal models and the lack of validated functional ECM end points relevant to fibrosis. In addition, drug discovery and development for fibrotic diseases has been challenging due to the lack of translatability from in vivo models to the clinic. Targeting growth factor signaling pathways such as transforming growth factor beta (TGFβ), platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF) are possible in simple recombinant cell models and the approval of the tyrosine kinase inhibitor, nintedanib (Ofev) is testament to the approach. However, drug targets directly impacting ECM synthesis, assembly or degradation have proven clinically intractable to date. The reasons for a lack of progress are many and include; non-traditional drug targets, lack of suitable high throughput screening assays and translational models, incomplete understanding of the role of the target. Here, we review the role of ECM in fibrosis, the challenges of ECM-targeted antifibrotic approaches, progress in the development of functional and biomarker-related ECM assays and where new translational models of fibrotic ECM remodeling could support drug discovery for fibrotic diseases.
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Affiliation(s)
- Pitchumani Sivakumar
- a Fibrosis Translational Research and Development , Bristol-Myers Squibb , Pennington , NJ , USA
| | - Christopher Kitson
- b Fibrosis Discovery Biology , Bristol-Myers Squibb , Pennington , NJ , USA
| | - Gabor Jarai
- a Fibrosis Translational Research and Development , Bristol-Myers Squibb , Pennington , NJ , USA
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Weiskirchen R. Hepatoprotective and Anti-fibrotic Agents: It's Time to Take the Next Step. Front Pharmacol 2016; 6:303. [PMID: 26779021 PMCID: PMC4703795 DOI: 10.3389/fphar.2015.00303] [Citation(s) in RCA: 56] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2015] [Accepted: 12/11/2015] [Indexed: 12/21/2022] Open
Abstract
Hepatic fibrosis and cirrhosis cause strong human suffering and necessitate a monetary burden worldwide. Therefore, there is an urgent need for the development of therapies. Pre-clinical animal models are indispensable in the drug discovery and development of new anti-fibrotic compounds and are immensely valuable for understanding and proofing the mode of their proposed action. In fibrosis research, inbreed mice and rats are by far the most used species for testing drug efficacy. During the last decades, several hundred or even a thousand different drugs that reproducibly evolve beneficial effects on liver health in respective disease models were identified. However, there are only a few compounds (e.g., GR-MD-02, GM-CT-01) that were translated from bench to bedside. In contrast, the large number of drugs successfully tested in animal studies is repeatedly tested over and over engender findings with similar or identical outcome. This circumstance undermines the 3R (Replacement, Refinement, Reduction) principle of Russell and Burch that was introduced to minimize the suffering of laboratory animals. This ethical framework, however, represents the basis of the new animal welfare regulations in the member states of the European Union. Consequently, the legal authorities in the different countries are halted to foreclose testing of drugs in animals that were successfully tested before. This review provides a synopsis on anti-fibrotic compounds that were tested in classical rodent models. Their mode of action, potential sources and the observed beneficial effects on liver health are discussed. This review attempts to provide a reference compilation for all those involved in the testing of drugs or in the design of new clinical trials targeting hepatic fibrosis.
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Affiliation(s)
- Ralf Weiskirchen
- Institute of Molecular Pathobiochemistry, Experimental Gene Therapy, and Clinical Chemistry, RWTH University Hospital Aachen Aachen, Germany
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8
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Nagel S, Talbot NP, Mecinović J, Smith TG, Buchan AM, Schofield CJ. Therapeutic manipulation of the HIF hydroxylases. Antioxid Redox Signal 2010; 12:481-501. [PMID: 19754349 DOI: 10.1089/ars.2009.2711] [Citation(s) in RCA: 70] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
The hypoxia-inducible factor (HIF) family of transcription factors is responsible for coordinating the cellular response to low oxygen levels in animals. By regulating the expression of a large array of target genes during hypoxia, these proteins also direct adaptive changes in the hematopoietic, cardiovascular, and respiratory systems. They also play roles in pathological processes, including tumorogenesis. In recent years, several oxygenases have been identified as key molecular oxygen sensors within the HIF system. The HIF hydroxylases regulate the stability and transcriptional activity of the HIF-alpha subunit by catalyzing hydroxylation of specific proline and asparaginyl residues, respectively. They require oxygen and 2-oxoglutarate (2OG) as co-substrates, and depend upon non-heme ferrous iron (Fe(II)) as a cofactor. This article summarizes current understanding of the biochemistry of the HIF hydroxylases, identifies targets for their pharmacological manipulation, and discusses their potential in the therapeutic manipulation of the HIF system.
