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1
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Wilhelmsen I, Combriat T, Dalmao-Fernandez A, Stokowiec J, Wang C, Olsen PA, Wik JA, Boichuk Y, Aizenshtadt A, Krauss S. The effects of TGF-β-induced activation and starvation of vitamin A and palmitic acid on human stem cell-derived hepatic stellate cells. Stem Cell Res Ther 2024; 15:223. [PMID: 39044210 PMCID: PMC11267759 DOI: 10.1186/s13287-024-03852-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2024] [Accepted: 07/14/2024] [Indexed: 07/25/2024] Open
Abstract
BACKGROUND Hepatic stellate cells (HSC) have numerous critical roles in liver function and homeostasis, while they are also known for their importance during liver injury and fibrosis. There is therefore a need for relevant in vitro human HSC models to fill current knowledge gaps. In particular, the roles of vitamin A (VA), lipid droplets (LDs), and energy metabolism in human HSC activation are poorly understood. METHODS In this study, human pluripotent stem cell-derived HSCs (scHSCs), benchmarked to human primary HSC, were exposed to 48-hour starvation of retinol (ROL) and palmitic acid (PA) in the presence or absence of the potent HSC activator TGF-β. The interventions were studied by an extensive set of phenotypic and functional analyses, including transcriptomic analysis, measurement of activation-related proteins and cytokines, VA- and LD storage, and cell energy metabolism. RESULTS The results show that though the starvation of ROL and PA alone did not induce scHSC activation, the starvation amplified the TGF-β-induced activation-related transcriptome. However, TGF-β-induced activation alone did not lead to a reduction in VA or LD stores. Additionally, reduced glycolysis and increased mitochondrial fission were observed in response to TGF-β. CONCLUSIONS scHSCs are robust models for activation studies. The loss of VA and LDs is not sufficient for scHSC activation in vitro, but may amplify the TGF-β-induced activation response. Collectively, our work provides an extensive framework for studying human HSCs in healthy and diseased conditions.
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Affiliation(s)
- Ingrid Wilhelmsen
- Department of Immunology and Transfusion Medicine, Oslo University Hospital, P.O. Box 4950, Nydalen, Oslo, 0424, Norway.
- Hybrid Technology Hub - Centre of Excellence, Institute of Basic Medical Sciences, University of Oslo, P.O. Box 1110, Blindern, Oslo, 0317, Norway.
| | - Thomas Combriat
- Hybrid Technology Hub - Centre of Excellence, Institute of Basic Medical Sciences, University of Oslo, P.O. Box 1110, Blindern, Oslo, 0317, Norway
| | - Andrea Dalmao-Fernandez
- Department of Immunology and Transfusion Medicine, Oslo University Hospital, P.O. Box 4950, Nydalen, Oslo, 0424, Norway
- Hybrid Technology Hub - Centre of Excellence, Institute of Basic Medical Sciences, University of Oslo, P.O. Box 1110, Blindern, Oslo, 0317, Norway
- Section for Pharmacology and Pharmaceutical Biosciences, Department of Pharmacy, University of Oslo, P.O. Box 1068, Blindern, Oslo, 0316, Norway
| | - Justyna Stokowiec
- Hybrid Technology Hub - Centre of Excellence, Institute of Basic Medical Sciences, University of Oslo, P.O. Box 1110, Blindern, Oslo, 0317, Norway
| | - Chencheng Wang
- Hybrid Technology Hub - Centre of Excellence, Institute of Basic Medical Sciences, University of Oslo, P.O. Box 1110, Blindern, Oslo, 0317, Norway
- Department of Transplantation Medicine, Institute for Surgical Research, Oslo University Hospital, P.O. Box 4950, Nydalen, Oslo, 0424, Norway
| | - Petter Angell Olsen
