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Sultana M, Islam MA, Khairnar R, Kumar S. A guide to pathophysiology, signaling pathways, and preclinical models of liver fibrosis. Mol Cell Endocrinol 2025; 598:112448. [PMID: 39755140 DOI: 10.1016/j.mce.2024.112448] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/28/2024] [Revised: 12/23/2024] [Accepted: 12/28/2024] [Indexed: 01/06/2025]
Abstract
Liver fibrosis is potentially a reversible form of liver disease that evolved from the early stage of liver scarring as a consequence of chronic liver injuries. Recurrent injuries in the liver without any appropriate medication cause the injuries to get intense and deeper, which gradually leads to the progression of irreversible cirrhosis or carcinoma. Unfortunately, there are no approved treatment strategies for reversing hepatic fibrosis, making it one of the significant risk factors for developing advanced liver disorders and liver disease-associated mortality. Consequently, the interpretation of the fundamental mechanisms, etiology, and pathogenesis is crucial for identifying the potential therapeutic target as well as evaluating novel anti-fibrotic therapy. However, despite innumerable research, the functional mechanism and disease characteristics are still obscure. To accelerate the understanding of underlying disease pathophysiology, molecular pathways and disease progression mechanism, it is crucial to mimic human liver disease through the formation of precise disease models. Although various in vitro and in vivo liver fibrotic models have emerged and developed already, a perfect clinical model replicating human liver diseases is yet to be established, which is one of the major challenges in discovering proper therapeutics. This review paper will shed light on pathophysiology, signaling pathways, preclinical models of liver fibrosis, and their limitations.
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Affiliation(s)
- Mehonaz Sultana
- Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John's University, Queens, NY, 11439, USA
| | - Md Asrarul Islam
- Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John's University, Queens, NY, 11439, USA
| | - Rhema Khairnar
- Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John's University, Queens, NY, 11439, USA
| | - Sunil Kumar
- Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John's University, Queens, NY, 11439, USA.
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2
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Ezhilarasan D, Najimi M. Quiescent hepatic stellate cell activation in liver fibrosis: Have we found the right trigger yet? Clin Res Hepatol Gastroenterol 2024; 48:102420. [PMID: 39002817 DOI: 10.1016/j.clinre.2024.102420] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/01/2024] [Accepted: 07/11/2024] [Indexed: 07/15/2024]
Affiliation(s)
- Devaraj Ezhilarasan
- Department of Pharmacology, Hepatology and Molecular Medicine Lab, Saveetha Dental College, Saveetha Institute of Medical and Technical Sciences, No.162, PH Rd, Chennai, Tamil Nadu 600 077, India.
| | - Mustapha Najimi
- Laboratory of Pediatric Hepatology and Cell Therapy, Institute of Experimental and Clinical Research (IREC), UCLouvain, Brussels, Belgium
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Cogliati B, Yashaswini CN, Wang S, Sia D, Friedman SL. Friend or foe? The elusive role of hepatic stellate cells in liver cancer. Nat Rev Gastroenterol Hepatol 2023; 20:647-661. [PMID: 37550577 PMCID: PMC10671228 DOI: 10.1038/s41575-023-00821-z] [Citation(s) in RCA: 60] [Impact Index Per Article: 30.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 07/06/2023] [Indexed: 08/09/2023]
Abstract
Liver fibrosis is a substantial risk factor for the development and progression of liver cancer, which includes hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA). Studies utilizing cell fate mapping and single-cell transcriptomics techniques have identified quiescent perisinusoidal hepatic stellate cells (HSCs) as the primary source of activated collagen-producing HSCs and liver cancer-associated fibroblasts (CAFs) in HCC and liver metastasis, complemented in iCCA by contributions from portal fibroblasts. At the same time, integrative computational analysis of single-cell, single-nucleus and spatial RNA sequencing data have revealed marked heterogeneity among HSCs and CAFs, with distinct subpopulations displaying unique gene expression signatures and functions. Some of these subpopulations have divergent roles in promoting or inhibiting liver fibrogenesis and carcinogenesis. In this Review, we discuss the dual roles of HSC subpopulations in liver fibrogenesis and their contribution to liver cancer promotion, progression and metastasis. We review the transcriptomic and functional similarities between HSC and CAF subpopulations, highlighting the pathways that either promote or prevent fibrosis and cancer, and the immunological landscape from which these pathways emerge. Insights from ongoing studies will yield novel strategies for developing biomarkers, assessing prognosis and generating new therapies for both HCC and iCCA prevention and treatment.
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Affiliation(s)
- Bruno Cogliati
- Division of Liver Diseases, Icahn School of Medicine at Mount Sinai, New York, NY, USA
- Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, SP, Brazil
| | | | - Shuang Wang
- Division of Liver Diseases, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Daniela Sia
- Division of Liver Diseases, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Scott L Friedman
- Division of Liver Diseases, Icahn School of Medicine at Mount Sinai, New York, NY, USA.
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4
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Meurer SK, Weiskirchen S, Tag CG, Weiskirchen R. Isolation, Purification, and Culture of Primary Murine Hepatic Stellate Cells: An Update. Methods Mol Biol 2023; 2669:1-32. [PMID: 37247051 DOI: 10.1007/978-1-0716-3207-9_1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/30/2023]
Abstract
In the healthy liver, quiescent hepatic stellate cells (HSCs) are found in the perisinusoidal space (i.e., the space of Dissé) in close proximity to endothelial cells and hepatocytes. HSCs represent 5-8% of the total number of liver cells and are characterized by numerous fat vacuoles that store vitamin A in the form of retinyl esters. Upon liver injury caused by different etiologies, HSCs become activated and acquire a myofibroblast (MFB) phenotype in a process called transdifferentiation. In contrast to quiescent HSC, MFB become highly proliferative and are characterized by an imbalance in extracellular matrix (ECM) homeostasis, by producing an excess of collagen and blocking its turnover by synthesis of protease inhibitors. This leads to a net accumulation of ECM during fibrosis. In addition to HSC, there are fibroblasts in the portal fields (pF), which also have the potency to acquire a myofibroblastic phenotype (pMF). The contributions of these two fibrogenic cell types (i.e., MFB and pMF) vary based on the etiology of liver damage (parenchymal vs. cholestatic). Based on their importance to hepatic fibrosis, the isolation and purification protocols of these primary cells are in great demand. Moreover, established cell lines may offer only limited information about the in vivo behavior of HSC/MFB and pF/pMF.Here we describe a method for high-purity isolation of HSC from mice. In the first step, the liver is digested with pronase and collagenase, and the cells are dissociated from the tissue. In the second step, HSCs are enriched by density gradient centrifugation of the crude cell suspension using a Nycodenz gradient. The resulting cell fraction can be further optionally purified by flow cytometric enrichment to generate ultrapure HSC.
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Affiliation(s)
- Steffen K Meurer
- Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry (IFMPEGKC), RWTH University Hospital Aachen, Aachen, Germany.
| | - Sabine Weiskirchen
- Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry (IFMPEGKC), RWTH University Hospital Aachen, Aachen, Germany.
| | - Carmen G Tag
- Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry (IFMPEGKC), RWTH University Hospital Aachen, Aachen, Germany
| | - Ralf Weiskirchen
- IInstitut für Molekulare Pathobiochemie, Experimentelle Gentherapie und Klinische Chemie (IFMPEGKC), Universitätsklinikum Aachen AöR, Aachen, Germany
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Kim KM, An HJ, Kim SH, Kim J, Sim C, Lee J, Park SH, Lee HI, Jang I, Lee S. Therapeutic Effect of Pericytes for Diabetic Wound Healing. Front Cardiovasc Med 2022; 9:868600. [PMID: 35647064 PMCID: PMC9135971 DOI: 10.3389/fcvm.2022.868600] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2022] [Accepted: 04/12/2022] [Indexed: 12/28/2022] Open
Abstract
Objective Numerous attempts have been made to devise treatments for ischemic foot ulcer (IFU), which is one of the most severe and fatal consequences of diabetes mellitus (DM). Pericytes, which are perivascular multipotent cells, are of interest as a treatment option for IFU because they play a critical role in forming and repairing various tissues. In this study, we want to clarify the angiogenic potential of pericytes in DM-induced wounds. Methods We evaluated pericyte stimulation capability for tube formation, angiogenesis, and wound healing (cell migration) in human umbilical vein endothelial cells (HUVECs) with in-vivo and in-vitro models of high glucose conditions. Results When HUVECs were co-cultured with pericytes, their tube-forming capacity and cell migration were enhanced. Our diabetic mouse model showed that pericytes promote wound healing via increased vascularization. Conclusion The findings of this study indicate that pericytes may enhance wound healing in high glucose conditions, consequently making pericyte transplantation suitable for treating IFUs.
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Affiliation(s)
- Kyeong Mi Kim
- Department of Laboratory Medicine, CHA Ilsan Medical Center, CHA University School of Medicine, Goyang-si, South Korea
| | - Hyun-Ju An
- Department of Orthopaedic Surgery, CHA Bundang Medical Center, CHA University School of Medicine, Seongnam-si, South Korea
| | - Sang-Hoon Kim
- Department of Cardiology, CHA Bundang Medical Center, CHA University School of Medicine, Gyeonggi-do, South Korea
| | - JuHee Kim
- Department of Orthopaedic Surgery, CHA Bundang Medical Center, CHA University School of Medicine, Seongnam-si, South Korea
| | - Changgon Sim
- CHA Graduate School of Medicine, Gyeonggi-do, South Korea
| | - Jaemin Lee
- Department of Orthopaedic Surgery, CHA Bundang Medical Center, CHA University School of Medicine, Seongnam-si, South Korea
| | - Sin Hyung Park
- Department of Orthopaedic Surgery, Bucheon Hospital, Soonchunhyang University School of Medicine, Gyeonggi-do, South Korea
| | - Hyun Il Lee
- Department of Orthopedic Surgery, Ilsan Paik Hospital, Inje University, Gyeonggi-do, South Korea
| | - Inseok Jang
- Department of Orthopaedic Surgery, CHA Bundang Medical Center, CHA University School of Medicine, Seongnam-si, South Korea
| | - Soonchul Lee
- Department of Orthopaedic Surgery, CHA Bundang Medical Center, CHA University School of Medicine, Seongnam-si, South Korea
- *Correspondence: Soonchul Lee
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Freise C, Lee H, Chronowski C, Chan D, Cziomer J, Rühl M, Dagdelen T, Lösekann M, Erben U, Catic A, Tegge W, Schuppan D, Somasundaram R, Sahin E. Alpha-single chains of collagen type VI inhibit the fibrogenic effects of triple helical collagen VI in hepatic stellate cells. PLoS One 2021; 16:e0254557. [PMID: 34473704 PMCID: PMC8412337 DOI: 10.1371/journal.pone.0254557] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2021] [Accepted: 06/29/2021] [Indexed: 11/18/2022] Open
Abstract
The interaction of extracellular matrix (ECM) components with hepatic stellate cells (HSCs) is thought to perpetuate fibrosis by stimulating signaling pathways that drive HSC activation, survival and proliferation. Consequently, disrupting the interaction between ECM and HSCs is considered a therapeutical avenue although respective targets and underlying mechanisms remain to be established. Here we have interrogated the interaction between type VI collagen (CVI) and HSCs based on the observation that CVI is 10-fold upregulated during fibrosis, closely associates with HSCs in vivo and promotes cell proliferation and cell survival in cancer cell lines. We exposed primary rat HSCs and a rat hepatic stellate cell line (CFSC) to soluble CVI and determined the rate of proliferation, apoptosis and fibrogenesis in the absence of any additional growth factors. We find that CVI in nanomolar concentrations prevents serum starvation-induced apoptosis. This potent anti-apoptotic effect is accompanied by induction of proliferation and acquisition of a pronounced pro-fibrogenic phenotype characterized by increased α-smooth muscle actin, TGF-β, collagen type I and TIMP-1 expression and diminished proteolytic MMP-13 expression. The CVI-HSC interaction can be disrupted with the monomeric α2(VI) and α3(VI) chains and abrogates the activating CVI effects. Further, functional relevant α3(VI)—derived 30 amino acid peptides lead to near-complete inhibition of the CVI effect. In conclusion, CVI serves as a potent mitogen and activating factor for HSCs. The antagonistic effects of the CVI monomeric chains and peptides point to linear peptide sequences that prevent activation of CVI receptors which may allow a targeted antifibrotic therapy.