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Affiliation(s)
- Simon Nagel
- Acute Stroke Programme, Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Headington, Oxford, United Kingdom
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Ye Z, Houssein HSH, Mahato RI. Bioconjugation of oligonucleotides for treating liver fibrosis. Oligonucleotides 2008; 17:349-404. [PMID: 18154454 DOI: 10.1089/oli.2007.0097] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023]
Abstract
Liver fibrosis results from chronic liver injury due to hepatitis B and C, excessive alcohol ingestion, and metal ion overload. Fibrosis culminates in cirrhosis and results in liver failure. Therefore, a potent antifibrotic therapy is urgently needed to reverse scarring and eliminate progression to cirrhosis. Although activated hepatic stellate cells (HSCs) remain the principle cell type responsible for liver fibrosis, perivascular fibroblasts of portal and central veins as well as periductular fibroblasts are other sources of fibrogenic cells. This review will critically discuss various treatment strategies for liver fibrosis, including prevention of liver injury, reduction of inflammation, inhibition of HSC activation, degradation of scar matrix, and inhibition of aberrant collagen synthesis. Oligonucleotides (ODNs) are short, single-stranded nucleic acids, which disrupt expression of target protein by binding to complementary mRNA or forming triplex with genomic DNA. Triplex forming oligonucleotides (TFOs) provide an attractive strategy for treating liver fibrosis. A series of TFOs have been developed for inhibiting the transcription of alpha1(I) collagen gene, which opens a new area for antifibrotic drugs. There will be in-depth discussion on the use of TFOs and how different bioconjugation strategies can be utilized for their site-specific delivery to HSCs or hepatocytes for enhanced antifibrotic activities. Various insights developed in individual strategy and the need for multipronged approaches will also be discussed.
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Affiliation(s)
- Zhaoyang Ye
- Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, Memphis, TN 38163, USA
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Kesteloot F, Desmoulière A, Leclercq I, Thiry M, Arrese JE, Prockop DJ, Lapière CM, Nusgens BV, Colige A. ADAM metallopeptidase with thrombospondin type 1 motif 2 inactivation reduces the extent and stability of carbon tetrachloride-induced hepatic fibrosis in mice. Hepatology 2007; 46:1620-31. [PMID: 17929299 DOI: 10.1002/hep.21868] [Citation(s) in RCA: 50] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/18/2023]
Abstract
UNLABELLED ADAMTS2 belongs to the "ADAM metallopeptidase with thrombospondin type 1 motif" (ADAMTS) family. Its primary function is to process collagen type I, II, III, and V precursors into mature molecules by excising the aminopropeptide. This process allows the correct assembly of collagen molecules into fibrils and fibers, which confers to connective tissues their architectural structure and mechanical resistance. To evaluate the impact of ADAMTS2 on the pathological accumulation of extracellular matrix proteins, mainly type I and III collagens, we evaluated carbon tetrachloride-induced liver fibrosis in ADAMTS2-deficient (TS2(-/-)) and wild-type (WT) mice. A single carbon tetrachloride injection caused a similar acute liver injury in deficient and WT mice. A chronic treatment induced collagen deposition in fibrous septa that were made of thinner and irregular fibers in TS2(-/-) mice. The rate of collagen deposition was slower in TS2(-/-) mice, and at an equivalent degree of fibrosis, the resorption of fibrous septa was slightly faster. Most of the genes involved in the development and reversion of the fibrosis were similarly regulated in TS2(-/-) and WT mice. CONCLUSION These data indicate that the extent of fibrosis is reduced in TS2(-/-) mice in comparison with their WT littermates. Inhibiting the maturation of fibrillar collagens may be a beneficial therapeutic approach to interfering with the development of fibrotic lesions.
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Affiliation(s)
- Frédéric Kesteloot
- Laboratory of Connective Tissues Biology, Interdisciplinary Cluster for Applied Genoproteomics/Center for Experimental Cancer Research, University of Liège, Liège, Belgium
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Gao XS, Cheng J, Zhen Z, Guo J, Zhang LY, Tao ML. Cloning of hepatitis B virus DNAPTP1 transactivating genes by suppression subtractive hybridization technique. Shijie Huaren Xiaohua Zazhi 2005; 13:2371-2374. [DOI: 10.11569/wcjd.v13.i19.2371] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [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 clone and identify the new target genes transactivated by DNAPTP1 of the hepatitis B virus with the suppression subtractive hybridization technique and bioinformatics.
METHODS: The mRNA was isolated from HepG2 cells transfected with pcDNA3.1 (-)-DNAPTP1, and then it was transcribed into the cDNA. The pcDNA3.1 (-) empty vector was used as control. After digested by Rsa I, the tester cDNA was then divided into two groups and ligated with two different adaptors. After the tester cDNA was hybridized twice with the driver cDNA and underwent two times of nested polymerase chain reaction (PCR), the amplified cDNA fragments were subcloned into pGEM-Teasy plasmid vectors to construct the subtractive library. The library was amplified by E. coli strain DH5a. The randomly picked cDNA was sequenced and analyzed in the GenBank after the PCR.
RESULTS: The cDNA subtractive library of HBV PTP1 transactivating genes was successfully constructed. The amplified library contained 60 positive clones, from which 32 inserts with 200-1?000 bp in length were obtained. Three differentially expressed protein genes and 4 sequences with unknown function were found by sequence analysis.