- Department of Immunology and Transfusion Medicine, Oslo University Hospital, P.O. Box 4950, Nydalen, Oslo, 0424, Norway
- Hybrid Technology Hub - Centre of Excellence, Institute of Basic Medical Sciences, University of Oslo, P.O. Box 1110, Blindern, Oslo, 0317, Norway
| | - Jonas Aakre Wik
- Department of Immunology and Transfusion Medicine, Oslo University Hospital, P.O. Box 4950, Nydalen, Oslo, 0424, Norway
- Hybrid Technology Hub - Centre of Excellence, Institute of Basic Medical Sciences, University of Oslo, P.O. Box 1110, Blindern, Oslo, 0317, Norway
| | - Yuliia Boichuk
- Hybrid Technology Hub - Centre of Excellence, Institute of Basic Medical Sciences, University of Oslo, P.O. Box 1110, Blindern, Oslo, 0317, Norway
| | - Aleksandra Aizenshtadt
- Department of Immunology and Transfusion Medicine, Oslo University Hospital, P.O. Box 4950, Nydalen, Oslo, 0424, Norway
- Hybrid Technology Hub - Centre of Excellence, Institute of Basic Medical Sciences, University of Oslo, P.O. Box 1110, Blindern, Oslo, 0317, Norway
| | - Stefan Krauss
- Department of Immunology and Transfusion Medicine, Oslo University Hospital, P.O. Box 4950, Nydalen, Oslo, 0424, Norway
- Hybrid Technology Hub - Centre of Excellence, Institute of Basic Medical Sciences, University of Oslo, P.O. Box 1110, Blindern, Oslo, 0317, Norway
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2
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Wilhelmsen I, Amirola Martinez M, Stokowiec J, Wang C, Aizenshtadt A, Krauss S. Characterization of human stem cell-derived hepatic stellate cells and liver sinusoidal endothelial cells during extended in vitro culture. Front Bioeng Biotechnol 2023; 11:1223737. [PMID: 37560536 PMCID: PMC10408301 DOI: 10.3389/fbioe.2023.1223737] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2023] [Accepted: 07/05/2023] [Indexed: 08/11/2023] Open
Abstract
Background: There is a significant need for predictive and stable in vitro human liver representations for disease modeling and drug testing. Hepatic stellate cells (HSCs) and liver sinusoidal endothelial cells (LSECs) are important non-parenchymal cell components of the liver and are hence of relevance in a variety of disease models, including hepatic fibrosis. Pluripotent stem cell- (PSC-) derived HSCs (scHSCs) and LSECs (scLSECs) offer an attractive alternative to primary human material; yet, the suitability of scHSCs and scLSECs for extended in vitro modeling has not been characterized. Methods: In this study, we describe the phenotypic and functional development of scHSCs and scLSECs during 14 days of 2D in vitro culture. Cell-specific phenotypes were evaluated by cell morphology, immunofluorescence, and gene- and protein expression. Functionality was assessed in scHSCs by their capacity for intracellular storage of vitamin A and response to pro-fibrotic stimuli induced by TGF-β. scLSECs were evaluated by nitric oxide- and factor VIII secretion as well as endocytic uptake of bioparticles and acetylated low-density lipoprotein. Notch pathway inhibition and co-culturing scHSCs and scLSECs were separately tested as options for enhancing long-term stability and maturation of the cells. Results and Conclusion: Both scHSCs and scLSECs exhibited a post-differentiation cell type-specific phenotype and functionality but deteriorated during extended culture with PSC line-dependent variability. Therefore, the choice of PSC line and experimental timeframe is crucial when designing in vitro platforms involving scHSCs and scLSECs. Notch inhibition modestly improved long-term monoculture in a cell line-dependent manner, while co-culturing scHSCs and scLSECs provides a strategy to enhance phenotypic and functional stability.
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Affiliation(s)
- Ingrid Wilhelmsen
- Department of Immunology and Transfusion Medicine, Oslo University Hospital, Oslo, Norway
- Hybrid Technology Hub—Centre of Excellence, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway
| | - Mikel Amirola Martinez
- Hybrid Technology Hub—Centre of Excellence, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway
| | - Justyna Stokowiec
- Hybrid Technology Hub—Centre of Excellence, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway
| | - Chencheng Wang
- Hybrid Technology Hub—Centre of Excellence, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway
| | - Aleksandra Aizenshtadt
- Hybrid Technology Hub—Centre of Excellence, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway
| | - Stefan Krauss
- Department of Immunology and Transfusion Medicine, Oslo University Hospital, Oslo, Norway
- Hybrid Technology Hub—Centre of Excellence, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway
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3
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Cho SS, Yang JH, Lee JH, Baek JS, Ku SK, Cho IJ, Kim KM, Ki SH. Ferroptosis contribute to hepatic stellate cell activation and liver fibrogenesis. Free Radic Biol Med 2022; 193:620-637. [PMID: 36370962 DOI: 10.1016/j.freeradbiomed.2022.11.011] [Citation(s) in RCA: 28] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/14/2022] [Revised: 11/04/2022] [Accepted: 11/08/2022] [Indexed: 11/11/2022]
Abstract
Ferroptosis is a widely known regulator of cell death in connection with the redox state as a consequence of the depletion of glutathione or accumulation of lipid peroxidation. Hepatic stellate cells (HSCs) play a pivotal role in the progression of hepatic fibrosis by increasing the production and secretion of the extracellular matrix. However, the role of ferroptosis in HSC activation and liver fibrogenesis has not been clearly elucidated. The ferroptosis inducer RAS-selective lethal 3 (RSL3) or erastin treatment in HSCs caused cell death. This effect was suppressed only after exposure to ferroptosis inhibitors. We observed induction of ferroptosis by RSL3 treatment in HSCs supported by decreased glutathione peroxidase 4, glutathione deficiency, reactive oxygen species generation, or lipid peroxidation. Interestingly, RSL3 treatment upregulated the expression of plasminogen activator inhibitor-1, a representative fibrogenic marker of HSCs. In addition, treatment with ferroptosis-inducing compounds increased c-JUN phosphorylation and activator protein 1 luciferase activity but did not alter Smad phosphorylation and Smad-binding element luciferase activity. Chronic administration of iron dextran to mice causes ferroptosis of liver in vivo. The expression of fibrosis markers, such as alpha-smooth muscle actin and plasminogen activator inhibitor-1, was increased in the livers of mice with iron accumulation. Hepatic injury accompanying liver fibrosis was observed based on the levels of alanine aminotransferase, aspartate aminotransferase, and hematoxylin and eosin staining. Furthermore, we found that both isolated primary hepatocyte and HSCs undergo ferroptosis. Consistently, cirrhotic liver tissue of patients indicated glutathione peroxidase 4 downregulation in fibrotic region. In conclusion, our results suggest that ferroptosis contribute to HSC activation and the progression of hepatic fibrosis.
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Affiliation(s)
- Sam Seok Cho
- College of Pharmacy, Chosun University, Gwangju, 61452, Republic of Korea
| | - Ji Hye Yang
- College of Korean Medicine, Dongshin University, Naju, Jeollanam-do, 58245, Republic of Korea
| | - Ji Hyun Lee
- College of Pharmacy, Chosun University, Gwangju, 61452, Republic of Korea
| | - Jin Sol Baek
- College of Pharmacy, Chosun University, Gwangju, 61452, Republic of Korea
| | - Sae Kwang Ku
- College of Korean Medicine, Daegu Haany University, Gyeongsan, Gyeongsangbuk-do, 38610, Republic of Korea
| | - Il Je Cho
- College of Korean Medicine, Daegu Haany University, Gyeongsan, Gyeongsangbuk-do, 38610, Republic of Korea
| | - Kyu Min Kim
- College of Pharmacy, Chosun University, Gwangju, 61452, Republic of Korea; Department of Biomedical Science, College of Natural Science, Chosun University, Gwangju, 61452, Republic of Korea.
| | - Sung Hwan Ki
- College of Pharmacy, Chosun University, Gwangju, 61452, Republic of Korea.