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Affiliation(s)
- Christian Freise
- Department of Gastroenterology, Infectious Diseases and Rheumatology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany
- Department of Radiology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany
| | - Hyunho Lee
- Huffington Center On Aging, Baylor College of Medicine, Houston, Texas, United States of America
| | - Christopher Chronowski
- Huffington Center On Aging, Baylor College of Medicine, Houston, Texas, United States of America
| | - Doug Chan
- Department of Cell Biology, Baylor College of Medicine, Houston, Texas, United States of America
| | - Jessica Cziomer
- Department of Gastroenterology, Infectious Diseases and Rheumatology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany
| | - Martin Rühl
- Department of Gastroenterology, Infectious Diseases and Rheumatology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany
| | - Tarkan Dagdelen
- Department of Gastroenterology, Infectious Diseases and Rheumatology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany
| | - Maik Lösekann
- Department of Gastroenterology, Infectious Diseases and Rheumatology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany
| | - Ulrike Erben
- Department of Gastroenterology, Infectious Diseases and Rheumatology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany
| | - Andre Catic
- Huffington Center On Aging, Baylor College of Medicine, Houston, Texas, United States of America
- Department of Cell Biology, Baylor College of Medicine, Houston, Texas, United States of America
| | - Werner Tegge
- Department of Chemical Biology, Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany
| | - Detlef Schuppan
- Division of Gastroenterology and Hepatology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, United States of America
- Institute of Translational Immunology and Research Center for Immune Therapy, University Medical Center, Mainz, Germany
| | - Rajan Somasundaram
- Department of Gastroenterology, Infectious Diseases and Rheumatology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany
- Department of Emergency Medicine, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany
| | - Ergun Sahin
- Huffington Center On Aging, Baylor College of Medicine, Houston, Texas, United States of America
- Department of Physiology and Biophysics, Baylor College of Medicine, Houston, Texas, United States of America
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7
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Liao J, Zhang Z, Yuan Q, Liu Q, Kuang J, Fang Y, Hu X. A lncRNA Gpr137b-ps/miR-200a-3p/CXCL14 axis modulates hepatic stellate cell (HSC) activation. Toxicol Lett 2021; 336:21-31. [PMID: 33069761 DOI: 10.1016/j.toxlet.2020.10.001] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2020] [Revised: 08/31/2020] [Accepted: 10/02/2020] [Indexed: 01/08/2023]
Abstract
Hepatic fibrosis is the wound healing response upon the liver tissue damage caused by multiple stimuli. Targeting activated hepatic stellate cells (HSCs), the major extracellular matrix (ECM)-producing cells within the damaged liver, has been regarded as one of the main treatments for hepatic fibrosis. In the present study, we performed preliminary bioinformatics analysis attempting to identify possible factors related to hepatic fibrosis and found that lncRNA G protein-coupled receptor 137B (Gpr137b-ps) and C-X-C motif chemokine ligand 14 (CXCL14) showed to be markedly upregulated within carbon tetrachloride (CCl4)-caused hepatic fibrotic mice tissue samples and activated HSCs. CXCL14 The silencing of lncRNA Gpr137b-ps or CXCL14 alone could significantly improve CCl4-induced fibrotic changes in mice liver in vivo and collagen I and III release by HSCs and HSC proliferation in vitro. miR-200a-3p directly targeted lncRNA Gpr137b-ps and CXCL14, respectively. LncRNA Gpr137b-ps relieved miR-200a-3p-induced inhibition on CXCL14 expression via acting as a ceRNA. In HSCs, the effects of lncRNA Gpr137b-ps silencing on collagen I and III release by HSCs and HSC proliferation were significantly reversed by miR-200a-3p inhibition, and the effects of miR-200a-3p inhibition were reversed by CXCL14 silencing. In conclusion, we demonstrated a lncRNA Gpr137b-ps/miR-200a-3p/CXCL14 axis that modulates HSC activation and might exert an effect on the pathogenesis of liver fibrosis.
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Affiliation(s)
- Jinmao Liao
- Department of Hepatopathy, The Hunan Provincial People's Hospital, The First Affiliated Hospital of Hunan Normal University, Changsha 410005, Hunan, China
| | - Zheng Zhang
- Department of Hepatopathy, The Hunan Provincial People's Hospital, The First Affiliated Hospital of Hunan Normal University, Changsha 410005, Hunan, China
| | - Qi Yuan
- Department of Hepatopathy, The Hunan Provincial People's Hospital, The First Affiliated Hospital of Hunan Normal University, Changsha 410005, Hunan, China
| | - Qiong Liu
- Department of Hepatopathy, The Hunan Provincial People's Hospital, The First Affiliated Hospital of Hunan Normal University, Changsha 410005, Hunan, China
| | - Jia Kuang
- Department of Hepatopathy, The Hunan Provincial People's Hospital, The First Affiliated Hospital of Hunan Normal University, Changsha 410005, Hunan, China
| | - Yuan Fang
- Department of Hepatopathy, The Hunan Provincial People's Hospital, The First Affiliated Hospital of Hunan Normal University, Changsha 410005, Hunan, China
| | - Xiaoxuan Hu
- Department of Hepatopathy, The Hunan Provincial People's Hospital, The First Affiliated Hospital of Hunan Normal University, Changsha 410005, Hunan, China.
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8
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Wagner C, Hois V, Pajed L, Pusch LM, Wolinski H, Trauner M, Zimmermann R, Taschler U, Lass A. Lysosomal acid lipase is the major acid retinyl ester hydrolase in cultured human hepatic stellate cells but not essential for retinyl ester degradation. Biochim Biophys Acta Mol Cell Biol Lipids 2020; 1865:158730. [PMID: 32361002 PMCID: PMC7279957 DOI: 10.1016/j.bbalip.2020.158730] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2019] [Revised: 04/22/2020] [Accepted: 04/25/2020] [Indexed: 02/07/2023]
Abstract
Vitamin A is stored as retinyl esters (REs) in lipid droplets of hepatic stellate cells (HSCs). To date, two different pathways are known to facilitate the breakdown of REs: (i) Hydrolysis of REs by neutral lipases, and (ii) whole lipid droplet degradation in autolysosomes by acid hydrolysis. In this study, we evaluated the contribution of neutral and acid RE hydrolases to the breakdown of REs in human HSCs. (R)-Bromoenol lactone (R-BEL), inhibitor of adipose triglyceride lipase (ATGL) and patatin-like phospholipase domain-containing 3 (PNPLA3), the hormone-sensitive lipase (HSL) inhibitor 76-0079, as well as the serine-hydrolase inhibitor Orlistat reduced neutral RE hydrolase activity of LX-2 cell-lysates between 20 and 50%. Interestingly, in pulse-chase experiments, R-BEL, 76-0079, as well as Orlistat exerted little to no effect on cellular RE breakdown of LX-2 cells as well as primary human HSCs. In contrast, Lalistat2, a specific lysosomal acid lipase (LAL) inhibitor, virtually blunted acid in vitro RE hydrolase activity of LX-2 cells. Accordingly, HSCs isolated from LAL-deficient mice showed RE accumulation and were virtually devoid of acidic RE hydrolase activity. In pulse-chase experiments however, LAL-deficient HSCs, similar to LX-2 cells and primary human HSCs, were not defective in degrading REs. In summary, results demonstrate that ATGL, PNPLA3, and HSL contribute to neutral RE hydrolysis of human HSCs. LAL is the major acid RE hydrolase in HSCs. Yet, LAL is not limiting for RE degradation under serum-starvation. Together, results suggest that RE breakdown of HSCs is facilitated by (a) so far unknown, non-Orlistat inhibitable RE-hydrolase(s).
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Affiliation(s)
- Carina Wagner
- Institute of Molecular Biosciences, NAWI Graz, University of Graz, Heinrichstraße 31/II, A-8010 Graz, Austria
| | - Victoria Hois
- Institute of Molecular Biosciences, NAWI Graz, University of Graz, Heinrichstraße 31/II, A-8010 Graz, Austria
| | - Laura Pajed
- Institute of Molecular Biosciences, NAWI Graz, University of Graz, Heinrichstraße 31/II, A-8010 Graz, Austria
| | - Lisa-Maria Pusch
- Institute of Molecular Biosciences, NAWI Graz, University of Graz, Heinrichstraße 31/II, A-8010 Graz, Austria
| | - Heimo Wolinski
- Institute of Molecular Biosciences, NAWI Graz, University of Graz, Heinrichstraße 31/II, A-8010 Graz, Austria
| | - Michael Trauner
- Hans Popper Laboratory of Molecular Hepatology, Division of Gastroenterology and Hepatology, Department of Medicine III, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria
| | - Robert Zimmermann
- Institute of Molecular Biosciences, NAWI Graz, University of Graz, Heinrichstraße 31/II, A-8010 Graz, Austria; BioTechMed-Graz, Graz, Austria
| | - Ulrike Taschler
- Institute of Molecular Biosciences, NAWI Graz, University of Graz, Heinrichstraße 31/II, A-8010 Graz, Austria.
| | - Achim Lass
- Institute of Molecular Biosciences, NAWI Graz, University of Graz, Heinrichstraße 31/II, A-8010 Graz, Austria; BioTechMed-Graz, Graz, Austria.
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9
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Dobie R, Wilson-Kanamori JR, Henderson BEP, Smith JR, Matchett KP, Portman JR, Wallenborg K, Picelli S, Zagorska A, Pendem SV, Hudson TE, Wu MM, Budas GR, Breckenridge DG, Harrison EM, Mole DJ, Wigmore SJ, Ramachandran P, Ponting CP, Teichmann SA, Marioni JC, Henderson NC. Single-Cell Transcriptomics Uncovers Zonation of Function in the Mesenchyme during Liver Fibrosis. Cell Rep 2019; 29:1832-1847.e8. [PMID: 31722201 PMCID: PMC6856722 DOI: 10.1016/j.celrep.2019.10.024] [Citation(s) in RCA: 267] [Impact Index Per Article: 44.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2019] [Revised: 08/26/2019] [Accepted: 10/07/2019] [Indexed: 12/11/2022] Open
Abstract
Iterative liver injury results in progressive fibrosis disrupting hepatic architecture, regeneration potential, and liver function. Hepatic stellate cells (HSCs) are a major source of pathological matrix during fibrosis and are thought to be a functionally homogeneous population. Here, we use single-cell RNA sequencing to deconvolve the hepatic mesenchyme in healthy and fibrotic mouse liver, revealing spatial zonation of HSCs across the hepatic lobule. Furthermore, we show that HSCs partition into topographically diametric lobule regions, designated portal vein-associated HSCs (PaHSCs) and central vein-associated HSCs (CaHSCs). Importantly we uncover functional zonation, identifying CaHSCs as the dominant pathogenic collagen-producing cells in a mouse model of centrilobular fibrosis. Finally, we identify LPAR1 as a therapeutic target on collagen-producing CaHSCs, demonstrating that blockade of LPAR1 inhibits liver fibrosis in a rodent NASH model. Taken together, our work illustrates the power of single-cell transcriptomics to resolve the key collagen-producing cells driving liver fibrosis with high precision.
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Affiliation(s)
- Ross Dobie
- Centre for Inflammation Research, The Queen's Medical Research Institute, Edinburgh BioQuarter, University of Edinburgh, Edinburgh EH16 4TJ, UK
| | - John R Wilson-Kanamori
- Centre for Inflammation Research, The Queen's Medical Research Institute, Edinburgh BioQuarter, University of Edinburgh, Edinburgh EH16 4TJ, UK
| | - Beth E P Henderson
- Centre for Inflammation Research, The Queen's Medical Research Institute, Edinburgh BioQuarter, University of Edinburgh, Edinburgh EH16 4TJ, UK
| | - James R Smith
- Centre for Inflammation Research, The Queen's Medical Research Institute, Edinburgh BioQuarter, University of Edinburgh, Edinburgh EH16 4TJ, UK
| | - Kylie P Matchett
- Centre for Inflammation Research, The Queen's Medical Research Institute, Edinburgh BioQuarter, University of Edinburgh, Edinburgh EH16 4TJ, UK
| | - Jordan R Portman
- Centre for Inflammation Research, The Queen's Medical Research Institute, Edinburgh BioQuarter, University of Edinburgh, Edinburgh EH16 4TJ, UK
| | - Karolina Wallenborg
- Karolinska Institutet (KI), Science for Life Laboratory, Tomtebodavägen 23, Solna 171 65, Sweden
| | - Simone Picelli
- Karolinska Institutet (KI), Science for Life Laboratory, Tomtebodavägen 23, Solna 171 65, Sweden
| | | | | | | | | | | | | | - Ewen M Harrison
- Clinical Surgery, University of Edinburgh, Royal Infirmary of Edinburgh, Edinburgh EH16 4SA, UK
| | - Damian J Mole
- Centre for Inflammation Research, The Queen's Medical Research Institute, Edinburgh BioQuarter, University of Edinburgh, Edinburgh EH16 4TJ, UK; Clinical Surgery, University of Edinburgh, Royal Infirmary of Edinburgh, Edinburgh EH16 4SA, UK
| | - Stephen J Wigmore
- Centre for Inflammation Research, The Queen's Medical Research Institute, Edinburgh BioQuarter, University of Edinburgh, Edinburgh EH16 4TJ, UK; Clinical Surgery, University of Edinburgh, Royal Infirmary of Edinburgh, Edinburgh EH16 4SA, UK
| | - Prakash Ramachandran
- Centre for Inflammation Research, The Queen's Medical Research Institute, Edinburgh BioQuarter, University of Edinburgh, Edinburgh EH16 4TJ, UK
| | - Chris P Ponting
- MRC Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine at the University of Edinburgh, Edinburgh EH4 2XU, UK; Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge CB10 1SA, UK
| | - Sarah A Teichmann
- Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge CB10 1SA, UK; European Molecular Biology Laboratory, European Bioinformatics Institute (EMBL-EBI), Hinxton, Cambridge CB10 1SD, UK; Theory of Condensed Matter Group, The Cavendish Laboratory, University of Cambridge, Cambridge CB3 0HE, UK
| | - John C Marioni
- Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge CB10 1SA, UK; European Molecular Biology Laboratory, European Bioinformatics Institute (EMBL-EBI), Hinxton, Cambridge CB10 1SD, UK; Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Cambridge CB2 0RE, UK
| | - Neil C Henderson
- Centre for Inflammation Research, The Queen's Medical Research Institute, Edinburgh BioQuarter, University of Edinburgh, Edinburgh EH16 4TJ, UK.
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10
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Wilson KJ, Xiao J, Chen CZ, Huang Z, Agoulnik IU, Ferrer M, Southall N, Hu X, Zheng W, Xu X, Wang A, Myhr C, Barnaeva E, George ER, Agoulnik AI, Marugan JJ. Optimization of the first small-molecule relaxin/insulin-like family peptide receptor (RXFP1) agonists: Activation results in an antifibrotic gene expression profile. Eur J Med Chem 2018; 156:79-92. [PMID: 30006176 PMCID: PMC6102074 DOI: 10.1016/j.ejmech.2018.06.008] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2018] [Revised: 05/25/2018] [Accepted: 06/02/2018] [Indexed: 01/08/2023]
Abstract
A dose responsive quantitative high throughput screen (qHTS) of >350,000 compounds against a human relaxin/insulin-like family peptide receptor (RXFP1) transfected HEK293 cell line identified 2-acetamido-N-phenylbenzamides 1 and 3 with modest agonist activity. An extensive structure-activity study has been undertaken to optimize the potency, efficacy, and physical properties of the series, resulting in the identification of compound 65 (ML-290), which has excellent in vivo PK properties with high levels of systemic exposure. This series, exemplified by 65, has produced first-in-class small-molecule agonists of RXFP1 and is a potent activator of anti-fibrotic genes.
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Affiliation(s)
- Kenneth J. Wilson
- NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville, MD 20850
| | - Jingbo Xiao
- NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville, MD 20850
| | - Catherine Z. Chen
- NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville, MD 20850
| | - Zaohua Huang
- Department of Human and Molecular Genetics, Herbert Wertheim College of Medicine, Florida International University, 11200 SW 8th Street, HLSI 419B, Miami, FL 33199
| | - Irina U. Agoulnik
- Department of Cellular Biology and Pharmacology, Herbert Wertheim College of Medicine, Florida International University, 11200 SW 8th Street, HLSI 419B, Miami, FL 33199
| | - Marc Ferrer
- NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville, MD 20850
| | - Noel Southall
- NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville, MD 20850
| | - Xin Hu
- NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville, MD 20850
| | - Wei Zheng
- NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville, MD 20850
| | - Xin Xu
- NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville, MD 20850
| | - Amy Wang
- NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville, MD 20850
| | - Courtney Myhr
- Department of Cellular Biology and Pharmacology, Herbert Wertheim College of Medicine, Florida International University, 11200 SW 8th Street, HLSI 419B, Miami, FL 33199
| | - Elena Barnaeva
- NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville, MD 20850
| | - Emmett R. George
- NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville, MD 20850
| | - Alexander I. Agoulnik
- Department of Human and Molecular Genetics, Herbert Wertheim College of Medicine, Florida International University, 11200 SW 8th Street, HLSI 419B, Miami, FL 33199
| | - Juan J. Marugan
- NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville, MD 20850
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11
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Abstract
Stellate cells are resident lipid-storing cells of the pancreas and liver that transdifferentiate to a myofibroblastic state in the context of tissue injury. Beyond having roles in tissue homeostasis, stellate cells are increasingly implicated in pathological fibrogenic and inflammatory programs that contribute to tissue fibrosis and that constitute a growth-permissive tumor microenvironment. Although the capacity of stellate cells for extracellular matrix production and remodeling has long been appreciated, recent research efforts have demonstrated diverse roles for stellate cells in regulation of epithelial cell fate, immune modulation, and tissue health. Our present understanding of stellate cell biology in health and disease is discussed here, as are emerging means to target these multifaceted cells for therapeutic benefit.
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Affiliation(s)
- Mara H Sherman
- Department of Cell, Developmental & Cancer Biology, Oregon Health & Science University, Portland, Oregon 97201, USA;
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12
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Ezhilarasan D, Sokal E, Najimi M. Hepatic fibrosis: It is time to go with hepatic stellate cell-specific therapeutic targets. Hepatobiliary Pancreat Dis Int 2018; 17:192-197. [PMID: 29709350 DOI: 10.1016/j.hbpd.2018.04.003] [Citation(s) in RCA: 101] [Impact Index Per Article: 14.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/26/2017] [Accepted: 03/29/2018] [Indexed: 02/06/2023]
Abstract
Hepatic fibrosis is a pathological lesion, characterized by the progressive accumulation of extracellular matrix (ECM) in the perisinusoidal space and it is a major problem in chronic liver diseases. Phenotypic activation of hepatic stellate cells (HSC) plays a central role in the progression of hepatic fibrosis. Retardation of proliferation and clearance of activated HSCs from the injured liver is an appropriate therapeutic strategy for the resolution and treatment of hepatic fibrosis. Clearance of activated HSCs from the injured liver by autophagy inhibitors, proapoptotic agents and senescence inducers with the high affinity toward the activated HSCs may be the novel therapeutic strategy for the treatment of hepatic fibrosis in the near future.
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Affiliation(s)
- Devaraj Ezhilarasan
- Biomedical Research Unit and Laboratory Animal Centre, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences, Chennai 600 077, Tamil Nadu, India.
| | - Etienne Sokal
- Institut de Recherche Expérimentale et Clinique (IREC), Laboratory of Pediatric Hepatology and Cell Therapy, Université Catholique de Louvain, Brussels 1200, Belgium
| | - Mustapha Najimi
- Institut de Recherche Expérimentale et Clinique (IREC), Laboratory of Pediatric Hepatology and Cell Therapy, Université Catholique de Louvain, Brussels 1200, Belgium
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13
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Abstract
Hepatic stellate cells (HSCs) are found in the perisinusoidal space of the liver (i.e., the space of Dissé). They represent 5-8% of the total number of liver cells. In normal liver, these cells have a quiescent phenotype and are characterized by numerous fat vacuoles that store vitamin A in a form of retinyl ester. In injured liver, these cells transdifferentiate into a myofibroblast phenotype, become highly proliferative and are responsible for excess collagen synthesis and deposition during fibrosis. Due to their exceptional pathophysiological relevance, several isolation and purification protocols of primary HSCs have been established that provide the basis for studying HSC biology in vitro. We here describe a method for high-purity isolation of HSCs from mice. This protocol includes the enzymatic digestion of the liver tissue by pronase and collagenase, cellular enrichment by centrifugation of the crude cell suspension through a Nycodenz density gradient, and a final (optional) flow cytometric enrichment that allows generating ultrapure HSC fractions.
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14
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3D in vitro models of liver fibrosis. Adv Drug Deliv Rev 2017; 121:133-146. [PMID: 28697953 DOI: 10.1016/j.addr.2017.07.004] [Citation(s) in RCA: 101] [Impact Index Per Article: 12.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2017] [Revised: 06/27/2017] [Accepted: 07/06/2017] [Indexed: 02/07/2023]
Abstract
Animal testing is still the most popular preclinical assessment model for liver fibrosis. To develop efficient anti-fibrotic therapies, robust and representative in vitro models are urgently needed. The most widely used in vitro fibrosis model is the culture-induced activation of primary rodent hepatic stellate cells. While these cultures have contributed greatly to the current understanding of hepatic stellate cell activation, they seem to be inadequate to cover the complexity of this regenerative response. This review summarizes recent progress towards the development of 3D culture models of liver fibrosis. Thus far, only a few hepatic culture systems have successfully implemented hepatic stellate cells (or other non-parenchymal cells) into hepatocyte cultures. Recent advances in bioprinting, spheroid- and precision-cut liver slice cultures and the use of microfluidic bioreactors will surely lead to valid 3D in vitro models of liver fibrosis in the near future.
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15
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Metastasis 'systems' biology: how are macro-environmental signals transmitted into microenvironmental cues for disseminated tumor cells? Curr Opin Cell Biol 2017; 48:79-86. [PMID: 28715713 DOI: 10.1016/j.ceb.2017.06.002] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2017] [Revised: 06/07/2017] [Accepted: 06/08/2017] [Indexed: 01/01/2023]
Abstract
Disseminated breast tumor cells reside on or near stable microvascular endothelium. Currently, the cues that disrupt DTC dormancy and facilitate outgrowth are largely unknown. This article explores the hypothesis that specific patient lifestyle exposures (e.g., alcohol abuse) may disrupt the microenvironments that maintain disseminated tumor cell (DTC) dormancy in a tissue-specific fashion. We suggest that such exposures are 'transmitted' to the dormant niche in the form of injury. Thus, we discuss the relationship between wound healing and metastasis using liver as an example to illustrate how injury steers the phenotype of liver endothelium and perivascular hepatic stellate cells to a potentially pro-metastatic one. We posit further that non-steroidal anti-inflammatory drugs (NSAIDs) - recently shown to prevent metastatic relapse - may act by preserving the dormant niche. We conclude by suggesting that maintenance of the dormant niche - either through patient lifestyle or via development of therapeutics that mimic local molecular cues/responses that coincide with a healthy lifestyle - is a means to prevent metastatic relapse, and should be the subject of far greater research.
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16
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The stellate cell system (vitamin A-storing cell system). Anat Sci Int 2017; 92:387-455. [PMID: 28299597 DOI: 10.1007/s12565-017-0395-9] [Citation(s) in RCA: 73] [Impact Index Per Article: 9.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2016] [Accepted: 02/15/2017] [Indexed: 01/18/2023]
Abstract
Past, present, and future research into hepatic stellate cells (HSCs, also called vitamin A-storing cells, lipocytes, interstitial cells, fat-storing cells, or Ito cells) are summarized and discussed in this review. Kupffer discovered black-stained cells in the liver using the gold chloride method and named them stellate cells (Sternzellen in German) in 1876. Wake rediscovered the cells in 1971 using the same gold chloride method and various modern histological techniques including electron microscopy. Between their discovery and rediscovery, HSCs disappeared from the research history. Their identification, the establishment of cell isolation and culture methods, and the development of cellular and molecular biological techniques promoted HSC research after their rediscovery. In mammals, HSCs exist in the space between liver parenchymal cells (PCs) or hepatocytes and liver sinusoidal endothelial cells (LSECs) of the hepatic lobule, and store 50-80% of all vitamin A in the body as retinyl ester in lipid droplets in the cytoplasm. SCs also exist in extrahepatic organs such as pancreas, lung, and kidney. Hepatic (HSCs) and extrahepatic stellate cells (EHSCs) form the stellate cell (SC) system or SC family; the main storage site of vitamin A in the body is HSCs in the liver. In pathological conditions such as liver fibrosis, HSCs lose vitamin A, and synthesize a large amount of extracellular matrix (ECM) components including collagen, proteoglycan, glycosaminoglycan, and adhesive glycoproteins. The morphology of these cells also changes from the star-shaped HSCs to that of fibroblasts or myofibroblasts.
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17
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Fullár A, Firneisz G, Regős E, Dudás J, Szarvas T, Baghy K, Ramadori G, Kovalszky I. Response of Hepatic Stellate Cells to TGFB1 Differs from the Response of Myofibroblasts. Decorin Protects against the Action of Growth Factor. Pathol Oncol Res 2016; 23:287-294. [PMID: 27495255 DOI: 10.1007/s12253-016-0095-0] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/30/2016] [Accepted: 07/27/2016] [Indexed: 12/15/2022]
Abstract
Regardless to the exact nature of damage, hepatic stellate cells (HSCs) and other non-parenchymal liver cells transform to activated myofibroblasts, synthesizing the accumulating extracellular matrix (ECM) proteins, and transforming growth factor-β1 (TGF-β1) plays a crucial role in this process. Later it was discovered that decorin, member of the small leucin rich proteoglycan family is able to inhibit this action of TGF-β1. The aim of our present study was to clarify whether HSCs and activated myofibroblasts of portal region exert identical or different response to TGF-β1 exposure, and the inhibitory action of decorin against the growth factor is a generalized phenomenon on myofibroblast of different origin? To this end we measured mRNA expression and production of major collagen components (collagen type I, III and IV) of the liver after stimulation and co-stimulation with TGF-β1 and decorin in primary cell cultures of HSCs and myofibroblasts (MFs). Production of matrix proteins, decorin and members of the TGF-β1 signaling pathways were assessed on Western blots. Messenger RNA expression of collagens and TIEG was quantified by real-time RT-PCR. HSCs and MFs responded differently to TGF-β1 exposure. In contrast to HSCs in which TGF-β1 stimulated the synthesis of collagen type I, type III, and type IV, only the increase of collagen type IV was detected in portal MFs. However, in a combined treatment, decorin seemed to interfere with TGF-β1 and its stimulatory effect was abolished. The different mode of TGF-β1 action is mirrored by the different activation of signaling pathways in activated HSCs and portal fibroblasts. In HSCs the activation of pSMAD2 whereas in myofibroblasts the activation of MAPK pathway was detected. The inhibitory effect of decorin was neither related to the Smad-dependent nor to the Smad-independent signaling pathways.
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Affiliation(s)
- Alexandra Fullár
- 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Üllői út 26, Budapest, H-1085, Hungary
| | - Gábor Firneisz
- 2nd Department of Internal Medicine, Semmelweis University, Budapest, Hungary
| | - Eszter Regős
- 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Üllői út 26, Budapest, H-1085, Hungary
| | - József Dudás
- Department of Otorhinolaryngology, Medical University Innsbruck, Innsbruck, Austria
- Department of Gastroenterology and Endocrinology, George August University, Göttingen, Germany
| | - Tibor Szarvas
- 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Üllői út 26, Budapest, H-1085, Hungary
| | - Kornélia Baghy
- 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Üllői út 26, Budapest, H-1085, Hungary
| | - Giuliano Ramadori
- Department of Gastroenterology and Endocrinology, George August University, Göttingen, Germany
| | - Ilona Kovalszky
- 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Üllői út 26, Budapest, H-1085, Hungary.
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18
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Meyer J, Gonelle-Gispert C, Morel P, Bühler L. Methods for Isolation and Purification of Murine Liver Sinusoidal Endothelial Cells: A Systematic Review. PLoS One 2016; 11:e0151945. [PMID: 26992171 PMCID: PMC4798180 DOI: 10.1371/journal.pone.0151945] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2015] [Accepted: 03/07/2016] [Indexed: 12/14/2022] Open
Abstract
To study the biological functions of liver sinusoidal endothelial cells (LSEC) and to identify their interplay with blood or liver cells, techniques allowing for the isolation and purification of LSEC have been developed over the last decades. The objective of the present review is to summarize and to compare the efficiency of existing methods for isolating murine LSEC. Toward this end, the MEDLINE database was searched for all original articles describing LSEC isolation from rat and mouse livers. Out of the 489 publications identified, 23 reported the main steps and outcomes of the procedure and were included in our review. Here, we report and analyse the technical details of the essential steps of the techniques used for LSEC isolation. The correlations between the prevalence of some steps and the efficiency of LSEC isolation were also identified. We found that centrifugal elutriation, selective adherence and, more recently, magnetic-activated cell sorting were used for LSEC purification. Centrifugal elutriation procured high yields of pure LSEC (for rats 30-141.9 million cells for 85-98% purities; for mice 9-9.25 million cells for >95% purities), but the use of this method remained limited due to its high technical requirements. Selective adherence showed inconsistent results in terms of cell yields and purities in rats (5-100 million cells for 73.7-95% purities). In contrast, magnetic-activated cell sorting allowed for the isolation of highly pure LSEC, but overall lower cell yields were reported (for rats 10.7 million cells with 97.6% purity; for mice 0.5-9 million cells with 90-98% purities). Notably, the controversies regarding the accuracy of several phenotypic markers for LSEC should be considered and their use for both magnetic sorting and characterization remain doubtful. It appears that more effort is needed to refine and standardize the procedure for LSEC isolation, with a focus on the identification of specific antigens. Such a procedure is required to identify the molecular mechanisms regulating the function of LSEC and to improve our understanding of their role in complex cellular processes in the liver.
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Affiliation(s)
- Jeremy Meyer
- Division of Digestive and Transplantation Surgery, University Hospitals of Geneva, Rue Gabrielle-Perret-Gentil 4, 1211, Genève 14, Switzerland
- Unit of Surgical Research, University of Geneva, Rue Michel-Servet 1, 1206, Genève, Switzerland
| | - Carmen Gonelle-Gispert
- Unit of Surgical Research, University of Geneva, Rue Michel-Servet 1, 1206, Genève, Switzerland
| | - Philippe Morel
- Division of Digestive and Transplantation Surgery, University Hospitals of Geneva, Rue Gabrielle-Perret-Gentil 4, 1211, Genève 14, Switzerland
- Unit of Surgical Research, University of Geneva, Rue Michel-Servet 1, 1206, Genève, Switzerland
| | - Léo Bühler
- Division of Digestive and Transplantation Surgery, University Hospitals of Geneva, Rue Gabrielle-Perret-Gentil 4, 1211, Genève 14, Switzerland
- Unit of Surgical Research, University of Geneva, Rue Michel-Servet 1, 1206, Genève, Switzerland
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19
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Isolation and time lapse microscopy of highly pure hepatic stellate cells. Anal Cell Pathol (Amst) 2015; 2015:417023. [PMID: 26258009 PMCID: PMC4519541 DOI: 10.1155/2015/417023] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2015] [Revised: 06/23/2015] [Accepted: 06/24/2015] [Indexed: 12/11/2022] Open
Abstract
Hepatic stellate cells (HSC) are the main effector cells for liver fibrosis. We aimed at optimizing HSC isolation by an additional step of fluorescence-activated cell sorting (FACS) via a UV laser. HSC were isolated from livers of healthy mice and animals subjected to experimental fibrosis. HSC isolation by iohexol- (Nycodenz) based density centrifugation was compared to a method with subsequent FACS-based sorting. We assessed cellular purity, viability, morphology, and functional properties like proliferation, migration, activation marker, and collagen expression. FACS-augmented isolation resulted in a significantly increased purity of stellate cells (>99%) compared to iohexol-based density centrifugation (60–95%), primarily by excluding doublets of HSC and Kupffer cells (KC). Importantly, this method is also applicable to young animals and mice with liver fibrosis. Viability, migratory properties, and HSC transdifferentiation in vitro were preserved upon FACS-based isolation, as assessed using time lapse microscopy. During maturation of HSC in culture, we did not observe HSC cell division using time lapse microscopy. Strikingly, FACS-isolated, differentiated HSC showed very limited molecular and functional responses to LPS stimulation. In conclusion, isolating HSC from mouse liver by additional FACS significantly increases cell purity by removing contaminations from other cell populations especially KC, without affecting HSC viability, migration, or differentiation.
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20
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Greenhalgh SN, Conroy KP, Henderson NC. Cre-ativity in the liver: transgenic approaches to targeting hepatic nonparenchymal cells. Hepatology 2015; 61:2091-9. [PMID: 25412828 PMCID: PMC4657490 DOI: 10.1002/hep.27606] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/12/2014] [Accepted: 11/08/2014] [Indexed: 01/08/2023]
Abstract
Rapid evolution in transgenic (Tg) mouse technology now permits cell-specific and temporal control of fluorescent cell-labeling and gene inactivation. Here, we discuss the principal strategies that have been utilized to target, label, and manipulate hepatic nonparenchymal cells, with emphasis on the utility of constitutive and inducible Cre-lox systems. We summarize key findings of studies employing Tg technology to target hepatic stellate cells, myofibroblasts, liver sinusoidal endothelial cells, and macrophages to illustrate the power of these approaches in identifying cell-specific molecular mechanisms critical to the pathophysiology of liver disease. Increasing adoption of Tg techniques will help to answer fundamental questions regarding the pathogenesis of hepatic diseases and provide the mechanistic rationale to allow identification of novel drug targets, ultimately translating into effective therapies for patients with liver disease.
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Affiliation(s)
- Stephen N Greenhalgh
- MRC Center for Inflammation Research, The Queen’s Medical Research Institute, University of EdinburghEdinburgh, UK
| | - Kylie P Conroy
- MRC Center for Inflammation Research, The Queen’s Medical Research Institute, University of EdinburghEdinburgh, UK
| | - Neil C Henderson
- MRC Center for Inflammation Research, The Queen’s Medical Research Institute, University of EdinburghEdinburgh, UK,
Address reprint requests to: Neil Henderson, B.Sc. (Hons.), M.B.Ch.B., Ph.D., MRC Center for Inflammation Research, The Queen’s Medical Research Institute, University of Edinburgh, 47 Little France Crescent, Edinburgh, EH16 4TJ, UK. E-mail: ; fax: +44(0)131 2429101
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21
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Proliferative and oxidative response of hepatocytes (Hep) and hepatic stellate cells (HSC) isolated from rats exposed to ketogenic diet. Pol J Vet Sci 2015; 17:703-11. [PMID: 25638985 DOI: 10.2478/pjvs-2014-0102] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
Abstract
Ketogenic diet (KD) is considered in the context of its anti-epileptic effects, but its influence on liver dysfunction has not been elucidated yet. The study was aimed to investigate the activity of hepatocytes (Hep) and hepatic stellate cells (HSC) isolated from rats fed with KD, in respect of NO and superoxide generation by these cells as well as their proliferative activity in vitro. We also sought to characterize the plasma FFA profiles in control and ketogenic rats. Hep and HSC were isolated by the collagenase perfusion method and separated by the Percoll gradient centrifugation. After the 4th, 8th and 12th day of incubation, the media were collected for further analysis. NO generation increased within the time of incubation both in Hep and HSC isolated from KD-rats. In HSC group NO production raised significantly from 2.65 ± 0.07 μM/10(6) cells on 4th day of incubation to 5.49 ± 1.2 μM/10(6) cells on 12th day of incubation. In respect to O2⁻· generation experimental Hep and HSC provide considerably higher quantities of this free radical until 12th day of incubation (2.5 ± 0.07 and 3.2 ± 0.3 nM/10(6) cells, respectively). Although KD exerts anti-proliferative effect on hepatocytes, in respect to HSC it intensifies their proliferative activity. Furthermore, as we estimated on the basis of NO and O2⁻. generation both Hep and HSC exposed to KD are the source of free radicals.
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22
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Birbrair A, Zhang T, Wang ZM, Messi ML, Mintz A, Delbono O. Pericytes at the intersection between tissue regeneration and pathology. Clin Sci (Lond) 2015; 128:81-93. [PMID: 25236972 PMCID: PMC4200531 DOI: 10.1042/cs20140278] [Citation(s) in RCA: 177] [Impact Index Per Article: 17.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Perivascular multipotent cells, pericytes, contribute to the generation and repair of various tissues in response to injury. They are heterogeneous in their morphology, distribution, origin and markers, and elucidating their molecular and cellular differences may inform novel treatments for disorders in which tissue regeneration is either impaired or excessive. Moreover, these discoveries offer novel cellular targets for therapeutic approaches to many diseases. This review discusses recent studies that support the concept that pericyte subtypes play a distinctive role in myogenesis, neurogenesis, adipogenesis, fibrogenesis and angiogenesis.
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Affiliation(s)
- Alexander Birbrair
- Department of Internal Medicine-Gerontology, Wake Forest School of Medicine, Winston-Salem, North Carolina, Medical Center Boulevard, Winston Salem, NC 27157, U.S.A
- Neuroscience Program, Wake Forest School of Medicine, Winston-Salem, North Carolina, Medical Center Boulevard, Winston Salem, NC 27157, U.S.A
| | - Tan Zhang
- Department of Internal Medicine-Gerontology, Wake Forest School of Medicine, Winston-Salem, North Carolina, Medical Center Boulevard, Winston Salem, NC 27157, U.S.A
| | - Zhong-Min Wang
- Department of Internal Medicine-Gerontology, Wake Forest School of Medicine, Winston-Salem, North Carolina, Medical Center Boulevard, Winston Salem, NC 27157, U.S.A
| | - Maria Laura Messi
- Department of Internal Medicine-Gerontology, Wake Forest School of Medicine, Winston-Salem, North Carolina, Medical Center Boulevard, Winston Salem, NC 27157, U.S.A
| | - Akiva Mintz
- Department of Neurosurgery, Wake Forest School of Medicine, Winston-Salem, North Carolina, Medical Center Boulevard, Winston Salem, NC 27157, U.S.A
| | - Osvaldo Delbono
- Department of Internal Medicine-Gerontology, Wake Forest School of Medicine, Winston-Salem, North Carolina, Medical Center Boulevard, Winston Salem, NC 27157, U.S.A
- Neuroscience Program, Wake Forest School of Medicine, Winston-Salem, North Carolina, Medical Center Boulevard, Winston Salem, NC 27157, U.S.A
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23
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Abstract
Hepatic stellate cells are resident perisinusoidal cells distributed throughout the liver, with a remarkable range of functions in normal and injured liver. Derived embryologically from septum transversum mesenchyme, their precursors include submesothelial cells that invade the liver parenchyma from the hepatic capsule. In normal adult liver, their most characteristic feature is the presence of cytoplasmic perinuclear droplets that are laden with retinyl (vitamin A) esters. Normal stellate cells display several patterns of intermediate filaments expression (e.g., desmin, vimentin, and/or glial fibrillary acidic protein) suggesting that there are subpopulations within this parental cell type. In the normal liver, stellate cells participate in retinoid storage, vasoregulation through endothelial cell interactions, extracellular matrix homeostasis, drug detoxification, immunotolerance, and possibly the preservation of hepatocyte mass through secretion of mitogens including hepatocyte growth factor. During liver injury, stellate cells activate into alpha smooth muscle actin-expressing contractile myofibroblasts, which contribute to vascular distortion and increased vascular resistance, thereby promoting portal hypertension. Other features of stellate cell activation include mitogen-mediated proliferation, increased fibrogenesis driven by connective tissue growth factor, and transforming growth factor beta 1, amplified inflammation and immunoregulation, and altered matrix degradation. Evolving areas of interest in stellate cell biology seek to understand mechanisms of their clearance during fibrosis resolution by either apoptosis, senescence, or reversion, and their contribution to hepatic stem cell amplification, regeneration, and hepatocellular cancer.
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Affiliation(s)
- Juan E Puche
- Division of Liver Diseases, Icahn School of Medicine at Mount Sinai Hospital, New York, New York, New York
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24
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Chiu YW, Chao PY, Tsai CC, Chiou HL, Liu YC, Hung CC, Shih HC, Lai TJ, Liu JY. Ocimum gratissimum is Effective in Prevention against Liver Fibrosis in Vivo and in Vitro. THE AMERICAN JOURNAL OF CHINESE MEDICINE 2014; 42:833-52. [DOI: 10.1142/s0192415x14500530] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Ocimum gratissimum is a traditional herb commonly found in tropical regions, which prevents free radical damage and protects the liver from oxidative stress. In this study, we tested in vivo and in vitro the effectiveness of O. gratissimum extracts (OGEs) in anti-hepatic fibrosis in rats. Male Wistar rats were administered with carbon tetrachloride (CCl4) by intraperitoneal injection and varying amounts of oral injection of OGE doses (0–40 mg/kg body weight) for 8 weeks. Our experiments showed that OGE significantly reduced liver damage, including steatosis and fibrosis, in a dose-dependent manner, as well as significantly decreased the elevation in plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT). It also inhibited the formation of lipid peroxidative products during CCl4 treatment. Moreover, OGE-inhibited CCl4-induced liver collagen accumulation and promoted the expression of catalase, an anti-oxidative enzyme. The inhibition of fibrosis factors α-SMA expression was also observed. In primary cultures, OGE significantly inhibited the serum-induced activation of hepatic stellate cells (HSCs), and the expression of α-SMA and collagen α (I). These data suggest that O. gratissimum possesses anti-hepatic fibrosis properties via its anti-oxidative components.
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Affiliation(s)
- Yung-Wei Chiu
- Emergency Department and Center of Hyperbaric Oxygen Therapy, Tungs' Taichung MetroHarbor Hospital, Wuchi Dist., Taichung 43503, Taiwan
- Institute of Medicine, Chung Shan Medical University, Taichung 40201, Taiwan
| | - Pei-Yu Chao
- Department of Leisure Industry Management, National Chin-Yi University of Technology, Taiping City, Taichung County 41111, Taiwan
- Graduate Institute of Basic Medical Science, China Medical University Taichung 40402, Taiwan
| | - Chin-Chiu Tsai
- Department of Physiology, School of Medicine, Chung-Shan Medical University, Taichung 40201, Taiwan
| | - Hui-Ling Chiou
- College of Medical Science and Technology, Chung-Shan Medical University, Taichung 40201, Taiwan
| | - Yuh-Chyan Liu
- Institute of Biochemistry and Biotechnology, Chung-Shan Medical University, Taichung 40201, Taiwan
| | - Chuan-Chen Hung
- Institute of Biochemistry and Biotechnology, Chung-Shan Medical University, Taichung 40201, Taiwan
| | - Hung-Che Shih
- Department of Pharmacology, School of Medicine, College of Medical, Chung-Shan Medical University, Taichung 40201, Taiwan
| | - Te-Jen Lai
- Institute of Medicine, Chung Shan Medical University, Taichung 40201, Taiwan
- Department of Psychiatry, Chung Shan Medical University Hospital, Taichung 40201, Taiwan
| | - Jer-Yuh Liu
- Graduate Institute of Cancer Biology, China Medical University Taichung 40402, Taiwan
- Center for Molecular Medicine, China Medical University Hospital, Taichung 40402, Taiwan
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25
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Henderson NC, Arnold TD, Katamura Y, Giacomini MM, Rodriguez JD, McCarty JH, Pellicoro A, Raschperger E, Betsholtz C, Ruminski PG, Griggs DW, Prinsen MJ, Maher JJ, Iredale JP, Lacy-Hulbert A, Adams RH, Sheppard D. Targeting of αv integrin identifies a core molecular pathway that regulates fibrosis in several organs. Nat Med 2013; 19:1617-24. [PMID: 24216753 PMCID: PMC3855865 DOI: 10.1038/nm.3282] [Citation(s) in RCA: 688] [Impact Index Per Article: 57.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2012] [Accepted: 06/17/2013] [Indexed: 02/06/2023]
Abstract
Myofibroblasts are the major source of extracellular matrix components that accumulate during tissue fibrosis, and hepatic stellate cells (HSCs) are believed to be the major source of myofibroblasts in the liver. To date, robust systems to genetically manipulate these cells have not been developed. We report that Cre under control of the promoter of Pdgfrb (Pdgfrb-Cre) inactivates loxP-flanked genes in mouse HSCs with high efficiency. We used this system to delete the gene encoding α(v) integrin subunit because various α(v)-containing integrins have been suggested as central mediators of fibrosis in multiple organs. Such depletion protected mice from carbon tetrachloride-induced hepatic fibrosis, whereas global loss of β₃, β₅ or β₆ integrins or conditional loss of β₈ integrins in HSCs did not. We also found that Pdgfrb-Cre effectively targeted myofibroblasts in multiple organs, and depletion of the α(v) integrin subunit using this system was protective in other models of organ fibrosis, including pulmonary and renal fibrosis. Pharmacological blockade of α(v)-containing integrins by a small molecule (CWHM 12) attenuated both liver and lung fibrosis, including in a therapeutic manner. These data identify a core pathway that regulates fibrosis and suggest that pharmacological targeting of all α(v) integrins may have clinical utility in the treatment of patients with a broad range of fibrotic diseases.
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Affiliation(s)
- Neil C Henderson
- Lung Biology Center, Department of Medicine, University of California, San Francisco, California, USA
- MRC Centre for Inflammation Research, The Queen’s Medical Research Institute, University of Edinburgh, Edinburgh, UK
| | - Thomas D Arnold
- Department of Pediatrics, University of California, San Francisco, California, USA
| | - Yoshio Katamura
- Lung Biology Center, Department of Medicine, University of California, San Francisco, California, USA
| | - Marilyn M Giacomini
- Lung Biology Center, Department of Medicine, University of California, San Francisco, California, USA
| | - Juan D Rodriguez
- Lung Biology Center, Department of Medicine, University of California, San Francisco, California, USA
| | - Joseph H McCarty
- Department of Cancer Biology, University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA
| | - Antonella Pellicoro
- MRC Centre for Inflammation Research, The Queen’s Medical Research Institute, University of Edinburgh, Edinburgh, UK
| | - Elisabeth Raschperger
- Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden
- Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden
| | - Christer Betsholtz
- Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden
- Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden
| | - Peter G Ruminski
- Center for World Health and Medicine, Saint Louis University, Edward A. Doisy Research Center, St. Louis, Missouri, USA
| | - David W Griggs
- Center for World Health and Medicine, Saint Louis University, Edward A. Doisy Research Center, St. Louis, Missouri, USA
| | - Michael J Prinsen
- Center for World Health and Medicine, Saint Louis University, Edward A. Doisy Research Center, St. Louis, Missouri, USA
| | - Jacquelyn J Maher
- The Liver Center, Department of Medicine, University of California, San Francisco, California, USA
| | - John P Iredale
- MRC Centre for Inflammation Research, The Queen’s Medical Research Institute, University of Edinburgh, Edinburgh, UK
| | - Adam Lacy-Hulbert
- Program of Developmental Immunology, Department of Pediatrics, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA
| | - Ralf H Adams
- Department of Tissue Morphogenesis, Faculty of Medicine, Max Planck Institute for Molecular Biomedicine, University of Münster, Münster, Germany
| | - Dean Sheppard
- Lung Biology Center, Department of Medicine, University of California, San Francisco, California, USA
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26
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Mezaki Y, Morii M, Hebiguchi T, Yoshikawa K, Yamaguchi N, Miura M, Imai K, Yoshino H, Senoo H. Differential increases in the expression of intermediate filament proteins and concomitant morphological changes of transdifferentiating rat hepatic stellate cells observed in vitro. Acta Histochem Cytochem 2013; 46:137-43. [PMID: 24194627 PMCID: PMC3813821 DOI: 10.1267/ahc.13007] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2013] [Accepted: 08/30/2013] [Indexed: 01/17/2023] Open
Abstract
The primary function of hepatic stellate cells (HSCs) is the storage of vitamin A. However, they are also responsible for liver fibrosis and are therapeutic targets for treatment of liver cirrhosis. Among the many molecular markers that define quiescent or activated states of HSCs, the characteristics of type III intermediate filaments are of particular interest. Whereas vimentin and desmin are upregulated in activated HSCs, glial fibrillary acidic protein is downregulated in activated HSCs. The functional differences between vimentin and desmin are poorly understood. By time-course quantifications of several molecular markers for HSC activation, we observed that the expression of vimentin preceded that of desmin during the transdifferentiation of HSCs. The immunoreactivity of vimentin in transdifferentiated HSCs was more intense in perinuclear regions compared to that of desmin. We propose that the delayed expression of desmin following the expression of vimentin and the peripheral localization of desmin compared to vimentin are both related to the more extended phenotype of transdifferentiating HSCs observed in vitro.
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Affiliation(s)
- Yoshihiro Mezaki
- Department of Cell Biology and Morphology, Akita University Graduate School of Medicine
| | - Mayako Morii
- Department of Pediatric Surgery, Akita University Graduate School of Medicine
| | - Taku Hebiguchi
- Department of Pediatric Surgery, Akita University Graduate School of Medicine
| | - Kiwamu Yoshikawa
- Department of Cell Biology and Morphology, Akita University Graduate School of Medicine
| | - Noriko Yamaguchi
- Department of Cell Biology and Morphology, Akita University Graduate School of Medicine
| | - Mitsutaka Miura
- Department of Cell Biology and Morphology, Akita University Graduate School of Medicine
| | - Katsuyuki Imai
- Department of Cell Biology and Morphology, Akita University Graduate School of Medicine
| | - Hiroaki Yoshino
- Department of Pediatric Surgery, Akita University Graduate School of Medicine
| | - Haruki Senoo
- Department of Cell Biology and Morphology, Akita University Graduate School of Medicine
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Rockey DC, Weymouth N, Shi Z. Smooth muscle α actin (Acta2) and myofibroblast function during hepatic wound healing. PLoS One 2013; 8:e77166. [PMID: 24204762 PMCID: PMC3812165 DOI: 10.1371/journal.pone.0077166] [Citation(s) in RCA: 136] [Impact Index Per Article: 11.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2013] [Accepted: 08/30/2013] [Indexed: 01/18/2023] Open
Abstract
Smooth muscle α actin (Acta2) expression is largely restricted to smooth muscle cells, pericytes and specialized fibroblasts, known as myofibroblasts. Liver injury, associated with cirrhosis, induces transformation of resident hepatic stellate cells into liver specific myofibroblasts, also known as activated cells. Here, we have used in vitro and in vivo wound healing models to explore the functional role of Acta2 in this transformation. Acta2 was abundant in activated cells isolated from injured livers but was undetectable in quiescent cells isolated from normal livers. Both cellular motility and contraction were dramatically increased in injured liver cells, paralleled by an increase in Acta2 expression, when compared with quiescent cells. Inhibition of Acta2 using several different techniques had no effect on cytoplasmic actin isoform expression, but led to reduced cellular motility and contraction. Additionally, Acta2 knockdown was associated with a significant reduction in Erk1/2 phosphorylation compared to control cells. The data indicate that Acta2 is important specifically in myofibroblast cell motility and contraction and raise the possibility that the Acta2 cytoskeleton, beyond its structural importance in the cell, could be important in regulating signaling processes during wound healing in vivo.
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Affiliation(s)
- Don C. Rockey
- Department of Internal Medicine, Medical University of South Carolina, Charleston, South Carolina, United States of America
- * E-mail:
| | - Nate Weymouth
- Division of Digestive and Liver Diseases, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America
| | - Zengdun Shi
- Department of Internal Medicine, Medical University of South Carolina, Charleston, South Carolina, United States of America
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28
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Greenhalgh SN, Iredale JP, Henderson NC. Origins of fibrosis: pericytes take centre stage. F1000PRIME REPORTS 2013; 5:37. [PMID: 24049641 PMCID: PMC3768328 DOI: 10.12703/p5-37] [Citation(s) in RCA: 61] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Pericytes are ubiquitous perivascular cells that have recently attracted interest as potential myofibroblast precursors. In turn, myofibroblasts are the major source of extracellular matrix components that accumulate during tissue fibrosis. Given the worldwide burden of fibrotic disease and paucity of therapeutic options available to halt its progression, elucidating the origins of myofibroblasts is of prime importance. The advent of genetic strategies that permit fate-mapping of specific cell populations through permanent and heritable expression of reporter proteins has begun to shed light on the source of the fibrogenic myofibroblast. Here we discuss recent studies in multiple organs that highlight the central role of pericytes in the origins of fibrosis.
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29
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Henderson NC, Sheppard D. Integrin-mediated regulation of TGFβ in fibrosis. BIOCHIMICA ET BIOPHYSICA ACTA 2013; 1832:891-6. [PMID: 23046811 PMCID: PMC3573259 DOI: 10.1016/j.bbadis.2012.10.005] [Citation(s) in RCA: 163] [Impact Index Per Article: 13.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/09/2012] [Revised: 10/01/2012] [Accepted: 10/03/2012] [Indexed: 12/26/2022]
Abstract
Fibrosis is a major cause of morbidity and mortality worldwide. Currently, therapeutic options for tissue fibrosis are severely limited, and organ transplantation is the only effective treatment for end-stage fibrotic disease. However, demand for donor organs greatly outstrips supply, and so effective anti-fibrotic treatments are urgently required. In recent years, the integrin family of cell adhesion receptors has gained prominence as key regulators of chronic inflammation and fibrosis. Fibrosis models in multiple organs have demonstrated that integrins have profound effects on the fibrotic process. There is now abundant in vivo data demonstrating critical regulatory roles for integrins expressed on different cell types during tissue fibrogenesis. In this review, we will examine the ways in which integrins regulate these processes and discuss how the manipulation of integrins using function blocking antibodies and small molecule inhibitors may have clinical utility in the treatment of patients with a broad range of fibrotic diseases. This article is part of a Special Issue entitled: Fibrosis: Translation of basic research to human disease.
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Affiliation(s)
- Neil C Henderson
- MRC Centre for Inflammation Research, The Queen’s Medical Research Institute, University of Edinburgh, Edinburgh, UK
| | - Dean Sheppard
- Lung Biology Center, Department of Medicine, University of California, San Francisco, California, USA
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30
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Abstract
The end point of liver fibrosis in almost all chronic liver diseases is cirrhosis. Progression to cirrhosis is accompanied by vascular remodeling and regeneration with important functional and hemodynamic consequences that include development of portal hypertension and eventually decompensation and death. Fibrosis can regress following successful treatment of the underlying disease. However, most common fibrosis scoring systems are not equipped for assessing this aspect. Nodule size, septal width and fibrosis area seem to correlate with disease severity and progression in cirrhosis. Classification systems based on nodule size, septal width, and fibrosis area need to be further validated.
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Affiliation(s)
- Pierre Bedossa
- Department of Pathology, Beaujon Hospital and Clichy, University Denis Diderot, Clichy, Paris 7, France.
| | - Guadalupe Garcia-Tsao
- Section of Digestive Diseases, Department of Medicine, Yale University School of Medicine, New Haven, CT, USA
| | - Dhanpat Jain
- Department of Pathology, Yale University School of Medicine, New Haven, CT 06520-8023, USA
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31
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Iredale JP, Thompson A, Henderson NC. Extracellular matrix degradation in liver fibrosis: Biochemistry and regulation. Biochim Biophys Acta Mol Basis Dis 2012; 1832:876-83. [PMID: 23149387 DOI: 10.1016/j.bbadis.2012.11.002] [Citation(s) in RCA: 195] [Impact Index Per Article: 15.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2012] [Revised: 10/31/2012] [Accepted: 11/01/2012] [Indexed: 02/06/2023]
Abstract
Fibrosis is a highly conserved wound healing response and represents the final common pathway of virtually all chronic inflammatory injuries. Over the past 3 decades detailed analysis of hepatic extracellular matrix synthesis and degradation using approaches incorporating human disease, experimental animal models and cell culture have highlighted the extraordinarily dynamic nature of tissue repair and remodelling in this solid organ. Furthermore emerging studies of fibrosis in other organs demonstrate that basic common mechanisms exist, suggesting that bidirectionality of the fibrotic process may not solely be the preserve of the liver. In this review we will examine the cellular and molecular mechanisms that govern extracellular matrix degradation and fibrosis resolution, and highlight how manipulation of these processes may result in the development of effective anti-fibrotic therapies. This article is part of a Special Issue entitled: Fibrosis: Translation of basic research to human disease.
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Affiliation(s)
- John P Iredale
- MRC Centre for Inflammation Research, The Queen's Medical Research Institute, University of Edinburgh, Edinburgh, UK.
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32
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Troeger JS, Mederacke I, Gwak GY, Dapito DH, Mu X, Hsu CC, Pradere JP, Friedman RA, Schwabe RF. Deactivation of hepatic stellate cells during liver fibrosis resolution in mice. Gastroenterology 2012; 143:1073-83.e22. [PMID: 22750464 PMCID: PMC3848328 DOI: 10.1053/j.gastro.2012.06.036] [Citation(s) in RCA: 393] [Impact Index Per Article: 30.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/29/2011] [Revised: 06/13/2012] [Accepted: 06/14/2012] [Indexed: 02/07/2023]
Abstract
BACKGROUND & AIMS Activated hepatic stellate cells (HSCs), the main fibrogenic cell type in the liver, undergo apoptosis after cessation of liver injury, which contributes to resolution of fibrosis. In this study, we investigated whether HSC deactivation constitutes an additional mechanism of liver fibrosis resolution. METHODS HSC activation and deactivation were investigated by single-cell PCR and genetic tracking in transgenic mice that expressed a tamoxifen-inducible CreER under control of the endogenous vimentin promoter (Vimentin-CreER). RESULTS Single-cell quantitative polymerase chain reaction demonstrated activation of almost the entire HSC population in fibrotic livers, and a gradual decrease of HSC activation during fibrosis resolution, indicating deactivation of HSCs. Vimentin-CreER marked activated HSCs, demonstrated by a 6- to 16-fold induction of a membrane-bound green fluorescent protein (mGFP) Cre-reporter after injection of carbon tetrachloride, in liver and isolated HSCs, and a shift in localization of mGFP-marked HSCs from peri-sinusoidal to fibrotic septa. Tracking of mGFP-positive HSCs revealed the persistence of 40%-45% of mGFP expression in livers and isolated HSCs 30-45 days after carbon tetrachloride was no longer administered, despite normalization of fibrogenesis parameters; these findings confirm reversal of HSC activation. After fibrosis resolution, mGFP expression was observed again in desmin-positive peri-sinusoidal HSCs; no mGFP expression was detected in hepatocytes or cholangiocytes, excluding mesenchymal-epithelial transition. Notably, reverted HSCs remained in a primed state, with higher levels of responsiveness to fibrogenic stimuli. CONCLUSIONS In mice, reversal of HSC activation contributes to termination of fibrogenesis during fibrosis resolution, but results in higher responsiveness of reverted HSCs to recurring fibrogenic stimulation.
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Affiliation(s)
- Juliane S. Troeger
- Department of Medicine, Columbia University, College of Physicians and Surgeons, New York, NY
| | - Ingmar Mederacke
- Department of Medicine, Columbia University, College of Physicians and Surgeons, New York, NY
| | - Geum-Youn Gwak
- Department of Medicine, Columbia University, College of Physicians and Surgeons, New York, NY
| | - Dianne H. Dapito
- Department of Medicine, Columbia University, College of Physicians and Surgeons, New York, NY
- Institute of Human Nutrition, Columbia University, New York, NY
| | - Xueru Mu
- Department of Medicine, Columbia University, College of Physicians and Surgeons, New York, NY
| | - Christine C. Hsu
- Department of Medicine, Columbia University, College of Physicians and Surgeons, New York, NY
| | - Jean-Philippe Pradere
- Department of Medicine, Columbia University, College of Physicians and Surgeons, New York, NY
| | - Richard A. Friedman
- Herbert Irving Comprehensive Cancer Center, Columbia University, New York, NY 10032, USA
- Center for Computational Biology and Bioinformatics, Columbia University, New York, NY 10032, USA
| | - Robert F. Schwabe
- Department of Medicine, Columbia University, College of Physicians and Surgeons, New York, NY
- Institute of Human Nutrition, Columbia University, New York, NY
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Chouteau P, Defer N, Florimond A, Caldéraro J, Higgs M, Gaudin A, Mérour E, Dhumeaux D, Lerat H, Pawlotsky JM. Hepatitis C virus (HCV) protein expression enhances hepatic fibrosis in HCV transgenic mice exposed to a fibrogenic agent. J Hepatol 2012; 57:499-507. [PMID: 22613003 DOI: 10.1016/j.jhep.2012.04.019] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/17/2011] [Revised: 03/18/2012] [Accepted: 04/02/2012] [Indexed: 02/06/2023]
Abstract
BACKGROUND & AIMS During chronic HCV infection, activation of fibrogenesis appears to be principally related to local inflammation. However, the direct role of hepatic HCV protein expression in fibrogenesis remains unknown. METHODS We used transgenic mice expressing the full length HCV open reading frame exposed to a 'second hit' of the fibrogenic agent carbon tetrachloride (CCl(4)). Both acute and chronic liver injuries were induced in these mice by CCl(4) injections. Liver injury, expression of matrix re-modeling genes, reactive oxygen species (ROS), inflammation, hepatocyte proliferation, ductular reaction and hepatic progenitor cells (HPC) expansion were examined. RESULTS After CCl(4) treatment, HCV transgenic mice exhibited enhanced liver fibrosis, significant changes in matrix re-modeling genes and increased ROS production compared to wild type littermates despite no differences in the degree of local inflammation. This increase was accompanied by a decrease in hepatocyte proliferation, which appeared to be due to delayed hepatocyte entry into the S phase. A prominent ductular reaction and hepatic progenitor cell compartment expansion were observed in transgenic animals. These observations closely mirror those previously made in HCV-infected individuals. CONCLUSIONS Together, these results demonstrate that expression of the HCV proteins in hepatocytes contributes to the development of hepatic fibrosis in the presence of other fibrogenic agents. In the presence of CCl(4), HCV transgenic mice display an intra-hepatic re-organization of several key cellular actors in the fibrogenic process.
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EASL recognition awardee 2012: Professor Scott L. Friedman. J Hepatol 2012; 57:244-5. [PMID: 22682672 DOI: 10.1016/j.jhep.2012.05.003] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/08/2012] [Accepted: 05/08/2012] [Indexed: 12/04/2022]
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A nanofiber membrane maintains the quiescent phenotype of hepatic stellate cells. Dig Dis Sci 2012; 57:1152-62. [PMID: 22359192 DOI: 10.1007/s10620-012-2084-9] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/03/2011] [Accepted: 02/06/2012] [Indexed: 12/27/2022]
Abstract
BACKGROUND Hepatic stellate cells (HSC) play a major role in the progression of liver fibrosis. AIM The aim of our study was to investigate whether rat HSC cultured on a nanofiber membrane (NM) retain their quiescent phenotype during both short- and long-term culture and whether activated HSC revert to a quiescent form when re-cultured on NM. METHODS Rat HSC cultured for 1 day on plastic plates (PP) were used as quiescent HSC, while cells cultured for 1 week on PP were considered to be activated HSC. Quiescent or activated HSC were subsequently plated on PP or NM and cultured for an additional 4 days at which time their gene expression, stress fiber development, and growth factor production were determined. For long-term culture, HSC were grown on NM for 20 days and the cells then replated on PP and cultured for another 10 days. RESULTS Expression of marker genes for HSC activation, stress fiber development, and growth factor production were significantly lower in both quiescent and activated HSC cultured on NM than in those cultured on PP. After long-term culture on NM, activation marker gene expression and stress fiber development were still significantly lower in HSC than in PP, and HSC still retained the ability to activate when replated onto PP. CONCLUSIONS HSC cultured on NM retained quiescent characteristics after both short- and long-term culture while activated HSC reverted toward a quiescent state when cultured on NM. Cultures of HSC grown on NM are a useful in vitro model to investigate the mechanisms of activation and deactivation.
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Tacke F, Weiskirchen R. Update on hepatic stellate cells: pathogenic role in liver fibrosis and novel isolation techniques. Expert Rev Gastroenterol Hepatol 2012; 6:67-80. [PMID: 22149583 DOI: 10.1586/egh.11.92] [Citation(s) in RCA: 149] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/18/2023]
Abstract
Hepatic stellate cells (HSCs), also called Ito cells or lipocytes, are vitamin A-storing cells located in the Dissé space between hepatocytes and sinusoidal endothelial cells. Upon liver injury, these cells transdifferentiate into extracellular matrix-producing, highly proliferative myofibroblasts that promote hepatic fibrogenesis. Other possible collagen-producing cells in liver fibrosis include portal fibroblasts, bone marrow-derived cells (mesenchymal stem cells, fibrocytes and hematopoietic cells) and parenchymal cells undergoing epithelial-to-mesenchymal transition. Important factors and signaling pathways for HSC activation, as well as different functions of HSC during homeostasis and fibrosis, such as collagen production, secretion of cytokines and chemokines, immune modulation and changes in contractile features, as well as vitamin A storage capacity, have been identified in vitro and in vivo. Novel isolation techniques, specifically HSC sorting by FACS via autofluorescence and antibodies, will provide us with further opportunities to advance our understanding of HSC biology in health and disease.
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Affiliation(s)
- Frank Tacke
- Department of Medicine III RWTH, University Hospital Aachen, Aachen, Germany.
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38
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Tarrats N, Moles A, Morales A, García-Ruiz C, Fernández-Checa JC, Marí M. Critical role of tumor necrosis factor receptor 1, but not 2, in hepatic stellate cell proliferation, extracellular matrix remodeling, and liver fibrogenesis. Hepatology 2011; 54:319-27. [PMID: 21523796 PMCID: PMC3125435 DOI: 10.1002/hep.24388] [Citation(s) in RCA: 106] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
UNLABELLED Tumor necrosis factor (TNF) has been implicated in the progression of many chronic liver diseases leading to fibrosis; however, the role of TNF in fibrogenesis is controversial and the specific contribution of TNF receptors to hepatic stellate cell (HSC) activation remains to be established. Using HSCs from wild-type, TNF-receptor-1 (TNFR1) knockout, TNF-receptor-2 (TNFR2) knockout, or TNFR1/R2 double-knockout (TNFR-DKO) mice, we show that loss of both TNF receptors reduced procollagen-α1(I) expression, slowed down HSC proliferation, and impaired platelet-derived growth factor (PDGF)-induced promitogenic signaling in HSCs. TNFR-DKO HSCs exhibited decreased AKT phosphorylation and in vitro proliferation in response to PDGF. These effects were reproduced in TNFR1 knockout, but not TNFR2 knockout, HSCs. In addition, matrix metalloproteinase 9 (MMP-9) expression was dependent on TNF binding to TNFR1 in primary mouse HSCs. These results were validated in the human HSC cell line, LX2, using neutralizing antibodies against TNFR1 and TNFR2. Moreover, in vivo liver damage and fibrogenesis after bile-duct ligation were reduced in TNFR-DKO and TNFR1 knockout mice, compared to wild-type or TNFR2 knockout mice. CONCLUSION TNF regulates HSC biology through its binding to TNFR1, which is required for HSC proliferation and MMP-9 expression. These data indicate a regulatory role for TNF in extracellular matrix remodeling and liver fibrosis, suggesting that targeting TNFR1 may be of benefit to attenuate liver fibrogenesis.
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MESH Headings
- Animals
- Antibodies/immunology
- Antibodies/pharmacology
- Bile Ducts/physiopathology
- Cell Line
- Cell Proliferation
- Collagen Type I/metabolism
- Collagen Type I, alpha 1 Chain
- Disease Models, Animal
- Extracellular Matrix/physiology
- Hepatic Stellate Cells/pathology
- Hepatic Stellate Cells/physiology
- Humans
- Ligation
- Liver Cirrhosis/metabolism
- Liver Cirrhosis/pathology
- Liver Cirrhosis/physiopathology
- Matrix Metalloproteinase 9/metabolism
- Mice
- Mice, Inbred C57BL
- Mice, Knockout
- Phosphorylation
- Proto-Oncogene Proteins c-akt/metabolism
- Receptors, Tumor Necrosis Factor, Type I/genetics
- Receptors, Tumor Necrosis Factor, Type I/immunology
- Receptors, Tumor Necrosis Factor, Type I/physiology
- Receptors, Tumor Necrosis Factor, Type II/genetics
- Receptors, Tumor Necrosis Factor, Type II/immunology
- Receptors, Tumor Necrosis Factor, Type II/physiology
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Affiliation(s)
- Núria Tarrats
- IDIBAPS, Liver Unit-Hospital Clínic, CIBEREHD, and Department of Cell Death and Proliferation, IIBB-CSIC, 08036-Barcelona, Spain
| | - Anna Moles
- IDIBAPS, Liver Unit-Hospital Clínic, CIBEREHD, and Department of Cell Death and Proliferation, IIBB-CSIC, 08036-Barcelona, Spain
| | - Albert Morales
- IDIBAPS, Liver Unit-Hospital Clínic, CIBEREHD, and Department of Cell Death and Proliferation, IIBB-CSIC, 08036-Barcelona, Spain
| | - Carmen García-Ruiz
- IDIBAPS, Liver Unit-Hospital Clínic, CIBEREHD, and Department of Cell Death and Proliferation, IIBB-CSIC, 08036-Barcelona, Spain
| | - José C. Fernández-Checa
- IDIBAPS, Liver Unit-Hospital Clínic, CIBEREHD, and Department of Cell Death and Proliferation, IIBB-CSIC, 08036-Barcelona, Spain
- Research Center for Alcoholic Liver and Pancreatic Diseases, Keck School of Medicine of the University of Southern California, Los Angeles, CA, USA
| | - Montserrat Marí
- IDIBAPS, Liver Unit-Hospital Clínic, CIBEREHD, and Department of Cell Death and Proliferation, IIBB-CSIC, 08036-Barcelona, Spain
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39
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Effectiveness of the PPARγ agonist, GW570, in liver fibrosis. Inflamm Res 2010; 59:1061-71. [DOI: 10.1007/s00011-010-0226-0] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2009] [Revised: 05/31/2010] [Accepted: 06/07/2010] [Indexed: 01/18/2023] Open
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40
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Bosselut N, Housset C, Marcelo P, Rey C, Burmester T, Vinh J, Vaubourdolle M, Cadoret A, Baudin B. Distinct proteomic features of two fibrogenic liver cell populations: hepatic stellate cells and portal myofibroblasts. Proteomics 2010; 10:1017-28. [PMID: 20049859 DOI: 10.1002/pmic.200900257] [Citation(s) in RCA: 52] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
In chronic liver diseases, the accumulation of extracellular matrix leading to fibrosis is caused by myofibroblasts, the origins of which are debatable. We performed a comparative proteomic study to identify markers and gain insight into distinct functions of myofibroblasts derived either from hepatic stellate cells (HSCs) or from portal mesenchymal cells. After isolation from normal liver and culture in similar conditions, myofibroblastic HSCs (MF-HSCs) presented enlarged cytoplasms whereas portal myofibroblasts (PMFs) were more proliferative, and formed more stress fibers. The two cell types were subjected to comparative analyses by 2-D MS/MS. Six proteins were overexpressed in PMFs, with myofibroblast-related typical functions. Among them, cofilin-1 showed the greatest difference in expression and a lower pI than expected. Immunoblot demonstrated higher levels of phosphorylation, a modification of the protein implicated in stress fiber formation. Eleven proteins, mostly involved in stress response, were overexpressed in MF-HSCs. Cytoglobin had the highest level of overexpression, as confirmed by reverse transcription quantitative real-time PCR, immunoblot and immunocytochemical analyses. These results identify cytoglobin as the best marker for distinguishing MF-HSCs from PMFs and suggest different functions for the two cell populations in the liver wound healing response, with a prominent role for PMFs in scar formation.
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Affiliation(s)
- Nelly Bosselut
- AP-HP, Hôpital Saint-Antoine, Biochimie A, Paris, France
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41
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Hardjo M, Miyazaki M, Sakaguchi M, Masaka T, Ibrahim S, Kataoka K, Huh NH. Suppression of carbon tetrachloride-induced liver fibrosis by transplantation of a clonal mesenchymal stem cell line derived from rat bone marrow. Cell Transplant 2009; 18:89-99. [PMID: 19476212 DOI: 10.3727/096368909788237140] [Citation(s) in RCA: 54] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Transplantation of hepatocytes or bone marrow-derived cells has been shown to ameliorate liver fibrosis in animal models, but no direct comparison of relative efficiency has been made. The aim of this study was to compare the efficiency of a bone marrow-derived clonal mesenchymal stem cell line established by us (rBM25/S3) with that of its adipogenic or hepatogenic differentiation derivative for suppression of rat liver fibrosis. After induction of differentiation of rBM25/S3 cells into adipogenic or hepatogenic cells in culture, we intrasplenically transplanted the three types of cells into rats (3 x 10(7) cells/rat) before and 4 weeks after initiation of carbon tetrachloride treatment (1 ml/kg body weight twice a week for 8 weeks) to induce liver fibrosis. Undifferentiated rBM25/S3 cells were the most effective for suppression of liver fibrosis, followed by the adipogenic cells and hepatogenic cells. Expression levels of MMP-2 and MMP-9 were also highest in undifferentiated rBM25/S3 cells. These results indicate that bone marrow-derived clonal mesenchymal stem cell lines are useful for further mechanistic studies on cell-mediated suppression of liver fibrosis and that such cell lines will provide information on an appropriate cell source for transplantation therapy for cirrhosis.
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Affiliation(s)
- Marhaen Hardjo
- Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8558, Japan
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42
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Desmet VJ. Alcoholic liver disease. Histological features and evolution. ACTA MEDICA SCANDINAVICA. SUPPLEMENTUM 2009; 703:111-26. [PMID: 3911738 DOI: 10.1111/j.0954-6820.1985.tb08909.x] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
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43
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Khan F, Peltekian KM, Peterson TC. Effect of interferon-alpha, ribavirin, pentoxifylline, and interleukin-18 antibody on hepatitis C sera-stimulated hepatic stellate cell proliferation. J Interferon Cytokine Res 2009; 28:643-51. [PMID: 18844579 DOI: 10.1089/jir.2007.0123] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
Chronic hepatitis C virus (HCV) infection is a major cause of liver fibrosis ultimately leading to cirrhosis. Hepatic stellate cell (HSC) proliferation is crucial in fibrosis development. Current antiviral treatment for HCV involves interferon-alpha (IFN-alpha) and Ribavirin combination therapy. IL-18, a novel cytokine of the IL-1 family of cytokines, is involved in inflammation and may be important in HCV-related inflammation. We hypothesize that block of one of the crucial events will block fibrosis due to HCV. The effect of HCV patient sera with and without IFN-alpha, ribavirin, and IL-18 antibody on HSC proliferation was assessed by [(3)H]-thymidine incorporation assays. Western analysis was used to assess the effect of pentoxifylline (PTX) on c-Jun immediate early gene phosphorylation (p-c-Jun formation). We demonstrate that HCV patient sera-stimulated HSC proliferation. Ribavirin with or without IFN-alpha significantly decreased HCV sera-stimulated HSC proliferation by 50%. Western analysis revealed that HCV serum increased p-c-Jun levels, which were decreased with Ribavirin and PTX. ELISA results showed an elevation of IL-18 levels in HCV sera when compared to normal sera. IL-18 did not stimulate HSC proliferation. However, IL-18 antibody significantly decreased patient sera-stimulated HSC proliferation. In conclusion, Ribavirin decreased HSC proliferation and may act by decreasing p-c-Jun levels in HSCs. IL-18 alone did not stimulate HSC proliferation but IL-18 antibody decreased stimulation, suggesting that IL-18 may work in conjunction with some other factor to increase HSC proliferation.
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Affiliation(s)
- Fareeha Khan
- Departments of Medicine and Pharmacology, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada
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44
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Camino AM, Atorrasagasti C, Maccio D, Prada F, Salvatierra E, Rizzo M, Alaniz L, Aquino JB, Podhajcer OL, Silva M, Mazzolini G. Adenovirus-mediated inhibition of SPARC attenuates liver fibrosis in rats. J Gene Med 2009; 10:993-1004. [PMID: 18615449 DOI: 10.1002/jgm.1228] [Citation(s) in RCA: 48] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
Abstract
BACKGROUND The interaction between fibrogenic cells and extracellular matrix plays a role in liver fibrosis, yet the mechanisms are largely unknown. Secreted protein, acidic and rich in cysteine (SPARC) is a matricellular glycoprotein that is expressed by hepatic stellate cells and is overexpressed in fibrotic livers. We investigated the in vivo role of SPARC in experimentally induced liver fibrosis in rats. METHODS A recombinant adenovirus carrying antisense SPARC was constructed (AdasSPARC). Advanced liver fibrosis was induced in Sprague-Dawley rats by prolonged intraperitoneal administration of thioacetamide. Animals received injections of AdasSPARC or Ad beta gal (control adenovirus) via the tail vein and directly into the liver 1 week after the first dose. The pathological changes in liver tissues and indices of fibrosis were assessed at eight weeks. Expression of SPARC, transforming growth factor (TGF)-beta and alpha-smooth muscle actin were evaluated by quantitative real-time polymerase chain reaction, western blotting, enzyme-linked immunosorbent assay and immunohistochemistry. RESULTS Hepatic SPARC expression significantly increased during the development of liver fibrosis. AdasSPARC markedly attenuated the development of hepatic fibrosis in rats treated with thiocetamide, as assessed by decreased collagen deposition, lower hepatic content of hydroxyproline and less advanced morphometric stage of fibrosis. AdasSPARC treatment reduced inflammatory activity (Knodell score) and suppressed transdifferentiation of hepatic stellate cell to the myofibroblasts like phenotype in vivo. Furthermore, in vitro inhibition of SPARC on hepatic stellate cells decreases the production of TGF-beta. CONCLUSIONS This is the first study to demonstrate that knockdown of hepatic SPARC expression ameliorates thioacetamide-induced liver fibrosis in rats with chronic liver injury. SPARC is a potential target for gene therapy in liver fibrosis.
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Affiliation(s)
- Alejandra M Camino
- Gene Therapy Laboratory, Liver Unit, School of Medicine, Austral University, Buenos Aires, Argentina
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45
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Oh SW, Kim DH, Ha JR, Kim DY. Anti-fibrotic Effects of a Methylenedioxybenzene Compound, CW209292 on Dimethylnitrosamine-Induced Hepatic Fibrosis in Rats. Biol Pharm Bull 2009; 32:1364-70. [DOI: 10.1248/bpb.32.1364] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Affiliation(s)
- Se-Woong Oh
- Department of Veterinary Pathology, College of Veterinary Medicine, Seoul National University
- Central Research Institute, Choongwae Pharma Corp
| | - Dae-Hoon Kim
- Central Research Institute, Choongwae Pharma Corp
| | - Jong-Ryul Ha
- Central Research Institute, Choongwae Pharma Corp
| | - Dae-Yong Kim
- Department of Veterinary Pathology, College of Veterinary Medicine, Seoul National University
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46
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Vinculin and cellular retinol-binding protein-1 are markers for quiescent and activated hepatic stellate cells in formalin-fixed paraffin embedded human liver. Histochem Cell Biol 2008; 131:313-25. [PMID: 19052772 DOI: 10.1007/s00418-008-0544-2] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 11/13/2008] [Indexed: 12/15/2022]
Abstract
Hepatic stellate cells (HSCs) have important roles in the pathogenesis of liver fibrosis and cirrhosis. As response to chronic injury HSCs are activated and change from quiescent into myofibroblast-like cells. Several HSC-specific markers have been described in rat or mouse models. The aim of our work was to identify the best marker(s) for human HSCs. To this end we used the automated high throughput NexES IHC staining device (Ventana Medical Systems) to incubate sections under standardized conditions. Formalin fixed paraffin embedded (FFPE) normal and diseased human livers were studied. With immunohistochemistry we examined the expression of synemin, desmin, vimentin, vinculin, neurotrophin-3 (NT-3), alpha-smooth muscle actin (alpha-SMA), cellular retinol-binding protein-1 (CRBP-1), glial fibrillary acidic protein (GFAP), cysteine- and glycine-rich protein 2 (CRP2), and cytoglobin/stellate cell activation-associated protein (cygb/STAP). This is the first study in which a series of HSC markers is compared on serial FFPE human tissues. CRBP-1 clearly stains lobular HSCs without reacting with smooth muscle cells (SMCs) and shows variable cholangiocyte positivity. Vinculin has a similar staining pattern as CRBP-1 but additionally stains SMCs, and (myo)fibroblasts. In conclusion, we therefore propose to use CRBP-1 and/or vinculin to stain HSCs in human liver tissues.
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47
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Lee JS, Kim JH. [The role of activated hepatic stellate cells in liver fibrosis, portal hypertension and cancer angiogenesis]. THE KOREAN JOURNAL OF HEPATOLOGY 2008; 13:309-19. [PMID: 17898548 DOI: 10.3350/kjhep.2007.13.3.309] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
Abstract
Although hepatic stellate cells, which are liver specific pericytes, have been recognized within the vasculature of the sinusoid for more than one hundred years, the biology and function of these cells is unclear. Recent studies have highlighted the key role of stellate cells in a number of fundamental processes that include wound healing/fibrosis, vasoregulation, and vascular remodeling/angiogenesis. In the liver, these processes are particularly important in the development of cirrhosis, portal hypertension and cancer. This article highlights the recent advances in our understanding of the biology of hepatic stellate cells and discusses some of the recently-ascribed functions that are relevant to liver fibrosis, portal hypertension and cancer angiogenesis.
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Affiliation(s)
- June Sung Lee
- Department of Internal Medicine, Ilsan Paik Hospital, Inje University College of Medicine, Goyang, Korea.
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48
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Abdeen SM, Olusi SO, Askar HA, Thalib L, Al-Azemi A, George S. The predictive value of CD38 positive hepatic stellate cell count for assessing disease activity and fibrosis in patients with chronic hepatitis. Acta Histochem 2008; 111:520-30. [PMID: 18829073 DOI: 10.1016/j.acthis.2008.04.008] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2008] [Revised: 04/03/2008] [Accepted: 04/03/2008] [Indexed: 01/19/2023]
Abstract
The activation of hepatic stellate cells (HSCs) is a critical event in hepatic fibrosis. The objectives of this study were to find out if cluster of differentiation 38 (CD38) can be demonstrated immunohistochemically on HSCs in liver biopsies from patients with chronic liver disease and if CD38 immunopositive HSC count is correlated with METAVIR inflammatory and fibrosis scores. Immunohistochemical labelling for CD38 was performed on 100 liver biopsies from patients with chronic liver disease. The CD38 immunopositive HSCs were identified and counted. The CD38 immunopositive HSC count was found to be associated with both the METAVIR score and the fibrosis scores. The CD38 immunopositive HSC count was able to discriminate between no fibrosis and stages 2, 3 or 4 fibrosis, but could not discriminate between no fibrosis and stage 1 fibrosis. Using receiver operating characteristic (ROC) curves, a cut-off point of 10 HSCs per 10 high power field (hpf), or 25 per 100 hepatocytes, is 80% sensitive and 70% specific for predicting fibrosis. The specificity rose to 100% in patients with hepatitis C viral (HCV) infection. We conclude that CD38 positive HSCs can be demonstrated immunohistochemically and that the count is highly predictive of moderate to severe hepatic fibrosis.
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49
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Henderson NC, Forbes SJ. Hepatic fibrogenesis: from within and outwith. Toxicology 2008; 254:130-5. [PMID: 18824072 DOI: 10.1016/j.tox.2008.08.017] [Citation(s) in RCA: 48] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2008] [Revised: 08/21/2008] [Accepted: 08/23/2008] [Indexed: 01/11/2023]
Abstract
Liver disease is now the fifth commonest cause of death in the United Kingdom and the incidence is increasing. Chronic injury to the liver typically due to toxic insult, viral infection, immunological or metabolic diseases usually results in a stereotypical response with both parenchymal regeneration and wound healing. Chronic hepatic injury results in liver fibrosis with eventual progression to cirrhosis and end stage liver disease. At this point the majority of the clinical complications arise such as portal hypertension and the development of liver cancer. If the causative disease can be effectively treated the liver can regenerate and at the least partial resolution of liver fibrosis may occur. Unfortunately, unless the primary disease can be eradicated there are no specific anti-fibrotic treatments in routine clinical use. This highlights the urgent need to both increase our understanding of the mechanisms of hepatic fibrogenesis and to develop novel therapies to arrest or reverse the fibrotic process. This article initially outlines the main cellular pathway of fibrogenesis within the liver-the activation of the quiescent hepatic stellate cell into an activated myofibroblast phenotype, resulting in the production of fibrillar collagen. We will then discuss newly emerging sources of scar forming cells during hepatic injury together with the role of hepatic macrophages which have a regulatory function in both the formation and resolution of liver fibrosis.
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Affiliation(s)
- Neil C Henderson
- The Queen's Medical Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ, UK.
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50
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Svegliati-Baroni G, De Minicis S, Marzioni M. Hepatic fibrogenesis in response to chronic liver injury: novel insights on the role of cell-to-cell interaction and transition. Liver Int 2008; 28:1052-64. [PMID: 18783548 DOI: 10.1111/j.1478-3231.2008.01825.x] [Citation(s) in RCA: 86] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/13/2023]
Abstract
Hepatic fibrosis represents the wound-healing response process of the liver to chronic injury, independently from aetiology. Advanced liver fibrosis results in cirrhosis that can lead to liver failure, portal hypertension and hepatocellular carcinoma. Currently, no effective therapies are available for hepatic fibrosis. After the definition of hepatic stellate cells (HSCs) as the main liver extracellular matrix-producing cells in the 1980s, the subsequent decade was dedicated to determine the role of specific cytokines and growth factors. Fibrotic progression of chronic liver diseases can be nowadays considered as a dynamic and highly integrated process of cellular response to chronic liver injury. The present review is dedicated to the novel mechanisms of cellular response to chronic liver injury leading to hepatic myofibroblasts' activation. The understanding of the cellular and molecular pathways regulating their function is crucial to counteract therapeutically the organ dysfunction caused by myofibroblasts' activation.
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Affiliation(s)
- Gianluca Svegliati-Baroni
- Department of Gastroenterology, Università Politecnica delle Marche and Ospedali Riuniti University Hospital, Ancona, Italy.
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