CONCLUSION: The obtained genes may code the proteins involved in the regulation of cell cycle, metabolism, immunity and cell apoptosis.
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Tsukada S, Parsons CJ, Rippe RA. Mechanisms of liver fibrosis. Clin Chim Acta 2005; 364:33-60. [PMID: 16139830 DOI: 10.1016/j.cca.2005.06.014] [Citation(s) in RCA: 283] [Impact Index Per Article: 14.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2005] [Revised: 06/07/2005] [Accepted: 06/08/2005] [Indexed: 01/18/2023]
Abstract
Liver fibrosis represents a significant health problem worldwide of which no acceptable therapy exists. The most characteristic feature of liver fibrosis is excess deposition of type I collagen. A great deal of research has been performed to understand the molecular mechanisms responsible for the development of liver fibrosis. The activated hepatic stellate cell (HSC) is the primary cell type responsible for the excess production of collagen. Following a fibrogenic stimulus, HSCs change from a quiescent to an activated, collagen-producing cell. Numerous changes in gene expression are associated with HSC activation including the induction of several intracellular signaling cascades, which help maintain the activated phenotype and control the fibrogenic and proliferative state of the cell. Detailed analyses in understanding the molecular basis of collagen gene regulation have revealed a complex process offering the opportunity for multiple potential therapeutic strategies. However, further research is still needed to gain a better understanding of HSC activation and how this cell maintains its fibrogenic nature. In this review we describe many of the molecular events that occur following HSC activation and collagen gene regulation that contribute to the fibrogenic nature of these cells and provide a review of therapeutic strategies to treat this disease.
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Affiliation(s)
- Shigeki Tsukada
- Division of Gastroenterology and Hepatology, Department of Medicine, University of North Carolina at Chapel Hill, NC 27599-7032, USA
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Stefanovic L, Stephens CE, Boykin D, Stefanovic B. Inhibitory effect of dicationic diphenylfurans on production of type I collagen by human fibroblasts and activated hepatic stellate cells. Life Sci 2005; 76:2011-26. [PMID: 15707883 DOI: 10.1016/j.lfs.2004.09.043] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2004] [Accepted: 09/23/2004] [Indexed: 11/19/2022]
Abstract
Excessive production of extracellular matrix is responsible for clinical manifestations of fibroproliferative disorders and drugs which can inhibit excessive synthesis of type I collagen are needed for the therapy. Several dicationic diphenylfurans were synthesized and were found to bind RNA. Two of these type compounds were able to reduce synthesis of type I collagen by human fibroblasts and human activated hepatic stellate cells (HSCs). Activated HSCs are responsible for collagen production in liver fibrosis. When added at 40 microM compound 588 reduced intracellular level and secretion of procollagen alpha1(I) by 50%, while compound 654 reduced these parameters by more than 80% at 20 microM. 654 also significantly reduced secretion of fibronectin. Toxic effects were observed at 80 microM for 588 and 40 microM for 654. 654 reduced expression of a reporter gene with collagen signal peptide, while expression of the same gene without signal peptide was unaffected. Also, expression of intracellular proteins tubulin and calnexin was unchanged. 654 accumulated inside the cell in the cytoplasm and did not change the steady-state level of collagen mRNAs. Treatment of cells with proteosome inhibitor MG132 did not change the inhibitory effect of 654, suggesting that 654 acts as suppressor of translation of proteins containing a signal peptide. Most secreted proteins of fibroblasts and activated HSCs are components of extracellular matrix. Therefore inhibition of their production, as shown here for procollagen alpha1(I) and fibronectin, may be a useful property of some of diphenylfurans, making these compounds a basis for development of antifibrotic drugs.
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Affiliation(s)
- Lela Stefanovic
- Department of Biomedical Science, Florida State University College of Medicine, Tallahassee, FL 32306, USA
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Abstract
Hepatic cirrhosis is a common disease that poses a serious threat to public health, and is characterized by chronic, progressive and diffuse hepatic lesions preceded by hepatic fibrosis regardless of the exact etiologies. In recent years, considerable achievements have been made in China in research of the etiopathogenesis, diagnosis and especially the treatment of hepatic fibrosis, resulting in much improved prognosis of hepatic fibrosis and cirrhosis. In this paper, the authors review the current status of research in hepatic fibrosis, cirrhosis and their major complications.
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Affiliation(s)
- Xi-Xian Yao
- Department of Gastroenterology of Internal Medicine, Second Hospital, Hebei Medical University, Shijiazhuang 050000, Hebei Province, China.
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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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Sakaida I, Hironaka K, Kimura T, Terai S, Yamasaki T, Okita K. Herbal medicine Sho-saiko-to (TJ-9) increases expression matrix metalloproteinases (MMPs) with reduced expression of tissue inhibitor of metalloproteinases (TIMPs) in rat stellate cell. Life Sci 2004; 74:2251-63. [PMID: 14987950 DOI: 10.1016/j.lfs.2003.09.059] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2003] [Accepted: 09/18/2003] [Indexed: 12/16/2022]
Abstract
We have reported that Sho-saiko-to (TJ-9) prevents liver fibrosis in vivo. To gain further insights into the effect of TJ-9, the matrix metalloproteinases (MMPs)/tissue inhibitors of metalloproteinases (TIMPs) balance was examined. Hepatic stellate cells (HSCs) were isolated from male Wistar rats and cultured with TJ-9 (0-1000 microg/ml) on uncoated plastic dishes for 4 days. To elucidate the effects on the MMPs/TIMPs balance by TJ-9, quantitative analysis of type IV collagen-degrading activity, gelatin zymography and reverse zymography were carried out. Northern blot analysis was performed to determine the expression of MMP-2, 13 and TIMP-1 mRNAs. TJ-9 treatment resulted in dose-dependent upregulation of MMP-2, 13 mRNA and downregulation of TIMP-1 mRNA up to 500 microg/ml. Gelatin zymography, reverse zymography and quantitative analysis of type IV collagen-degrading activity confirmed that TJ-9 increased MMP-2 activity and prevented TIMP-1, 2 activities in a dose-dependent manner. SB203580 diminished the reduction of mRNA as well as the activity of TIMP-1 by TJ-9 and induction of mRNA as well as the activity of MMP-2. These results show that TJ-9 increased MMP-2, 13 activity with reduced TIMP-1, 2 activities on HSCs possibly via P38 pathway.
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Affiliation(s)
- Isao Sakaida
- Department of Gastroenterology and Hepatology, Yamaguchi University, School of Medicine, Minami-Kogushi 1-1-1, Ube, Yamaguchi 755-8505, Japan.
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Liu SH, Tan DM, Hou J, Hu GL. Cloning and expression of tissue inhibitor of metalloproteinases-1 (TIMP-1) gene in Chinese. Shijie Huaren Xiaohua Zazhi 2003; 11:1164-1167. [DOI: 10.11569/wcjd.v11.i8.1164] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM To clone the Chinese gene of TIMP-1 and express the fusion protein MBP-TIMP-1.
METHODS The fragment of TIMP-1 gene from the liver tissue of indigenous Hunan residents was amplified by RT-nest-PCR and it was cloned, sequenced by gene recombinations techniques. Fusion protein MBP-TIMP-1 was expressed in E.coli, analysed by SDS-PAGE and Western blot and purified by affinity chromatography.
RESULTS The sequence of cloned Chinese TIMP-1 gene was 624 bp. It is homologous with the reported other human TIMP-1 gene sequence. Fusion protein MBP-TIMP-1 was 66 kd and is of the antigenicity of TIMP-1. The MBP-TIMP-1 was purified by affinity chromatography.
CONCLUSION The Chinese gene of TIMP-1 was cloned and MBP-TIMP-1 was expressed and purified successfully in vitro. It may be useful to the diagnosis of liver fibrosis.
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Affiliation(s)
- Shuang-Hu Liu
- Department of Infectious Diseases, the Affiliated Xiangya Hospital, Central-South University, Changsha 410008, Hunan Province, China
| | - De-Ming Tan
- Department of Infectious Diseases, the Affiliated Xiangya Hospital, Central-South University, Changsha 410008, Hunan Province, China
| | - Jue Hou
- Department of Infectious Diseases, the Affiliated Xiangya Hospital, Central-South University, Changsha 410008, Hunan Province, China
| | - Guo-Ling Hu
- Department of Infectious Diseases, the Affiliated Xiangya Hospital, Central-South University, Changsha 410008, Hunan Province, China
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Benlloch S, Beltrán B, Moreno R, Berenguer M. [Fibrogenesis and liver transplantation]. GASTROENTEROLOGIA Y HEPATOLOGIA 2003; 26:381-95. [PMID: 12809575 DOI: 10.1016/s0210-5705(03)70375-6] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/20/2023]
Affiliation(s)
- S Benlloch
- Servicio de Medicina Digestiva. Hospital La Fe. Valencia. España
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Nie QH, Xie YM, Zhou YX, Cheng YQ, Luo H, Luo XD. Expression of TIMP-1 in normal and fibrotic livers in rats. Shijie Huaren Xiaohua Zazhi 2003; 11:204-208. [DOI: 10.11569/wcjd.v11.i2.204] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the expression of TIMP-1 in normal and experimental fibrotic livers in rats.
METHODS: Hepatic fibrosis rat models were induced with human serum albumin, and normal rats were used as control. TIMP-1 mRNA and its related antigens in liver were examined with immunohistochemistry and in situ hybridization. Expression of TIMP-1 gene was detected with PCR.
RESULTS: The positive signal of TIMP-1 proteins was mainly located in cytoplasm rather than nucleus in hepatic myofibroblasts and fibroblasts, which dominated in portal areas and fibrous septums fibrotic rats. Such distribution of expression of TIMP-1 detected with immunohistochemistry was consistent with that in situ hybridization. The level of TIMP-1 expression in fibrotic rats was obviously higher than that in normal ones.
CONCLUSION: TIMP-1 was expressed dominantly in fibroblast and myofibroblast in fibrotic liver, the expression level increased with progression of liver injury and severity of hepatic fibrosis.
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20
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Liu WB, Yang CQ, Jiang W, Wang YQ, Guo JS, He BM, Wang JY. Inhibition on the production of collagen type I, III of activated hepatic stellate cells by antisense TIMP-1 recombinant plasmid. World J Gastroenterol 2003; 9:316-9. [PMID: 12532457 PMCID: PMC4611337 DOI: 10.3748/wjg.v9.i2.316] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/14/2002] [Revised: 10/04/2002] [Accepted: 10/18/2002] [Indexed: 02/06/2023] Open
Abstract
AIM To investigate the inhibition effects on the production of collagen type I, III secreted by activated rat hepatic stellate cells (rHSCs) by antisense tissue inhibitors of metalloproteinase 1 (TIMP-1) recombinant plasmid through elevating interstitial collagenase activity. METHODS rHSCs were extracted from normal rat liver by pronase and collagenase digestion and purified by centrifugal elutriation, and were cultured on plastic dishes until they were activated to a myofibroblastic phenotype after 7-10 days. RT-Nest-PCR and gene recombinant techniques were used to construct the rat antisense TIMP-1 recombinant plasmids which can express in eucaryotic cells. The recombinant plasmid and the pcDNA3 empty plasmid were transfected in rHSCs by Effectene (QIAGEN) separately. Cells were selected after growing in DMEM containing 400 microg/ml G418 for 2-3 weeks. Expression of exogenous gene was assessed by Northern blot, and expression of TIMP-1 in rHSCs was determined by Northern blot and Western blot. We tested the interstitial collagenase activity with FITC-labled type I collagen as substrate. Ultimately, we quantified the type I, III collagen by Western blot. RESULTS The exogenous antisense TIMP-1 recombinant plasmid could be expressed in rHSCs well, which could block the expression of TIMP-1 greatly, the ratio of TIMP-1/GAPDH was 0.67, 2.41, and 2.97 separately at mRNA level (P<0.05); the ratio of TIMP-1/beta-actin was 0.31, 0.98 and 1.32 separately at protein level (P<0.05); It might elevate active and latent interstitial collagenase activity, the collagenase activity was 0.3049, 0.1411 and 0.1196 respectively. (P<0.05), which led to promotion the degradation of type I, III collagen, the ratio of collagen I/beta-actin was 0.63, 1.78 and 1.92 separately (P<0.05); and the ratio of collagen III/beta-actin was 0.59, 1.81 and 1.98 separately (P<0.05). CONCLUSION These data shows that the antisense TIMP-1 recombinant plasmid has the inhibitory effects on the production of type I, III collagens secreted by activated rHSCs in vitro. It could be a novel method to reverse hepatic fibrosis in the future.
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Affiliation(s)
- Wen-Bin Liu
- Division of Gastroenterology, Zhongshan Hospital, Fudan University, Shanghai 200032, China
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21
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Sakaida I, Hironaka K, Terai S, Okita K. Gadolinium chloride reverses dimethylnitrosamine (DMN)-induced rat liver fibrosis with increased matrix metalloproteinases (MMPs) of Kupffer cells. Life Sci 2003; 72:943-59. [PMID: 12493575 DOI: 10.1016/s0024-3205(02)02342-1] [Citation(s) in RCA: 38] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
The aim of this study was to investigate whether matrix metalloproteinases (MMP-13, 9) of Kupffer cells induced by gadolinium chloride (GdCl(3)) treatment can reverse dimethylnitrosamine (DMN)-induced liver fibrosis (in vivo) and the effect of GdCl(3) on MAP kinase activity (in vitro). Male Wistar rats 6 weeks of age received DMN (10 mg/kg) three successive days a week for 4 weeks. Then two groups of rats (n = 6 each) were treated twice weekly with either GdCl(3) (7 mg/kg) or saline solution intravenously for the next 4 weeks. Animals were sacrificed to estimate the degree of liver fibrosis. Isolated Kuppfer cells were treated with GdCl(3) and the expressions of MMPs, MAP kinase activity (ERK, SAPK/JNK, P38) as well as apoptosis were also examined. Rats that received DMN for 4 weeks followed by GdCl(3) injection for 4 weeks showed an reduced liver hydroxyproline content compared to rats treated with DEN followed by saline (277 +/- 22 VS 348 +/- 34 microg/g, n = 6 each, P < 0.01). There were significantly increased MMP-13 mRNA levels in GdCl(3)-treated rats. However, no significant change was observed in procollagen type I mRNA levels. Isolated Kuppfer cells treated with GdCl(3) showed increased MAP kinase activity, especially P38 pathway as well as MMP-13, 9 mRNA and type I collagen-degrading activity leading to apoptosis. SB203580, inhibitor of P38 pathway diminished these activation and prevented apoptosis. These results suggest that Kuppfer cells can reverse liver fibrosis via the expression of MMPs mainly through P38 pathway.
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Affiliation(s)
- Isao Sakaida
- Department of Gastroenterology and Hepatology School of Medicine, Yamaguchi University, Yamaguchi-Pref. 755-8505, Ube, Japan.
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Abstract
Liver fibrosis represents a major worldwide healthcare burden. Current therapy is limited to removing the causal agent. This approach is successful in some diseases; particularly haemochromatosis and chronic viral hepatitis. However, for many patients treatment is not possible, while other patients present to medical attention at an advanced stage of fibrosis. There is therefore a great need for novel therapies for liver fibrosis. The hepatic stellate cell has been recognised to be responsible for most of the excess extracellular matrix observed in chronic liver fibrosis. The detailed understanding of hepatic stellate cell biology has allowed the rational design of novel antifibrotic therapies. This review describes for the general reader the novel emerging therapies for liver fibrosis.
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Affiliation(s)
- Frank Murphy
- Liver Research Group, Division of Infection, Inflammation & Repair, University of Southampton, Southampton General Hospital, Southampton, SO16 6YD, UK.
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Nie QH, Cheng YQ, Xie YM, Zhou YX, Bai XG, Cao YZ. Methodologic research on TIMP-1, TIMP-2 detection as a new diagnostic index for hepatic fibrosis and its significance. World J Gastroenterol 2002; 8:282-7. [PMID: 11925608 PMCID: PMC4658367 DOI: 10.3748/wjg.v8.i2.282] [Citation(s) in RCA: 19] [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: 11/02/2001] [Revised: 01/07/2002] [Accepted: 01/15/2002] [Indexed: 02/06/2023] Open
Abstract
AIM To set up a new method to detect tissue inhibitors of metalloproteinase-1 and -2(TIMP-1 and TIMP-2) in sera of patients with hepatic cirrhosis, and to investigate the expression and location of TIMP-1 and TIMP-2 in liver tissue of patients with hepatic cirrhosis, and the correlation between TIMPs in liver and those in sera so as to discuss whether TIMPs can be used as a diagnosis index of hepatic fibrosis. METHODS The monoclonal antibodies (McAbs) of TIMP-1 and TIMP-2 were used to sensitize erythrocytes, and solid-phase absorption to sensitized erythrocytes (SPASE) was used to detect TIMP-1 and TIMP-2 in the sera of patients with hepatic cirrhosis. Meanwhile, with the method of in situ hybridization and immunohistochemistry, we studied the mRNA expression and antigen location of TIMP-1 and TIMP-2 in the livers of 40 hepatic cirrhosis patients with pathologic diagnosis. RESULTS With SPASE, they were 16.4% higher in the acute hepatitis group, 33.3% higher in the chronic hepatitis group, and the positive rates were 73.6% and 61.2% respectively in sera of hepatic cirrhosis patients, which were remarkably higher than those in chronic hepatitis and acute hepatitis group (P<0.001). In 40 samples of hepatic cirrhosis tissues, all of them showed positive expression of TIMP-1 and TIMP-2 mRNA detected with immunohistochemistry or in situ hybridization (positive rate was 100%). Expression of TIMPs in different degrees could be found in liver tissue with cirrhosis. TIMPs were located in cytoplasm of liver cells of patients with hepatic cirrhosis. There was a significant correlation between serum TIMPs level and liver TIMPs level. CONCLUSION SPASE is a useful method to detect the TIMP-1 and TIMP-2 in sera of patients with hepatic cirrhosis, and TIMP-1 and TIMP-2 can be considered as a useful diagnostic index of hepatic fibrosis, especially TIMP-1.
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Affiliation(s)
- Qing-He Nie
- The Centre of Infectious Disease Diagnosis and Treatment of Chinese PLA, Tangdu Hospital, Fourth Military Medical University, Xi'an 710038, Shaanxi Province, China.
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Nie QH, Cheng YQ, Xie YM, Zhou YX, Cao YZ. Inhibiting effect of antisense oligonucleotides phosphorthioate on gene expression of TIMP-1 in rat liver fibrosis. World J Gastroenterol 2001; 7:363-9. [PMID: 11819791 PMCID: PMC4688723 DOI: 10.3748/wjg.v7.i3.363] [Citation(s) in RCA: 48] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/11/2001] [Revised: 02/03/2001] [Accepted: 02/12/2001] [Indexed: 02/06/2023] Open
Abstract
AIM To observe the inhibition of antisense oligonucleotides (asON) phosphorthioate to the tissue inhibitors metalloproteinase-1 (TIMP-1) gene and protein expression in the liver tissue of immunologically induced hepatic fibrosis rats. The possibility of reversing hepatic fibrosis through gene therapy was observed. METHODS Human serum albumin (HSA) was used to attack rats, as hepatic fibrosis model, in which asONs were used to block the gene and protein expressing TIMP-1. According to the analysis of modulator, structure protein, coding series of TIMP-1 genome, we designed four different asONs. These asONs were injected into the hepatic fibrosis models through coccygeal vein. The results was observed by RT-PCR for measuring TIMP-1 mRNA expression, immunohistochemistry and in situ hybridization for collagen I, II, special staining of collagen fiber, and electron microscopic examination. RESULTS Hepatic fibrosis could last within 363 days in our modified model. The expressing level of TIMP-1 was high during hepatic fibrosis process. It has been proved by the immunohistochemical and the electron microscopic examination that the asON phosphorthioate of TIMP-1 could exactly express in vivo. The effect of colchicine was demonstrated to inhibit the expressing level of mRNA and the content of collagen I, III in the liver of experimental hepatic fibrosis rats. However, the electron microscopy research and the pathologic grading of hepatic fibrosis showed that there was no significant difference between the treatment group and the model group (P> 0.05). CONCLUSION The experimental rat model of hepatic fibrosis is one of the preferable models to estimate the curative effect of anti-hepatic fibrosis drugs. The asON phosphorthioate of TIMP-1 could block the gene and protein expression of TIMP-1 in the liver of experimental hepatic fibrosis rats at the mRNA level. It is possible to reverse hepatic fibrosis, and it is expected to study a new drug of antihepatic fibrosis on the genetic level. Colchicine has very limited therapeutic effect on hepatic fibrosis, furthermore, its toxicity and side effects are obvious.
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Affiliation(s)
- Q H Nie
- The Center of Infectious Disease Diagnosis and Treatment of PLA, Tangdu Hospital, Forth Military Medical University, Xi'an 710038, Shaanxi Province, China.
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Sassi S, Sweetinburgh M, Erogul J, Zhang P, Teng-Umnuay P, West CM. Analysis of Skp1 glycosylation and nuclear enrichment in Dictyostelium. Glycobiology 2001; 11:283-95. [PMID: 11358877 DOI: 10.1093/glycob/11.4.283] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Skp1 is a subunit of SCF-E3 ubiquitin ligases and other protein complexes in the nucleus and cytoplasm of yeast and mammalian cells. In Dictyostelium, Skp1 is partially modified by an unusual pentasaccharide O-linked to hydroxyproline143. This modification was found to be susceptible to known prolyl hydroxylase inhibitors based on M(r)-shift analysis using SDS-polyacrylamide gel electrophoresis/Western blotting. In addition, Dictyostelium Skp1 consists of 2 genetic isoforms, Skp1A and Skp1B, which differ by a single amino acid and appear to be expressed throughout the life cycle based on reverse-transcription polymerase chain reactions. The significance of these structural variations was examined by expressing myc-tagged Skp1s and mutants that lacked the glycosylation site. Gel-based M(r)-shift studies showed that Skp1A and Skp1B are both nearly completely glycosylated during growth and early development, and mass spectrometry of glycopeptides showed that they were glycosylated similarly. Skp1 expressed later in prespore cells was not glycosylated, unlike bulk Skp1 persisting from earlier in development, but became glycosylated after return to growth medium. Skp1A and Skp1B were each concentrated in the nucleus and regions of the cytoplasm, based on immunofluorescence localization. However, when Skp1 glycosylation was blocked by mutation, prolyl hydroxylase inhibitors, or expression in prespore cells, nuclear concentration of Skp1 was not detected. Furthermore, nuclear concentration occurred in a mutant that attached only the core disaccharide to Skp1. Overall, there was no evidence for differential Skp1 isoform expression, glycosylation variants in the bulk Skp1 pool, or regulation of nuclear localization. However, these studies uncovered evidence that the glycosylation pathway is developmentally regulated and can function posttranslationally, and that core glycosylation is required for Skp1's nuclear concentration.
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Affiliation(s)
- S Sassi
- Department of Anatomy and Cell Biology, Box 100235, 1600 SW Archer Road, University of Florida College of Medicine, Gainesville FL 32610-0235, USA
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Beken S, De Smet K, Depreter M, Roels F, Vercruysse A, Rogiers V. Effects of L-proline on phase I and phase II xenobiotic biotransformation capacities of rat and human hepatocytes in long-term collagen gel cultures. Altern Lab Anim 2001; 29:35-53. [PMID: 11178573 DOI: 10.1177/026119290102900102] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
L-Proline supplementation of the medium for collagen gel cultures of hepatocytes has been shown to improve albumin secretion. A study was made as to whether L-proline is also essential for the maintenance of xenobiotic biotransformation capacities in collagen gel sandwich and immobilisation cultures of rat and human hepatocytes. Key phase I (cytochrome P450-dependent monooxygenase [CYP)] and microsomal epoxide hydrase [mEH]) and phase II (glutathione S-transferase [GST]) biotransformation enzyme activities and the secretion of albumin in the culture medium were assessed in the absence and presence of L-proline. CYP and mEH activities were not affected by the addition of L-proline, whereas phase II alpha-Class GST activity of rat hepatocytes in collagen cultures was decreased. Species differences were demonstrated, as human hepatocytes showed a better maintenance of GST activities than their rat counterparts in the presence of L-proline. Albumin secretion, often considered to be a marker for differentiated cell function, does not parallel the biotransformation capacities of the hepatocytes in culture. Additional results demonstrated an L-proline-mediated enhancement of the proliferation rate of contaminating stellate cells in conventional monolayer culture. Transdifferentiation of stellate cells to proliferating myofibroblasts, along with an increased albumin secretion and collagen synthesis, are characteristic of fibrotic liver. Since the last two phenomena have been observed in L-proline-supplemented collagen gel cultures, it can be concluded that when stable collagen gel cultures of rat hepatocytes are needed for long-term pharmacotoxicological studies, it is preferable to use an L-proline-free culture medium. Further studies on medium optimisation are required for hepatocytes from species other than rat.
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Affiliation(s)
- S Beken
- Department of Toxicology (FAFY), Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium
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Rockey DC. The cell and molecular biology of hepatic fibrogenesis. Clinical and therapeutic implications. Clin Liver Dis 2000; 4:319-55. [PMID: 11232195 DOI: 10.1016/s1089-3261(05)70113-6] [Citation(s) in RCA: 47] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Much has been learned in the past 2 decades about the cellular and molecular mechanisms underlying hepatic fibrogenesis and about potential therapeutic approaches in patients with liver disease. The central event in fibrogenesis seems to be the activation of hepatic stellate cells. Stellate cell activation is characterized by several important features, including enhanced matrix synthesis and a prominent contractile phenotype, processes that probably contribute to the physical distortion and dysfunction of the liver in advanced disease. It is important to emphasize that the factors controlling activation are multifactorial and complex. The extracellular matrix is a dynamic, active constituent of the fibrogenic response and undergoes active remodeling, including synthesis and degradation. Effective therapy for hepatic fibrogenesis will probably also be multifactorial, based on the basic mechanisms underlying the fibrogenic process. The most effective therapies will probably be directed at the stellate cell. Approaches that address matrix remodeling (i.e., by enhancing matrix degradation or by inhibiting factors that prevent matrix breakdown) may be effective.
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Affiliation(s)
- D C Rockey
- Departments of Medicine and Cell Biology, Liver Center, Duke University Medical Center, Durham, North Carolina, USA.
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Hironaka K, Sakaida I, Matsumura Y, Kaino S, Miyamoto K, Okita K. Enhanced interstitial collagenase (matrix metalloproteinase-13) production of Kupffer cell by gadolinium chloride prevents pig serum-induced rat liver fibrosis. Biochem Biophys Res Commun 2000; 267:290-5. [PMID: 10623612 DOI: 10.1006/bbrc.1999.1910] [Citation(s) in RCA: 58] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Hepatic fibrosis results from an imbalance between fibrogenesis and fibrolysis in the liver. It remains uninvestigated whether Kupffer cells produce matrix metalloproteinase-13 (MMP-13), which mainly hydrolyzes extracellular matrix (ECM). We sought to determine the role of Kupffer cells in fibrogenesis/fibrolysis. In vivo, we used the rat model of pig serum-induced liver fibrosis. A subset was treated with gadolinium chloride (GdCl(3)), which specifically acts on Kupffer cells. Administration of GdCl(3) remarkably decreased the hydroxyproline content of the liver and increased the expression of MMP-13 mRNA in the liver without a difference in procollagen type I and tissue inhibitors of metalloproteinase-1 (TIMP-1) mRNA expression on Northern blot analysis with the elimination of ED2-positive cells. In vitro, addition of GdCl(3) to isolated Kupffer cells showed increased type I collagen-degrading activity in a dose-dependent manner as well as MMP-13 mRNA expression on Northern blot analysis. It is concluded that Kupffer cells are a major source of MMP-13 and modulation of Kupffer cells by GdCl(3) prevents liver fibrosis with increased expression of MMP-13 mRNA and protein, whereas procollagen type I and TIMP-1 mRNA, which encode two major effectors of fibrogenesis, were unchanged. This is the first report showing that Kupffer cells produce interstitial collagenase (MMP-13) resulting in the reduction of ECM. This discovery may provide new insights into therapy for hepatic fibrosis.
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Affiliation(s)
- K Hironaka
- School of Medicine, Yamaguchi University, Ube, Yamaguchi Prefecture, 755-8505, Japan
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