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4
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Directed differentiation of human induced pluripotent stem cells to hepatic stellate cells. Nat Protoc 2021; 16:2542-2563. [PMID: 33864055 DOI: 10.1038/s41596-021-00509-1] [Citation(s) in RCA: 30] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2020] [Accepted: 01/21/2021] [Indexed: 12/20/2022]
Abstract
Hepatic stellate cells (HSCs) are nonparenchymal liver cells responsible for extracellular matrix homeostasis and are the main cells involved in the development of liver fibrosis following injury. The lack of reliable sources of HSCs has hence limited the development of complex in vitro systems to model liver diseases and toxicity. Here we describe a protocol to differentiate human induced pluripotent stem cells (iPSCs) into hepatic stellate cells (iPSC-HSCs). The protocol is based on the addition of several growth factors important for liver development sequentially over 12 d. iPSC-HSCs present phenotypic and functional characteristics of primary HSCs and can be expanded or frozen and used to perform high-throughput in vitro studies. We also describe how to coculture iPSC-HSCs with hepatocytes, which self-assemble into three-dimensional (3D) hepatic spheroids. This protocol enables the generation of HSC-like cells for in vitro modeling and drug screening studies.
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Abstract
Liver fibrosis is defined as excessive accumulation of extracellular matrix, and results from maladaptive wound healing processes that occur in response to chronic liver injury and inflammation. The main etiologies of liver fibrosis include nonalcoholic fatty liver disease (NAFLD), chronic viral hepatitis, as well as alcoholic and cholestatic liver disease. In patients, liver fibrosis typically develops over several decades and can progress to cirrhosis, and liver failure due to replacement of functional liver tissue with scar tissue. Additionally, advanced fibrosis and cirrhosis are associated with an increased risk for the development of hepatocellular carcinoma. On a cellular level, hepatic fibrosis is mediated by activated hepatic stellate cells, the primary fibrogenic cell type of the liver. Murine models are employed to recapitulate, understand, and therapeutically target mechanisms of fibrosis and hepatic stellate cell activation. Here, we summarize different mouse models of liver fibrosis focusing on the most commonly used models of toxic, biliary, and metabolically induced liver fibrosis, triggered by treatment with carbon tetrachloride (CCl4), thioacetamide (TAA), bile duct ligation (BDL), 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), and high-fat diets.
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Affiliation(s)
| | - Robert F Schwabe
- Department of Medicine, Columbia University, New York, NY, USA. .,Institute of Human Nutrition, Columbia University, 1130 St. Nicholas Avenue, ICRC 926, New York, NY, USA.
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6
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Hassan S, Sebastian S, Maharjan S, Lesha A, Carpenter AM, Liu X, Xie X, Livermore C, Zhang YS, Zarrinpar A. Liver-on-a-Chip Models of Fatty Liver Disease. Hepatology 2020; 71:733-740. [PMID: 31909504 PMCID: PMC7012755 DOI: 10.1002/hep.31106] [Citation(s) in RCA: 73] [Impact Index Per Article: 14.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/12/2019] [Accepted: 12/23/2019] [Indexed: 01/06/2023]
Abstract
As a consequence of the obesity epidemic and increasing incidence of metabolic syndrome fatty liver disease now affects a large portion of the world’s population. Left untreated, fatty liver disease can progress to more severe pathologic conditions such as cirrhosis and liver cancer. In an effort to probe the pathophysiology of fatty liver disease and its progression, research over the last decade has led to the engineering of in vitro models of the liver to aid in drug discovery and study of liver pathophysiology. In this review, we discuss advances in modeling liver tissue and the latest developments in understanding disease etiology and treatment from the perspective of engineered in vitro models spanning from conventional planar, static monolayer cultures to those based on the more recently developed bioprinted and liver-on-a-chip platforms. These technologies promise to transform basic biological research, the pharmaceutical industry, and clinical medicine of the liver.
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Affiliation(s)
- Shabir Hassan
- Division of Engineering in Medicine, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Cambridge, MA 02139, USA
| | - Shikha Sebastian
- Division of Engineering in Medicine, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Cambridge, MA 02139, USA
| | - Sushila Maharjan
- Division of Engineering in Medicine, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Cambridge, MA 02139, USA
| | - Ami Lesha
- Division of Engineering in Medicine, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Cambridge, MA 02139, USA
| | - Anne-Marie Carpenter
- Department of Surgery, College of Medicine, University of Florida, Gainesville, FL 32610, USA
| | - Xiuli Liu
- Department of Pathology, Immunology, and Laboratory Medicine, College of Medicine, University of Florida, Gainesville, FL 32610, USA
| | - Xin Xie
- Division of Engineering in Medicine, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Cambridge, MA 02139, USA
| | - Carol Livermore
- Micropower and Nanoengineering Lab, Department of Mechanical and Industrial Engineering, Northeastern University, Boston, MA 02115, USA
| | - Yu Shrike Zhang
- Division of Engineering in Medicine, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Cambridge, MA 02139, USA,Correspondence: (Y.S.Z.), (A.Z.)
| | - Ali Zarrinpar
- Department of Surgery, College of Medicine, University of Florida, Gainesville, FL 32610, USA,Department of Biochemistry and Molecular Biology, Department of Bioengineering, College of Medicine, University of Florida, Gainesville, FL 32610, USA,Correspondence: (Y.S.Z.), (A.Z.)
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7
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Wakashima T, Tanaka T, Fukui K, Komoda Y, Shinozaki Y, Kobayashi H, Matsuo A, Nangaku M. JTZ-951, an HIF prolyl hydroxylase inhibitor, suppresses renal interstitial fibroblast transformation and expression of fibrosis-related factors. Am J Physiol Renal Physiol 2019; 318:F14-F24. [PMID: 31630548 DOI: 10.1152/ajprenal.00323.2019] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Some preceding studies have provided evidence that hypoxia-inducible factor (HIF)-prolyl hydroxylase (PH) inhibitors have therapeutic potential against tubular interstitial fibrosis (TIF). Recently, transformation of renal interstitial fibroblasts (RIFs) into α-smooth muscle actin-positive myofibroblasts with loss of their hypoxia-inducible erythropoietin (EPO) expression has been hypothesized as the central mechanism responsible for TIF with renal anemia (the RIF hypothesis). These reports have suggested that HIF-PH inhibitors may suppress TIF via suppressing transformation of RIFs. However, the direct effect of HIF-PH inhibitors on transformation of RIFs has not been demonstrated because there has been no appropriate assay system. Here, we established a novel in vitro model of the transformation of RIFs. This model expresses key phenotypic changes such as transformation of RIFs accompanied by loss of their hypoxia-inducible EPO expression, as proposed by the RIF hypothesis. Using this model, we demonstrated that JTZ-951, a newly developed HIF-PH inhibitor, stabilized HIF protein in RIFs, suppressed transformation of RIFs, and maintained their hypoxia-inducible EPO expression. JTZ-951 also suppressed the expression of FGF2, FGF7, and FGF18, which are upregulated during transformation of RIFs. Furthermore, expression of Fgf2, Fgf7, and Fgf18 was correlated with TIF in an animal model of TIF. We also demonstrated that not only FGF2, which is a well-known growth-promoting factor, but also FGF18 promoted proliferation of RIFs. These data suggest that JTZ-951 has therapeutic potential against TIF with renal anemia. Furthermore, FGF2, FGF7, and FGF18, which faithfully reflect the anti-TIF effects of JTZ-951, have potential as TIF biomarkers.
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Affiliation(s)
- Takeshi Wakashima
- Division of Nephrology and Endocrinology, The University of Tokyo Graduate School of Medicine, Tokyo, Japan.,Biological and Pharmacological Laboratories, Central Pharmaceutical Research Institute, Japan Tobacco, Inc., Osaka, Japan
| | - Tetsuhiro Tanaka
- Division of Nephrology and Endocrinology, The University of Tokyo Graduate School of Medicine, Tokyo, Japan
| | - Kenji Fukui
- Division of Nephrology and Endocrinology, The University of Tokyo Graduate School of Medicine, Tokyo, Japan.,Biological and Pharmacological Laboratories, Central Pharmaceutical Research Institute, Japan Tobacco, Inc., Osaka, Japan
| | - Yasumasa Komoda
- Biological and Pharmacological Laboratories, Central Pharmaceutical Research Institute, Japan Tobacco, Inc., Osaka, Japan
| | - Yuichi Shinozaki
- Biological and Pharmacological Laboratories, Central Pharmaceutical Research Institute, Japan Tobacco, Inc., Osaka, Japan
| | - Hatsue Kobayashi
- Biological and Pharmacological Laboratories, Central Pharmaceutical Research Institute, Japan Tobacco, Inc., Osaka, Japan
| | - Akira Matsuo
- Biological and Pharmacological Laboratories, Central Pharmaceutical Research Institute, Japan Tobacco, Inc., Osaka, Japan
| | - Masaomi Nangaku
- Division of Nephrology and Endocrinology, The University of Tokyo Graduate School of Medicine, Tokyo, Japan
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8
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Yee C, Main NM, Terry A, Stevanovski I, Maczurek A, Morgan AJ, Calabro S, Potter AJ, Iemma TL, Bowen DG, Ahlenstiel G, Warner FJ, McCaughan GW, McLennan SV, Shackel NA. CD147 mediates intrahepatic leukocyte aggregation and determines the extent of liver injury. PLoS One 2019; 14:e0215557. [PMID: 31291257 PMCID: PMC6619953 DOI: 10.1371/journal.pone.0215557] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2019] [Accepted: 06/24/2019] [Indexed: 01/26/2023] Open
Abstract
Background Chronic inflammation is the driver of liver injury and results in progressive fibrosis and eventual cirrhosis with consequences including both liver failure and liver cancer. We have previously described increased expression of the highly multifunctional glycoprotein CD147 in liver injury. This work describes a novel role of CD147 in liver inflammation and the importance of leukocyte aggregates in determining the extent of liver injury. Methods Non-diseased, progressive injury, and cirrhotic liver from humans and mice were examined using a mAb targeting CD147. Inflammatory cell subsets were assessed by multiparameter flow cytometry. Results In liver injury, we observe abundant, intrahepatic leukocyte clusters defined as ≥5 adjacent CD45+ cells which we have termed “leukocyte aggregates”. We have shown that these leukocyte aggregates have a significant effect in determining the extent of liver injury. If CD147 is blocked in vivo, these leukocyte aggregates diminish in size and number, together with a marked significant reduction in liver injury including fibrosis. This is accompanied by no change in overall intrahepatic leukocyte numbers. Further, blocking of aggregation formation occurs prior to an appreciable increase in inflammatory markers or fibrosis. Additionally, there were no observed, “off-target” or unpredicted effects in targeting CD147. Conclusion CD147 mediates leukocyte aggregation which is associated with the development of liver injury. This is not a secondary effect, but a cause of injury as aggregate formation proceeds other markers of injury. Leukocyte aggregation has been previously described in inflammation dating back over many decades. Here we demonstrate that leukocyte aggregates determine the extent of liver injury.
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Affiliation(s)
- Christine Yee
- Centenary Institute of Cancer Medicine and Cell Biology, The University of Sydney, NSW, Australia
- Gastroenterology and Liver Laboratory, Ingham Institute for Applied Medical Research, Liverpool, NSW, Australia
| | - Nathan M. Main
- Gastroenterology and Liver Laboratory, Ingham Institute for Applied Medical Research, Liverpool, NSW, Australia
| | - Alexandra Terry
- Centenary Institute of Cancer Medicine and Cell Biology, The University of Sydney, NSW, Australia
- Gastroenterology and Liver Laboratory, Ingham Institute for Applied Medical Research, Liverpool, NSW, Australia
| | - Igor Stevanovski
- Gastroenterology and Liver Laboratory, Ingham Institute for Applied Medical Research, Liverpool, NSW, Australia
| | - Annette Maczurek
- Centenary Institute of Cancer Medicine and Cell Biology, The University of Sydney, NSW, Australia
| | - Alison J. Morgan
- Centenary Institute of Cancer Medicine and Cell Biology, The University of Sydney, NSW, