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Balaphas A, Meyer J, Gameiro C, Frobert A, Giraud MN, Egger B, Bühler LH, Gonelle-Gispert C. Optimized Isolation and Characterization of C57BL/6 Mouse Hepatic Stellate Cells. Cells 2022; 11:1379. [PMID: 35563686 PMCID: PMC9102395 DOI: 10.3390/cells11091379] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2022] [Revised: 03/17/2022] [Accepted: 03/29/2022] [Indexed: 02/04/2023] Open
Abstract
To obtain meaningful results of hepatic stellate cell (HSC) function, it is crucial to use highly pure HSC populations. Our aim was to optimize HSC isolation from mice livers without exploiting the characteristically transient vitamin A autofluorescence of HSC. HSCs were isolated from C57BL/6 mice using a two-step collagenase digestion and Nycodenz gradient separation followed by CD11b-negative sorting step in order to remove contaminating macrophages and dendritic cells. Isolated cells were analyzed for yield, viability, purity, and potential new markers using immunofluorescence and flow cytometry. We obtained a yield of 350,595 ± 100,773 HSC per mouse liver and a viability of isolated cells of 92.4 ± 3.1%. We observed a low macrophage/dendritic cell contamination of 1.22 ± 0.54%. Using flow cytometry, we demonstrated that CD38 was expressed at the surface of HSC subpopulations and that all expressed intracellular markers specific for HSC in the liver. This isolation method, avoiding fluorescent activated cell sorting (FACS), allowed isolation of HSCs with high purity. Further, flow cytometry analysis suggests that CD38 may be a reliable marker of HSCs and may include subpopulations of HSCs without retinoid droplets.
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Affiliation(s)
- Alexandre Balaphas
- Division of Digestive Surgery, University Hospitals of Geneva, 1205 Geneva, Switzerland; (A.B.); (J.M.)
- Department of Surgery, Clinical Medicine Section, Faculty of Medicine, University of Geneva, 1206 Geneva, Switzerland
| | - Jeremy Meyer
- Division of Digestive Surgery, University Hospitals of Geneva, 1205 Geneva, Switzerland; (A.B.); (J.M.)
| | - Cécile Gameiro
- Flow Cytometry Core Facility, Faculty of Medicine, University of Geneva, 1206 Geneva, Switzerland;
| | - Aurélien Frobert
- Surgical Research Unit, Faculty of Science and Medicine, Section of Medicine, University of Fribourg, 1700 Fribourg, Switzerland; (A.F.); (M.-N.G.); (B.E.); (L.H.B.)
| | - Marie-Noëlle Giraud
- Surgical Research Unit, Faculty of Science and Medicine, Section of Medicine, University of Fribourg, 1700 Fribourg, Switzerland; (A.F.); (M.-N.G.); (B.E.); (L.H.B.)
| | - Bernhard Egger
- Surgical Research Unit, Faculty of Science and Medicine, Section of Medicine, University of Fribourg, 1700 Fribourg, Switzerland; (A.F.); (M.-N.G.); (B.E.); (L.H.B.)
| | - Leo H. Bühler
- Surgical Research Unit, Faculty of Science and Medicine, Section of Medicine, University of Fribourg, 1700 Fribourg, Switzerland; (A.F.); (M.-N.G.); (B.E.); (L.H.B.)
| | - Carmen Gonelle-Gispert
- Surgical Research Unit, Faculty of Science and Medicine, Section of Medicine, University of Fribourg, 1700 Fribourg, Switzerland; (A.F.); (M.-N.G.); (B.E.); (L.H.B.)
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Shang L, Hosseini M, Liu X, Kisseleva T, Brenner DA. Human hepatic stellate cell isolation and characterization. J Gastroenterol 2018; 53:6-17. [PMID: 29094206 DOI: 10.1007/s00535-017-1404-4] [Citation(s) in RCA: 91] [Impact Index Per Article: 13.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/15/2017] [Accepted: 09/22/2017] [Indexed: 02/04/2023]
Abstract
The hepatic stellate cells (HSCs) localize at the space of Disse in the liver and have multiple functions. They are identified as the major contributor to hepatic fibrosis. Significant understanding of HSCs has been achieved using rodent models and isolated murine HSCs; as well as investigating human liver tissues and human HSCs. There is growing interest and need of translating rodent study findings to human HSCs and human liver diseases. However, species-related differences impose challenges on the translational research. In this review, we focus on the current information on human HSCs isolation methods, human HSCs markers, and established human HSC cell lines.
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Affiliation(s)
- Linshan Shang
- Department of Medicine, University of California, San Diego, La Jolla, USA
| | - Mojgan Hosseini
- Department of Pathology, University of California, San Diego, La Jolla, USA
| | - Xiao Liu
- Department of Surgery, University of California, San Diego, La Jolla, USA
| | - Tatiana Kisseleva
- Department of Surgery, University of California, San Diego, La Jolla, USA
| | - David Allen Brenner
- Department of Medicine, University of California, San Diego, La Jolla, USA.
- School of Medicine, UC San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0602, USA.
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Avolio E, Alvino VV, Ghorbel MT, Campagnolo P. Perivascular cells and tissue engineering: Current applications and untapped potential. Pharmacol Ther 2016; 171:83-92. [PMID: 27889329 PMCID: PMC5345698 DOI: 10.1016/j.pharmthera.2016.11.002] [Citation(s) in RCA: 55] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023]
Abstract
The recent development of tissue engineering provides exciting new perspectives for the replacement of failing organs and the repair of damaged tissues. Perivascular cells, including vascular smooth muscle cells, pericytes and other tissue specific populations residing around blood vessels, have been isolated from many organs and are known to participate to the in situ repair process and angiogenesis. Their potential has been harnessed for cell therapy of numerous pathologies; however, in this Review we will discuss the potential of perivascular cells in the development of tissue engineering solutions for healthcare. We will examine their application in the engineering of vascular grafts, cardiac patches and bone substitutes as well as other tissue engineering applications and we will focus on their extensive use in the vascularization of engineered constructs. Additionally, we will discuss the emerging potential of human pericytes for the development of efficient, vascularized and non-immunogenic engineered constructs.
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Affiliation(s)
- Elisa Avolio
- Division of Experimental Cardiovascular Medicine, Bristol Heart Institute, University of Bristol, United Kingdom
| | - Valeria V Alvino
- Division of Experimental Cardiovascular Medicine, Bristol Heart Institute, University of Bristol, United Kingdom
| | - Mohamed T Ghorbel
- Division of Congenital Heart Surgery, Bristol Heart Institute, University of Bristol, United Kingdom
| | - Paola Campagnolo
- School of Biosciences and Medicine, University of Surrey, Guildford, United Kingdom.
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Selimovic D, El-Khattouti A, Ghozlan H, Haikel Y, Abdelkader O, Hassan M. Hepatitis C virus-related hepatocellular carcinoma: An insight into molecular mechanisms and therapeutic strategies. World J Hepatol 2012; 4:342-55. [PMID: 23355912 PMCID: PMC3554798 DOI: 10.4254/wjh.v4.i12.342] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/21/2012] [Revised: 11/17/2012] [Accepted: 11/24/2012] [Indexed: 02/06/2023] Open
Abstract
Hepatitis C virus (HCV) infects more than 170 million people worldwide, and thereby becomes a series global health challenge. Chronic infection with HCV is considered one of the major causes of end-stage liver disease including cirrhosis and hepatocellular carcinoma. Although the multiple functions of the HCV proteins and their impacts on the modulation of the intracellular signaling transduction processes, the drive of carcinogenesis during the infection with HCV, is thought to result from the interactions of viral proteins with host cell proteins. Thus, the induction of mutator phenotype, in liver, by the expression of HCV proteins provides a key mechanism for the development of HCV-associated hepatocellular carcinoma (HCC). HCC is considered one of the most common malignancies worldwide with increasing incidence during the past decades. In many countries, the trend of HCC is attributed to several liver diseases including HCV infection. However, the development of HCC is very complicated and results mainly from the imbalance between tumor suppressor genes and oncogenes, as well as from the alteration of cellular factors leading to a genomic instability. Besides the poor prognosis of HCC patients, this type of tumor is quite resistance to the available therapies. Thus, understanding the molecular mechanisms, which are implicated in the development of HCC during the course of HCV infection, may help to design a general therapeutic protocol for the treatment and/or the prevention of this malignancy. This review summarizes the current knowledge of the molecular mechanisms, which are involved in the development of HCV-associated HCC and the possible therapeutic strategies.
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Affiliation(s)
- Denis Selimovic
- Denis Selimovic, Youssef Haikel, Mohamed Hassan, Institut National de la Santé et de la Recherche Médicale, U 977, 67000 Strasbourg, France
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Marinho CC, Bretas T, Voieta I, Queiroz LCD, Ruiz-Guevara R, Teixeira AL, Antunes CM, Prata A, Lambertucci JR. Serum hyaluronan and collagen IV as non-invasive markers of liver fibrosis in patients from an endemic area for schistosomiasis mansoni: a field-based study in Brazil. Mem Inst Oswaldo Cruz 2010; 105:471-8. [DOI: 10.1590/s0074-02762010000400020] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2009] [Accepted: 09/04/2009] [Indexed: 12/27/2022] Open
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Bae MA, Rhee SD, Jung WH, Ahn JH, Song BJ, Cheon HG. Selective inhibition of activated stellate cells and protection from carbon tetrachloride-induced liver injury in rats by a new PPARgamma agonist KR62776. Arch Pharm Res 2010; 33:433-42. [PMID: 20361309 PMCID: PMC3835440 DOI: 10.1007/s12272-010-0313-3] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2009] [Revised: 11/23/2009] [Accepted: 01/07/2010] [Indexed: 12/23/2022]
Abstract
Activated hepatic stellate cells (HSC) are the primary source of extracellular matrix proteins found in liver fibrosis/cirrhosis patients. Therefore, the prevention of HSC activation is an important strategy for treating severe liver injury. This study examined the effects of KR62776, a new peroxisome proliferator-activated receptor gamma (PPARgamma) agonist, on the rate of cell proliferation and expression of alpha-smooth muscle actin (alpha-SMA) in rat hepatic stellate HSC-T6 cells. In addition, its effects on the liver damage induced by carbon tetrachloride were investigated. KR62776 caused the apoptosis of activated HSC-T6 cells with the concomitant decrease in the alpha-smooth muscle actin levels in a time- and concentration-dependent manner. However, KR62776 did not cause the apoptosis of human HepG2 and rat McARH7777 hepatoma cells, suggesting that KR62776 has a specific effect on stellate cells. KR62776 increased the levels of Gadd45, p27, p21 and PPARgamma proteins but decreased the cell cyclerelated proteins, such as cdk2, cyclin B and cyclin D1. These changes were reversed by BADGE, a specific PPARgamma antagonist, indicating that the effects of KR62776 are, at least in part, PPARgamma-dependent. In addition, KR62776 administration showed some protection against carbon tetrachloride-induced hepatocellular damage in rats. Overall, these results suggest that KR62776 may have potential in the chemoprevention of liver fibrosis/cirrhosis.
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Affiliation(s)
- Myung-Ae Bae
- Drug Discovery Platform Technology Team, Medicinal Science Division, Korea Research Institute of Chemical Technology, Daejon, 305-600, Korea.
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Abstract
In this chapter we describe a reliable and reproducible method for the selective propagation and culture of renal fibroblasts derived from explantation of renal cortical tissue in vitro. The chapter outlines how primary renal interstitial fibroblasts are derived from explants grown in medium supplemented with foetal calf serum. The subculture of confluent cells and their ultimate characterisation as fibroblasts through immunohistochemical and immunocytochemical techniques are described in detail.
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Affiliation(s)
- Lauren Grimwood
- Department of Nephrology, The Royal Melbourne Hospital, Melbourne, Victoria, Australia
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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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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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Herrmann J, Gressner AM, Weiskirchen R. Immortal hepatic stellate cell lines: useful tools to study hepatic stellate cell biology and function? J Cell Mol Med 2007; 11:704-22. [PMID: 17760834 PMCID: PMC3823251 DOI: 10.1111/j.1582-4934.2007.00060.x] [Citation(s) in RCA: 85] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
At the cellular level, the activation and transdifferentiation of quiescent hepatic stellate cells (HSC) into myofibroblasts is the key process involved in hepatic fibrogenesis that is associated with an increased and altered deposition of extracellular matrix components in the liver. The temporal sequence of molecular events associated with stellate cell activation turned out to be appropriately mimicked when HSC isolated from normal livers are cultured on uncoated plastic surface. Therefore, cultured primary cells isolated from rodents and human beings are common in vitro models in investigations addressing these issues of hepatic stellate biology and function. However, the limited supply, cost-effective isolation procedure and the ever growing need have resulted in efforts to establish immortalized stellate cell lines having the advantage of virtually unlimited access. They allow rapid screening for disease-associated factors and restrict the necessary number of animal experiments. From the first description of an immortal HSC line in 1986, a huge number of studies were conducted with these established cell lines. However, differences in morphology, growth characteristics and anomalies of chromosome number and structure make the applications of these models questionable. Here, we summarize the history and cellular characteristics of respective cell lines and discuss the differences of continuous HSC lines and their primary counterparts.
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Affiliation(s)
- Jens Herrmann
- *Correspondence to: Prof. Dr R. WEISKIRCHEN Institute of Clinical Chemistry and Pathobiochemistry, RWTH University Hospital, D-52074 Aachen, Germany. Tel.: +49 24 1 80 88 68 3 Fax: +49 24 1 80 82 5 12 E-mail:
| | | | - Ralf Weiskirchen
- *Correspondence to: Prof. Dr R. WEISKIRCHEN Institute of Clinical Chemistry and Pathobiochemistry, RWTH University Hospital, D-52074 Aachen, Germany. Tel.: +49 24 1 80 88 68 3 Fax: +49 24 1 80 82 5 12 E-mail:
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Taras D, Blanc JF, Rullier A, Dugot-Senant N, Laurendeau I, Vidaud M, Rosenbaum J. Pravastatin reduces lung metastasis of rat hepatocellular carcinoma via a coordinated decrease of MMP expression and activity. J Hepatol 2007; 46:69-76. [PMID: 16935385 DOI: 10.1016/j.jhep.2006.06.015] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/02/2006] [Revised: 05/10/2006] [Accepted: 06/05/2006] [Indexed: 12/04/2022]
Abstract
BACKGROUND/AIMS Statins have beneficial effects in early pre-clinical models of hepatocellular carcinoma (HCC). Our aim was to test the efficacy of pravastatin on the progression of established HCC in rat, and to study its mechanisms. METHODS HCC was induced with diethylnitrosamine and N-nitrosomorpholine. After 14 weeks, all rats developed HCC and then received pravastatin or its solvent for 10 weeks (10 rats/group). RESULTS Liver tumor mass was lower in pravastatin group (PG) than control group (CG), as estimated from the number of liver tumors (p<0.004) and the liver weight/body weight ratio (p<0.04). Every CG rat surviving at 24 weeks (4/4) had lung metastasis, against only 5/8 in PG. Moreover, the percentage of lung surface occupied by metastasis was 10-fold smaller in PG than CG (p<0.016). Pravastatin decreased liver matrix metalloproteinase (MMP)-9 activity and mostly suppressed MMP-2 activation (p<0.004), likely because it decreased expression of MMP-14 and tissue inhibitor of matrix metalloproteinases-2 (p<0.01), required for MMP-2 activation. CONCLUSIONS Pravastatin reduces progression and limits metastatic diffusion of established HCC. This could be linked to the decreased MMP activity. These results, obtained in a very aggressive HCC model, further suggest the potential benefit of statins in human HCC.
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Si-Tayeb K, Monvoisin A, Mazzocco C, Lepreux S, Decossas M, Cubel G, Taras D, Blanc JF, Robinson DR, Rosenbaum J. Matrix metalloproteinase 3 is present in the cell nucleus and is involved in apoptosis. THE AMERICAN JOURNAL OF PATHOLOGY 2006; 169:1390-401. [PMID: 17003494 PMCID: PMC1780186 DOI: 10.2353/ajpath.2006.060005] [Citation(s) in RCA: 132] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Matrix metalloproteinase (MMP)-3 is a protease involved in cancer progression and tissue remodeling. Using immunofluorescence and immunoelectron microscopy, we identified nuclear localization of MMP-3 in several cultured cell types and in human liver tissue sections. Western blot analysis of nuclear extracts revealed two immunoreactive forms of MMP-3 at 35 and 45 kd, with the 35-kd form exhibiting caseinolytic activity. By transient transfection, we expressed active MMP-3 fused to the enhanced green fluorescent protein (EGFP/aMMP-3) in Chinese hamster ovary cells. We showed that EGFP/aMMP-3 translocates into the nucleus. A functional nuclear localization signal was demonstrated by the loss of nuclear translocation after site-directed mutagenesis of a putative nuclear localization signal and by the ability of the MMP-3 nuclear localization signal to drive a heterologous protein into the nucleus. Finally, expression by Chinese hamster ovary cells of EGFP/aMMP-3 induced a twofold increase of apoptosis rate, compared with EGFP/pro-MMP-3, which does not translocate to the nucleus. Increased apoptosis was abolished by site-directed mutagenesis of the catalytic site of MMP-3 or by using the MMP inhibitor GM6001. This study elucidates for the first time the mechanisms of nuclear localization of a MMP and shows that nuclear MMP-3 can induce apoptosis via its catalytic activity.
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Affiliation(s)
- Karim Si-Tayeb
- INSERM E362, Université Victor Segalen Bordeaux 2, 33076 Bordeaux, France
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Bandapalli OR, Geheeb M, Kobelt D, Kuehnle K, Elezkurtaj S, Herrmann J, Gressner AM, Weiskirchen R, Beule D, Blüthgen N, Herzel H, Franke C, Brand K. Global analysis of host tissue gene expression in the invasive front of colorectal liver metastases. Int J Cancer 2005; 118:74-89. [PMID: 16080196 DOI: 10.1002/ijc.21307] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Host cell reactions are a crucial determinant for tumor invasion. We analyzed on a genomewide scale gene expression differences between microdissected tissues taken from unaffected liver tissue of a human colorectal tumor (LS174) growing in the livers of nude mice and tissue from the host part of the invasive front. Due to the low degree of interspecies cross-hybridization of 15% as determined on Affymetrix microarrays, our xenograft model allowed for the distinction of genes of murine versus human origin even if the respective tissues could not be isolated separately. Using the gene ontology (GO) classification, we were able to determine patterns of up- and downregulated genes in the liver part of the invasive front. We observed a pronounced overrepresentation, e.g., of the GO terms "extracellular matrix," "cell communication," "response to biotic stimulus," "structural molecule activity" and "cell growth," indicating a very pronounced host cell response to tumor invasion. On the single gene level, hepatic stellate cell (HSC) activation markers were overrepresented in the liver part of the invasion front. Immunohistochemistry and qPCR confirmed an activation of HSC as well as an increased number of HSC in the invasive front as compared to the noninvaded liver tissue. In summary, our data demonstrate the feasibility of an interspecies differential gene expression approach on a genomewide scale.
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Affiliation(s)
- Obul Reddy Bandapalli
- Institute of Biology, Humboldt University Berlin, Max Delbrück Center for Molecular Medicine, Berlin, Germany
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Lorena D, Darby IA, Reinhardt DP, Sapin V, Rosenbaum J, Desmoulière A. Fibrillin-1 expression in normal and fibrotic rat liver and in cultured hepatic fibroblastic cells: modulation by mechanical stress and role in cell adhesion. J Transl Med 2004; 84:203-12. [PMID: 14661032 DOI: 10.1038/labinvest.3700023] [Citation(s) in RCA: 61] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023] Open
Abstract
Fibrillin-1, together with elastin, is the main component of elastic fibers found throughout the extracellular space and responsible for the biomechanical properties of most tissues and organs. In this work, fibrillin-1 expression and modulation were explored in experimental rat liver fibrosis and in vitro; furthermore, the role of fibrillin-1 fragments on cell adhesion was analyzed. Fibrosis was induced by subjecting rats to common bile duct ligation for 72 h and 7 days or carbon tetrachloride (CCl(4)) treatment for 2 and 6 weeks. Immunohistochemistry showed that, after bile duct ligation, fibrillin-1, elastin, and alpha-smooth muscle actin colocalized in the developing portal connective tissue. In CCl(4)-treated animals, a similar colocalization was observed in septa; however, elastin deposition was not observed around activated alpha-smooth muscle actin-positive stellate cells of the parenchyma. Treatment with the profibrogenic mediator transforming growth factor-beta1 (TGF-beta1) greatly increased the fibrillin-1 expression of cultured liver fibroblasts. The level of fibrillin-1 expression was significantly higher in cells grown in restrained (stressed) collagen lattices compared with those grown in unrestrained collagen lattices. Cell adhesion on the C-terminal fragment of fibrillin-1 containing the RGD sequence (rF6H) slightly increased (between 0.3 and 2.5 microg/ml) and decreased at higher concentrations, while adhesion on the N-terminal fragment of fibrillin-1 (rF16) was dose-dependently decreased. In addition, the rF16 fragment decreased cell adhesion to fibronectin. In conclusion, our study illustrates the important deposition of fibrillin-1 that occurs in two mechanistically distinct settings of liver fibrogenesis. Furthermore, the induction of fibrillin-1 expression by TGF-beta1 and mechanical stress, and the antiadhesive properties of fibrillin-1 fragments suggest important implications for physiological and pathological fibrillin-1 catabolism during tissue remodeling.
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Affiliation(s)
- Dionne Lorena
- Groupe de Recherches pour l'Etude du Foie, INSERM E0362, and Institut Fédératif de Recherche 66, Pathologies Infectieuses et Cancers, Université Victor Segalen Bordeaux 2, Bordeaux, France
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Neaud V, Duplantier JG, Mazzocco C, Kisiel W, Rosenbaum J. Thrombin up-regulates tissue factor pathway inhibitor-2 synthesis through a cyclooxygenase-2-dependent, epidermal growth factor receptor-independent mechanism. J Biol Chem 2003; 279:5200-6. [PMID: 14623891 DOI: 10.1074/jbc.m306679200] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The serine proteinase inhibitor tissue factor pathway inhibitor-2 (TFPI-2) inhibits the tissue factor-factor VIIa complex and thereby impairs factor Xa and subsequently thrombin generation. Here we show that thrombin itself up-regulates TFPI-2 mRNA and protein expression in human liver myofibroblasts, a cell type shown to express high levels of TFPI-2 (Neaud, V., Hisaka, T., Monvoisin, A., Bedin, C., Balabaud, C., Foster, D. C., Desmoulière, A., Kisiel, W., and Rosenbaum, J. (2000) J. Biol. Chem. 275, 35565-35569). This effect required thrombin catalytic activity, as shown by its abolition with hirudin. Although the thrombin effect could be mimicked by agonists of both protease-activated receptor (PAR)-1 and PAR-4, it was largely blocked by a PAR-1 blocking antibody. Transactivation of the epidermal growth factor (EGF) receptor has been reported as a common event in thrombin signaling. However, thrombin did not detectably transactivate the EGF receptor in liver myofibroblasts, and blocking the EGF receptor did not affect TFPI-2 induction. On the other hand, thrombin increased the expression of cyclooxygenase-2 (COX-2) mRNA via a MAPK-dependent pathway, and a specific COX-2 inhibitor abolished the effect of thrombin on TFPI-2 expression. Thus, thrombin, through PAR-1 signaling, up-regulates the synthesis of TFPI-2 via a MAPK/COX-2-dependent pathway. The up-regulation of TFPI-2 expression by thrombin could in turn down-regulate thrombin generation and contribute to limit blood coagulation.
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Affiliation(s)
- Véronique Neaud
- Groupe de Recherches pour l'Etude du Foie, INSERM E362 and IFR66, Université Victor Segalen Bordeaux 2, 33076 Bordeaux, France
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Tan K, Guibert C, Neaud V, Rosenbaum J. Hepatitis C virus proteins do not directly trigger fibrogenic events in cultured human liver myofibroblasts. J Viral Hepat 2003; 10:427-32. [PMID: 14633175 DOI: 10.1046/j.1365-2893.2003.00460.x] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
Although liver fibrosis is the major complication of hepatitis C virus (HCV) infection, the mechanisms of fibrogenesis in this setting are not completely understood. The aim of this study was to test the direct effect of HCV proteins on signalling- and fibrosis-related events in cultured human liver myofibroblasts, the effector cells of liver fibrogenesis. Cultured myofibroblasts were exposed to recombinant HCV core, a structural protein, and nonstructural proteins (NS) 3, NS 4 and NS 5. HCV proteins did not significantly increase DNA synthesis in myofibroblasts. We then examined if these proteins affected early signalling events. None of the HCV proteins affected the phosphorylation of the mitogen activated protein kinases/extracellular regulated kinases 1 and 2, or of the phosphatidylinositol 3-kinase target, Akt. HCV proteins had also no effect on intracellular calcium concentration. In other experiments, fibrogenesis-related parameters were measured. None of the HCV proteins had any effect on the secretion of type I collagen, tissue inhibitor of matrix metalloproteinases type 1, gelatinase or urokinase. Alpha-smooth muscle actin expression was also not modified. In summary, our experiments do not support a direct effect of these HCV proteins on fibrogenic cells.
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Affiliation(s)
- K Tan
- Groupe de Recherches pour l'Etude du Foie, INSERM E 0362 and IFR 66, Université Victor Segalen Bordeaux 2, Bordeaux, France
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17
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Cheng J, Imanishi H, Liu W, Iwasaki A, Ueki N, Nakamura H, Hada T. Inhibition of the expression of alpha-smooth muscle actin in human hepatic stellate cell line, LI90, by a selective cyclooxygenase 2 inhibitor, NS-398. Biochem Biophys Res Commun 2002; 297:1128-34. [PMID: 12372403 DOI: 10.1016/s0006-291x(02)02301-x] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Cyclooxygenase 2 (COX-2) has been thought to be associated with liver fibrosis whereas it is well known that hepatic stellate cells (HSC) play a central role in the pathogenesis of liver fibrosis. There is little evidence of how COX-2 regulates the activation of human HSC or the mechanism involved. In this study, we investigated the effect of a COX-2 inhibitor, NS-398, on a line of human HSC, LI90. Our findings demonstrated that alpha-smooth muscle actin (alpha-SMA) protein expression was inhibited in a dose-dependent manner by treatment with NS-398. Proliferation cell nuclear antigen (PCNA) expression and cell growth were partially down-regulated. The generation of PGE2, IL-8, IL-6, and hyaluronan in the cultured medium was also inhibited. In conclusion, our findings imply that a selective COX-2 inhibitor might be a potential drug for the chemoprevention and treatment of liver fibrosis by inhibiting the activation of HSC.
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Affiliation(s)
- Jidong Cheng
- Division of Hepatobiliary and Pancreatic Disease, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawacho, Nishinomiya, 663, Hyogo, Japan.
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18
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Chen WX, Li YM, Yu CH, Cai WM, Zheng M, Chen F. Quantitative analysis of transforming growth factor beta 1 mRNA in patients with alcoholic liver disease. World J Gastroenterol 2002; 8:379-381. [PMID: 11925630 PMCID: PMC4658389 DOI: 10.3748/wjg.v8.i2.379] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/28/2001] [Revised: 10/05/2001] [Accepted: 10/12/2001] [Indexed: 02/06/2023] Open
Abstract
AIM To investigate the expression of the transforming growth factor beta 1(TGF-beta 1) mRNA in different stages of alcoholic liver disease (ALD) and its clinical value. METHODS One hundred and seven male alcoholics were grouped by clinical findings into four groups: alcohol abusers without liver impairment (n =22), alcoholic steatosis (n =30); alcoholic hepatitis (n=31); and alcoholic cirrhosis(n=24). Using peripheral blood mononuclear cells (PBMC) as samples the gene expression of TGF-beta 1 was examined quantitatively by reverse transcription polymerase chain reaction (RT-PCR) and dot blot. There are 34 healthy subjects served as control. RESULTS The expression of TGF-beta 1 from all ALD patients was significantly greater than that in controls (1.320 +/- 1.162 vs 0.808 +/- 0.276, P<0.001). The differences of the expressions were significant between the patients from each groups (alcoholic steatosis, alcoholic hepatitis and alcoholic cirrhosis) and the controls (1.168 +/- 0.852, 1.462 +/- 1.657, 1.329 +/- 0.610 vs 0.808 +/- 0.276, P<0.050). No significant differences of TGF -beta 1 mRNA expression were observed between alcohol abusers without liver impairment and controls. The expressions in patients with alcoholic hepatitis and alcoholic cirrhosis were significantly greater than that in alcohol abusers respectively (1.462 +/- 1.657, 1.329 +/- 0.610 vs 0.841 +/- 0.706, P<0.050). No significant differences of TGF-beta 1 mRNA expression were observed between alcoholic fatty liver men and alcohol abusers. CONCLUSION TGF-beta 1 expression level can be a risk factor for alcoholic liver disease and might be related to the inflammatory activity and fibrosis of the liver in patients.
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Affiliation(s)
- Wei-Xing Chen
- Department of Gastroenterology, The First Affiliated Hospital, Medical College of Zhejiang University, Hangzhou 310003, Zhejiang Province, China.
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19
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Monvoisin A, Bisson C, Si-Tayeb K, Balabaud C, Desmoulière A, Rosenbaum J. Involvement of matrix metalloproteinase type-3 in hepatocyte growth factor-induced invasion of human hepatocellular carcinoma cells. Int J Cancer 2002; 97:157-62. [PMID: 11774258 DOI: 10.1002/ijc.1595] [Citation(s) in RCA: 62] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
Intra-hepatic invasion is a key feature of hepatocellular carcinoma (HCC) progression. We have shown that human liver myofibroblasts induce invasion of HCC cells through Matrigel, via the secretion of hepatocyte growth factor (HGF). In our study, we investigated the role of matrix metalloproteinases (MMP) in HGF-induced HCC cells invasion. Marimastat, a synthetic MMP inhibitor, dose-dependently decreased HGF-induced invasion of HepG2 cells with a maximum of 82.7 +/- 13.3% at 20 microM. TIMP-2, a natural inhibitor, decreased invasion up to 51.2 +/- 11.2% at 200 ng/ml. To determine the target for these inhibitors, we examined MMP expression using RT-PCR. MMPs 1, 7-9 and 10 were not expressed in HepG2 cells either in the absence or in the presence of HGF. MMP-2 and MMP-13 transcripts were detected in unstimulated cells but their expression was unchanged after exposition to HGF. MMP-3 transcripts were undetectable in unstimulated HepG2 cells. They became clearly expressed in HGF-stimulated cells, however, and this was confirmed by Northern blot. By Western blot, HGF dose-dependently stimulated the secretion of pro-MMP-3 in the culture medium. The role of MMP-3 in HGF-induced invasion was directly confirmed by using an antibody to MMP-3, that blocked invasion. Finally, RT-PCR demonstrated MMP-3 expression in 10/16 human HCCs tested, but not in normal liver. In conclusion, our data demonstrate that MMPs, most likely MMP-3, mediate HGF-induced invasion of HCC cells. The in vivo expression of MMP-3 in HCC suggests a role for this protease in HCC progression.
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Affiliation(s)
- Arnaud Monvoisin
- Groupe de Recherches pour l'Etude du Foie (GREF), INSERM E9917, Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux cedex, France
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20
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Schneider E, Schmid-Kotsas A, Zhao J, Weidenbach H, Schmid RM, Menke A, Adler G, Waltenberger J, Grünert A, Bachem MG. Identification of mediators stimulating proliferation and matrix synthesis of rat pancreatic stellate cells. Am J Physiol Cell Physiol 2001; 281:C532-43. [PMID: 11443052 DOI: 10.1152/ajpcell.2001.281.2.c532] [Citation(s) in RCA: 174] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The aim of this study was to identify fibrogenic mediators stimulating activation, proliferation, and/or matrix synthesis of rat pancreatic stellate cells (PSC). PSC were isolated from the pancreas of normal Wistar rats and from rats with cerulein pancreatitis. Cell activation was demonstrated by immunofluorescence microscopy of smooth muscle alpha-actin (SMA) and real-time quantitative RT-PCR of SMA, fibronectin, and transforming growth factor (TGF)-beta(1). Proliferation was measured by bromodeoxyuridine incorporation. Matrix synthesis was demonstrated on the protein and mRNA level. Within a few days in primary culture, PSC changed their phenotype from fat-storing to SMA-positive myofibroblast-like cells expressing platelet-derived growth factor (PDGF) alpha- and PDGF beta-receptors. TGF-beta(1) and tumor necrosis factor (TNF)-alpha accelerated the change in the cells' phenotype. Addition of 50 ng/ml PDGF and 5 ng/ml basic fibroblast growth factor (bFGF) to cultured PSC significantly stimulated cell proliferation (4.37 +/- 0.49- and 2.96 +/- 0.39-fold of control). Fibronectin synthesis calculated on the basis of DNA was stimulated by 5 ng/ml bFGF (3.44 +/- 1.13-fold), 5 ng/ml TGF-beta(1) (2.46 +/- 0.89-fold), 20 ng/ml PDGF (2.27 +/- 0.68-fold), and 50 ng/ml TGF-alpha (1.87 +/- 0.19-fold). As shown by RT-PCR, PSC express predominantly the splice variant EIII-A of fibronectin. Immunofluorescence microscopy and Northern blot confirmed that in particular bFGF and TGF-beta(1) stimulated the synthesis of fibronectin and collagens type I and III. In conclusion, our data demonstrate that 1) TGF-beta(1) and TNF-alpha accelerate the change in the cell phenotype, 2) PDGF represents the most effective mitogen, and 3) bFGF, TGF-beta(1), PDGF, and, to a lesser extent, TGF-alpha stimulate extracellular matrix synthesis of cultured rat PSC.
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Affiliation(s)
- E Schneider
- Department of Clinical Chemistry and Pathobiochemistry, University of Ulm, 89070 Ulm, Germany
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21
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Mangasser-Stephan K, Gartung C, Lahme B, Gressner AM. Expression of isoforms and splice variants of the latent transforming growth factor beta binding protein (LTBP) in cultured human liver myofibroblasts. LIVER 2001; 21:105-13. [PMID: 11318979 DOI: 10.1034/j.1600-0676.2001.021002105.x] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
BACKGROUND/AIMS The activation of hepatic stellate cells (HSC) to extracellular matrix (ECM) producing myofibroblasts (MFB) is the key pathogenetic event in human liver fibrogenesis. Latent transforming growth factor beta binding protein (LTBP), a component of the profibrogenic large latent transforming growth factor (TGF)-beta complex, is suggested to be important for secretion, latency, storage and activation of TGF-beta in the ECM. This study was performed to identify the expression profile of all hitherto known LTBP isoforms and LTBP splice variants in conjunction with that of TGF-beta isoforms in cultured human liver MFB. METHODS Cultured human MFB were analyzed for TGF-beta and LTBP using reverse-transcription polymerase chain reaction (RT-PCR), sequence analysis, immunofluorescence staining, metabolic labeling, immunoprecipitation, and enzyme-linked immunosorbent assay (ELISA). RESULTS Transcripts of all three TGF-beta isoforms, of all four LTBP isoforms and of nearly all splice variants of LTBP-1 and LTBP-4 so far known were detected. Metabolic labeling followed by immunoprecipitation with anti-LTBP-1 antibody revealed the synthesis of LTBP proteins. Secretion of free LTBP and LTBP integrated into the large latent TGF-beta complex was demonstrated by size-exclusion chromatography. Co-localization of LTBP-1 and -2 with fibronectin and collagen type I was observed by double immunofluorescence staining. CONCLUSION The expression of a complete profile of hitherto known LTBP proteins by cultured human MFB suggests a role in modulating the bioactivity of TGF-beta in the diseased liver.
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Affiliation(s)
- K Mangasser-Stephan
- Institute of Clinical Chemistry and Pathobiochemistry, Central Laboratory, RWTH-University Hospital, Aachen, Germany
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22
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Neaud V, Hisaka T, Monvoisin A, Bedin C, Balabaud C, Foster DC, Desmoulière A, Kisiel W, Rosenbaum J. Paradoxical pro-invasive effect of the serine proteinase inhibitor tissue factor pathway inhibitor-2 on human hepatocellular carcinoma cells. J Biol Chem 2000; 275:35565-9. [PMID: 10954721 DOI: 10.1074/jbc.m006101200] [Citation(s) in RCA: 37] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
We have previously shown that human liver myofibroblasts promote in vitro invasion of human hepatocellular carcinoma (HCC) cells through a hepatocyte growth factor (HGF)/urokinase/plasmin-dependent mechanism. In this study, we demonstrate that myofibroblasts synthesize the serine proteinase inhibitor tissue factor pathway inhibitor-2 (TFPI-2). Despite the fact that recombinant TFPI-2 readily inhibits plasmin, we show that it potentiates HGF-induced invasion of HCC cells and is capable of inducing invasion on its own. Furthermore, HCC cells stably transfected with a TFPI-2 expression vector became spontaneously invasive. HCC cells express tissue factor and specifically factor VII. Addition of an antibody to factor VII abolished the pro-invasive effect of TFPI-2. We suggest that TFPI-2 induces invasion following binding to a tissue factor-factor VIIa complex preformed on HCC cells. Our data thus demonstrate an original mechanism of cell invasion that may be specific for liver tumor cells.
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Affiliation(s)
- V Neaud
- Groupe de Recherches pour l'Etude du Foie, INSERM E9917, Université Victor Segalen Bordeaux 2, 33076 Bordeaux, France
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23
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Godichaud S, Krisa S, Couronné B, Dubuisson L, Mérillon JM, Desmoulière A, Rosenbaum J. Deactivation of cultured human liver myofibroblasts by trans-resveratrol, a grapevine-derived polyphenol. Hepatology 2000; 31:922-31. [PMID: 10733549 DOI: 10.1053/he.2000.5848] [Citation(s) in RCA: 87] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Liver myofibroblasts are major actors in the development of liver fibrosis and cancer progression. There is a large interest in drugs that might deactivate these cells. Many studies have shown that the grapevine-derived polyphenol, trans-resveratrol, and other stilbenes have therapeutic potential in some diseases. In this work, we have studied the effect of grapevine polyphenols on cultured human liver myofibroblasts. We have shown that trans-resveratrol profoundly affects myofibroblast phenotype. Trans-resveratrol induced morphological modifications. It markedly reduced proliferation of myofibroblasts in a dose-dependent manner. Trans-resveratrol also decreased the expression of alpha smooth muscle actin (alpha-SMA) without affecting vimentin or beta-cytoplasmic actin expression. It decreased myofibroblast migration in a monolayer wounding assay. We also showed that trans-resveratrol inhibited the messenger RNA (mRNA) expression of type I collagen. Finally, it decreased the secretion of matrix metalloproteinase 2 (MMP-2). We conclude that trans-resveratrol can deactivate human liver myofibroblasts. In the second part of this study, we have shown that neither trans-piceid (a glycosylated analog) nor trans-piceatannol (a hydroxylated analog) reproduces trans-resveratrol effects on liver myofibroblasts. We finally show that, although trans-resveratrol decreases the proliferation of skin fibroblast and vascular smooth muscle cells, it does not affect their expression of alpha-SMA, which indicates some cell specificity.
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Affiliation(s)
- S Godichaud
- Groupe de Recherches pour l'Etude du Foie INSERM E9917, Faculté des Sciences Pharmaceutiques, Université Victor Segalen Bordeaux 2, Bordeaux, France
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24
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Knittel T, Kobold D, Saile B, Grundmann A, Neubauer K, Piscaglia F, Ramadori G. Rat liver myofibroblasts and hepatic stellate cells: different cell populations of the fibroblast lineage with fibrogenic potential. Gastroenterology 1999; 117:1205-21. [PMID: 10535885 DOI: 10.1016/s0016-5085(99)70407-5] [Citation(s) in RCA: 282] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
BACKGROUND & AIMS Hepatic stellate cells (HSCs) are considered the principal matrix-producing cells of the damaged liver. However, other cell types of the fibroblast lineage that have not yet been characterized are also involved in liver tissue repair and fibrogenesis. METHODS We established cultures of cells of the fibroblast lineage, termed rat liver myofibroblasts, and analyzed their phenotypical and functional properties in comparison with HSCs. RESULTS HSCs and rat liver myofibroblasts were discernible by morphological criteria and growth behavior. Prolonged subcultivation of rat liver myofibroblasts was achieved, but HSCs were maintained in culture at maximum until second passage. HSCs were characterized by expression of glial fibrillary acidic protein, desmin, and vascular cell adhesion molecule 1, which were almost completely absent in rat liver myofibroblasts. For synthetic properties, HSCs and rat liver myofibroblasts displayed mostly overlapping properties with 4 striking differences. The complement-activating protease P100 and the protease inhibitor alpha(2)-macroglobulin were preferentially expressed by HSCs, whereas interleukin 6-coding messenger RNAs and the extracellular matrix protein fibulin 2 were almost exclusively detectable in rat liver myofibroblasts. CONCLUSIONS The data show that morphologically and functionally different fibroblastic populations, HSCs and rat liver myofibroblasts, can be derived from liver tissue. HSCs may not represent the single matrix-producing cell type of the fibroblast lineage in the liver.
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Affiliation(s)
- T Knittel
- Section of Gastroenterology, Department of Internal Medicine, University of Göttingen, Göttingen, Germany
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25
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26
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Préaux AM, Mallat A, Nhieu JT, D'Ortho MP, Hembry RM, Mavier P. Matrix metalloproteinase-2 activation in human hepatic fibrosis regulation by cell-matrix interactions. Hepatology 1999; 30:944-50. [PMID: 10498646 DOI: 10.1002/hep.510300432] [Citation(s) in RCA: 96] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/18/2023]
Abstract
Matrix metalloproteinase-2 (MMP-2) is involved in extracellular matrix remodeling. It is secreted as a proenzyme and activated by membrane type-MMPs (MT-MMP), such as MT1-MMP. In liver fibrosis, MMP-2 is highly expressed in myofibroblasts and may have a profibrogenic role. The mechanisms of its activation in the liver are still unclear. The aim of this work was to show that pro-MMP-2 is efficiently activated in human fibrotic liver and to investigate the role of cell-matrix interactions in this process. Liver specimens obtained from patients with active cirrhosis were compared to normal liver specimens. Human hepatic myofibroblasts were cultured either on plastic, fibronectin, laminin, or on collagen I gels. MMP-2 activity was visualized by gelatin zymography. MMP-2 active form (59 kd) was detected in active cirrhosis but not in normal liver. Myofibroblasts cultured on plastic, fibronectin, or laminin predominantly expressed inactive pro-MMP-2 (66 kd). In contrast, myofibroblasts cultured on collagen I markedly activated the enzyme. Similar results were obtained using membrane fractions from cells previously cultured on collagen or plastic. Activation was inhibited by the tissue inhibitor of metalloproteinases-2 but not by tissue inhibitor of metalloproteinases-1, implicating a MT-MMP-mediated process. Culture on collagen I up-regulated MT1-MMP protein detected by Western blotting, but decreased MT1-MMP mRNA. This study shows that MMP-2 is activated in fibrotic liver. It suggests that interactions between collagen I and myofibroblasts promote this process through a post-translational increase of MT1-MMP expression in these cells.
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Affiliation(s)
- A M Préaux
- INSERM U99, Hôpital Henri Mondor, AP-HP, Créteil, France
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27
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Lamireau T, Le Bail B, Boussarie L, Fabre M, Vergnes P, Bernard O, Gautier F, Bioulac-Sage P, Rosenbaum J. Expression of collagens type I and IV, osteonectin and transforming growth factor beta-1 (TGFbeta1) in biliary atresia and paucity of intrahepatic bile ducts during infancy. J Hepatol 1999; 31:248-55. [PMID: 10453937 DOI: 10.1016/s0168-8278(99)80221-9] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/04/2022]
Abstract
BACKGROUND/AIMS Biliary atresia and paucity of intrahepatic bile ducts are the main causes of neonatal cholestasis leading to hepatic fibrosis. Fibrotic evolution is slow in paucity of bile ducts as compared to the rapid progression to biliary cirrhosis in biliary atresia when cholestasis persists despite hepatoportoenterostomy. Our aim was to compare the expression of collagens type I and IV, alpha-smooth muscle actin, osteonectin and transforming growth factor beta1 in biliary atresia and paucity of bile ducts. METHODS Liver biopsies were obtained in 12 children with biliary atresia and in five with paucity of bile ducts. Collagens type I and IV, alpha-smooth muscle actin were detected with immunostaining. Collagens type I and IV, osteonectin and transforming growth factor beta1 mRNAs were detected by in situ hybridization. RESULTS Expression of mRNA and proteins was roughly parallel. In ductular proliferation areas of biliary atresia: (1) the expression of collagens type I and IV and osteonectin was increased, and was localized to periductular myofibroblasts; (2) transforming growth factor beta1 was expressed around biliary ductules, probably in inflammatory cells, and also in biliary cells. Osteonectin expression was also increased in the lobules. In paucity of bile ducts, there was no overexpression of collagens type I and IV and transforming growth factor beta1, except in the only child with marked fibrosis. However, osteonectin expression was enhanced at the periphery of the lobules, even when fibrosis was mild or absent. CONCLUSIONS These findings suggest that in biliary atresia ductular proliferation areas are the site of a marked production of extracellular matrix proteins in periductular myofibroblasts, probably secondary to transforming growth factor beta1 production by inflammatory cells and by biliary cells. The weak expression of transforming growth factor beta1 could explain the slow progression of fibrosis in paucity of bile ducts.
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MESH Headings
- Actins/analysis
- Bile Duct Diseases/metabolism
- Bile Duct Diseases/pathology
- Bile Ducts, Intrahepatic/abnormalities
- Bile Ducts, Intrahepatic/chemistry
- Biliary Atresia/metabolism
- Biliary Atresia/pathology
- Collagen/analysis
- Female
- Humans
- Immunohistochemistry
- In Situ Hybridization
- Infant
- Infant, Newborn
- Infant, Newborn, Diseases/metabolism
- Infant, Newborn, Diseases/pathology
- Male
- Muscle, Smooth, Vascular/metabolism
- Muscle, Smooth, Vascular/pathology
- Osteonectin/analysis
- RNA, Messenger/analysis
- Transforming Growth Factor beta/analysis
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Affiliation(s)
- T Lamireau
- Groupe de Recherches pour l'Etude du Foie, Université Victor Ségalen Bordeaux, France
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28
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Neaud V, Faouzi S, Guirouilh J, Monvoisin A, Rosenbaum J. Hepatocyte growth factor secreted by human liver myofibroblasts increases invasiveness of hepatocellular carcinoma cells. CURRENT TOPICS IN PATHOLOGY. ERGEBNISSE DER PATHOLOGIE 1999; 93:195-203. [PMID: 10339912 DOI: 10.1007/978-3-642-58456-5_20] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/12/2023]
Affiliation(s)
- V Neaud
- Groupe de Recherches pour l'Etude du Foie, Université Victor Segalen, Bordeaux, France
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29
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Guerret S, Desmoulière A, Chossegros P, Costa AM, Badid C, Trépo C, Grimaud JA, Chevallier M. Long-term administration of interferon-alpha in non-responder patients with chronic hepatitis C: follow-up of liver fibrosis over 5 years. J Viral Hepat 1999; 6:125-33. [PMID: 10607223 DOI: 10.1046/j.1365-2893.1999.00148.x] [Citation(s) in RCA: 37] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
In chronic hepatitis C, previous data have shown that short-term treatment with interferon-alpha (IFN-alpha) can reduce collagen deposition in the liver independently of the viral response. The aim of this work was to determine, in non-responder patients, the long-term effect of IFN-alpha on liver fibrosis according to the total administered dose and the fibrotic stage. Fibrosis was investigated on liver biopsies from 24 non-responder patients with chronic hepatitis C retreated with successive courses of IFN-alpha. The degree of liver fibrosis was assessed on three successive biopsies, performed before IFN-alpha treatment and 1 and 5 years later, in 13 and 11 patients, respectively, treated for less (mean: 7.5 months, 313 MU) and more (mean: 21.8 months, 791 MU) than 1 year. For each biopsy, fibrosis was assessed using a histological semiquantitative fibrosis scoring system and by morphometry after picrosirius red staining. Regardless of the dose and duration of IFN-alpha therapy, a slight decrease of fibrosis was observed in patients 5 years after starting treatment. In cirrhotic patients, a short treatment induced an improvement followed by a relapse of fibrosis in 57%, and only 43% of patients showed constant collagen regression over the 5 years of follow-up. On the contrary, after prolonged therapy, a progressive and significant decrease occurred throughout the follow-up period in all patients (P = 0.045). Long-term treatment with IFN-alpha is therefore associated with regression of liver fibrosis, particularly in cirrhotic patients. These promising results need to be confirmed in a larger series of patients.
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Affiliation(s)
- S Guerret
- Laboratoire d'Anatomie et Cytologie Pathologiques, Laboratoire Marcel Mérieux, Lyon, France
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30
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Monvoisin A, Neaud V, De Lédinghen V, Dubuisson L, Balabaud C, Bioulac-Sage P, Desmoulière A, Rosenbaum J. Direct evidence that hepatocyte growth factor-induced invasion of hepatocellular carcinoma cells is mediated by urokinase. J Hepatol 1999; 30:511-8. [PMID: 10190737 DOI: 10.1016/s0168-8278(99)80113-5] [Citation(s) in RCA: 53] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/04/2022]
Abstract
BACKGROUND/AIMS We have shown that hepatocyte growth factor secreted by human hepatic myofibroblasts increased the in vitro invasion of the hepatocarcinoma cell line HepG2 through Matrigel. Our aim in this study was to evaluate the role of urokinase in this process. METHODS Expression of urokinase in HepG2 cells was measured by Northern blot and zymography, and plasminogen activation was shown by a chromogenic substrate assay. Cell invasion was assayed on Matrigel-coated filters. Urokinase and urokinase receptor transcripts in hepatocarcinoma were detected by reverse transcription-polymerase chain reaction. Activated hepatocyte growth factor was detected by Western blot with a hepatocyte growth factor-beta chain-specific antibody. RESULTS HepG2 cells expressed urokinase mRNA and secreted active urokinase. Urokinase expression was enhanced by hepatocyte growth factor at the protein and mRNA level. Notably, cell-surface-associated urokinase was increased 22-fold by hepatocyte growth factor. Hepatocyte growth factor also increased urokinase receptor mRNA expression. B428, a urokinase inhibitor, decreased by up to 70% HepG2 invasion induced by myofibroblasts and by 90% that induced by recombinant hepatocyte growth factor. This was not due to a decrease in the generation of activated hepatocyte growth factor by myofibroblasts. Finally, all 17 hepatocarcinoma samples tested expressed urokinase and urokinase receptor transcripts. CONCLUSION Hepatocyte growth factor-dependent, myofibroblasts-induced invasion of HepG2 cells is secondary to the induction of urokinase expression on tumor cells.
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Affiliation(s)
- A Monvoisin
- Groupe de Recherches pour l'Etude du Foie, Université Victor Segalen Bordeaux 2, France
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Faouzi S, Le Bail B, Neaud V, Boussarie L, Saric J, Bioulac-Sage P, Balabaud C, Rosenbaum J. Myofibroblasts are responsible for collagen synthesis in the stroma of human hepatocellular carcinoma: an in vivo and in vitro study. J Hepatol 1999; 30:275-84. [PMID: 10068108 DOI: 10.1016/s0168-8278(99)80074-9] [Citation(s) in RCA: 91] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/04/2022]
Abstract
BACKGROUND/AIMS Marked changes in extracellular matrix occur in the stroma of hepatocellular carcinoma, as compared to normal or cirrhotic liver. The cell types responsible for extracellular matrix synthesis within hepatocellular carcinoma have not been clearly identified. METHODS In vivo collagen synthesis was studied by in situ hybridization and immunohistochemistry for types I, IV, V and VI collagen, together with immunolabeling of alpha-smooth muscle actin, a myofibroblast marker, and CD34, an endothelial cell marker. In vitro, extracellular matrix deposition by cultured myofibroblasts was studied by reticulin staining, immunocytochemistry and RNase protection. RESULTS All collagens studied were expressed in the stroma of the tumor, with a higher level of type VI and IV collagens than of type I and V. The majority of the cells expressing collagen transcripts in human hepatocellular carcinoma stroma were alpha-actin positive and CD 34 negative. In vitro experiments demonstrated that the hepatocellular carcinoma cell lines HepG2, HuH7 and Hep3B markedly increased extracellular matrix deposition by human liver myofibroblasts. This increase was mediated by a soluble mediator present in tumor cell conditioned medium. It was not explained by an increase in mRNA levels of extracellular matrix components, nor by a decrease in the secretion of matrix-degrading proteinases by myofibroblasts. CONCLUSIONS Myofibroblasts are the main source of collagens in the stroma of hepatocellular carcinoma. Our data also indicate that tumoral hepatocytes increase extracellular matrix deposition by cultured myofibroblasts, probably by post-transcriptional mechanisms. The generation of hepatocellular carcinoma stroma by myofibroblasts could thus be under control of tumoral cells.
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Affiliation(s)
- S Faouzi
- Groupe de Recherches pour l'Etude du Foie and Laboratoire d'Anatomie Pathologique, Université Victor Segalen Bordeaux 2, France
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Bachem MG, Schneider E, Gross H, Weidenbach H, Schmid RM, Menke A, Siech M, Beger H, Grünert A, Adler G. Identification, culture, and characterization of pancreatic stellate cells in rats and humans. Gastroenterology 1998; 115:421-32. [PMID: 9679048 DOI: 10.1016/s0016-5085(98)70209-4] [Citation(s) in RCA: 778] [Impact Index Per Article: 28.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
BACKGROUND & AIMS Until now, the basic matrix-producing cell type responsible for pancreas fibrosis has not been identified. In this report, retinoid-containing pancreatic stellate cells (PSCs) in rat and human pancreas are described, and morphological and biochemical similarities to hepatic stellate cells are shown. METHODS Electron and immunofluorescence microscopy (collagen types I and III, fibronectin, laminin, alpha-actin, and desmin) was performed using pancreatic tissue and cultured PSCs. Extracellular matrix synthesis was shown using quantitative immunoassay and Northern blot analysis. RESULTS PSCs are located in interlobular areas and in interacinar regions. Early primary cultured PSCs contain retinol and fatty acid retinyl-esters. Addition of retinol to passaged cells resulted in retinol uptake and esterification. During primary culture, the cells changed from a quiescent fat-storing phenotype to a highly synthetic myofibroblast-like cell expressing iso-alpha-smooth muscle actin (>90%) and desmin (20%-40%) and showing strong positive staining with antibodies to collagen types I and III, fibronectin, and laminin. As determined on protein and messenger RNA level, serum growth factors stimulated the synthesis of collagen type I and fibronectin. CONCLUSIONS The identification of PSCs, particularly in fibrotic areas, and the similarities of these cells to hepatic stellate cells suggest that PSCs participate in the development of pancreas fibrosis.
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Affiliation(s)
- M G Bachem
- Department of Clinical Chemistry and Pathobiochemistry, University Hospital Ulm, Ulm, Germany
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Nayak NC, Junaid TA, Sathar SA, al-Agha MA. Immunohistochemical study of proliferating mesenchymal spindle cells in heterogenous soft tissue lesions. Acta Histochem 1998; 100:315-27. [PMID: 9717569 DOI: 10.1016/s0065-1281(98)80018-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Proliferation of mesenchymal spindle cells is a main event in a variety of lesions with similar morphological features but widely divergent biological behaviour. In order to identify criteria for precise histological diagnosis, 60 human soft tissue lesions, divided into 40 cases of fibroblastic cell proliferation, 10 smooth muscle cell tumours and 10 nerve sheath cell tumours, were examined for the immunohistochemical profile of the main lesional cell in addition to other histological features. The three groups could be differentiated by determining the lineage of the constituent spindle cell on the pattern of expression of vimentin, alpha-smooth muscle actin (ASMA) and macrophage antigen CD68 (MA-CD68). Smooth muscle cells expressed ASMA and vimentin but not MA-CD68, while nerve sheath cells were negative for ASMA but positive for vimentin and MA-CD68. The fibroblastic cell lesions as a group were easily differentiated on the basis of positive reactivity for all three markers but individual lesions could only be distinguished by additional assessment of histological features. Because of consistent co-expression of ASMA, vimentin and MA-CD68 in the spindle mesenchymal cell present in all varieties of lesions in this heterogeneous group, we suggest that this proliferating "fibroblastic" cell is phenotypically a fibromyohistiocyte.
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Affiliation(s)
- N C Nayak
- Department of Pathology, Faculty of Medicine Kuwait University, Safat, Kuwait
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Demirci G, Nashan B, Pichlmayr R. Fibrosis in chronic rejection of human liver allografts: expression patterns of transforming growth factor-TGFbeta1 and TGF-beta3. Transplantation 1996; 62:1776-83. [PMID: 8990362 DOI: 10.1097/00007890-199612270-00016] [Citation(s) in RCA: 38] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Abstract
Activation and transformation of lipocytes (Ito cells, stellate cells) into alpha-actin-positive myofibroblast-like cells is an essential step in the initiation of liver fibrosis. Transforming growth factor-beta (TGF-beta) is considered an important mediator of this process. In order to determine mechanisms of fibrotic deposition in a hepatic transplant setting, we analyzed 10 chronically rejected human liver allografts for the expression of extracellular matrix (ECM) molecules, myofibroblast-like cells (alpha-actin), macrophages, and TGF-beta1 and -beta3. Using single- and double-immunohistochemical staining techniques, all specimens investigated showed increased deposition of the ECM proteins fibronectin, tenascin, undulin, and collagen VI with a characteristic densification especially in pericentral areas. Likewise, strong accumulation of alpha-actin-positive cells and TGF-beta1-expressing macrophages was observed in the same fields, supporting the concept of lipocyte activation/transformation and subsequent ECM production fostered by macrophage-derived TGF-beta1. In contrast, TGF-beta3 was found to be mainly expressed by a markedly increased number of lipocytes. Interestingly, distribution of TGF-beta3 corresponded to that of tenascin, an ECM molecule known to be involved in early matrix organization, suggesting that TGF-beta3 may likewise act mainly in early stages of fibrogenesis. Furthermore, TGF-beta3 restriction to high numbers of a single cell type (i.e., lipocytes) implied a possible role in cell proliferation through autocrine loops. In conclusion, fibrosis in chronic rejection seems to follow similar mechanisms as in non-transplanted livers but additionally suggests differential temporal and functional roles for the TGF-beta isoforms 1 and 3 in the course of a multistep process leading to lipocyte transformation and ECM production.
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Affiliation(s)
- G Demirci
- Klinik für Abdominal- und Transplantationschirurgie, Medizinische Hochschule Hannover, Germany
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Abstract
The presence of myofibroblasts in granulation tissue and various fibrotic settings is well established. Recent work on this cell has shown that myofibroblasts derive mainly from local fibroblasts, but also from pericytes and smooth muscle cells as well as from specialized cells such as perisinusoidal stellate cells of the liver and mesangial cells of the kidney glomerulus. During the healing of an open wound, myofibroblasts disappear by means of apoptosis when the wound is closed and granulation tissue gradually transforms into scar tissue. The possibility exists that an altered regulation of this process leads to the development of a hypertrophic scar.
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Affiliation(s)
- G Gabbiani
- CMU-Department of Pathology, University of Geneva, Switzerland.
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Rosenbaum J, Blazejewski S, Préaux AM, Mallat A, Dhumeaux D, Mavier P. Fibroblast growth factor 2 and transforming growth factor beta 1 interactions in human liver myofibroblasts. Gastroenterology 1995; 109:1986-96. [PMID: 7498665 DOI: 10.1016/0016-5085(95)90767-x] [Citation(s) in RCA: 62] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
BACKGROUND & AIMS During liver fibrogenesis, myofibroblastic liver cells proliferate and synthesize components of fibrosis. Fibroblast growth factor 2 (FGF-2) is expressed in vivo in myofibroblastic liver cells (MFLCs) during fibrogenesis, and exogenous FGF-2 is mitogenic for MFLCs. The aim of this study was to study the expression and role of endogenous FGF-2 in cultured human MFLCs. METHODS FGF-2 and FGF-2 receptors were studied using immunoblotting. All RNA studies used ribonuclease protection. Growth of MFLCs was studied using [3H]thymidine incorporation and direct cell counting. RESULTS MFLCs expressed FGF-2 and its receptors FGF receptor 1 and FGF receptor 2. An antibody to FGF-2 blocked the mitogenic effect of transforming growth factor beta 1 (TGF-beta 1) for MFLCs but not TGF-beta 1-induced increase in cellular fibronectin messenger RNA (mRNA). TGF-beta 1 increased levels of FGF-2 and FGF receptor mRNAs in MFLCs. We have previously shown that TGF-beta 1 also increased platelet-derived growth factor (PDGF) A chain mRNA in these cells and that anti-PDGF antibody blunted the mitogenic effect of TGF-beta 1. The present results show that anti-FGF-2 and anti-PDGF-AA are not additive and that FGF-2 and PDGF-AA are not sequentially induced by TGF-beta 1. CONCLUSIONS FGF-2 mediates the mitogenic but not the profibrogenic effect of TGF-beta 1 for human MFLCs, and autocrine FGF-2 and PDGF-A interact in the mediation of the mitogenic effect of TGF-beta 1.
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Affiliation(s)
- J Rosenbaum
- INSERM Unité 99, Hôpital Henri Mondor, Créteil, France
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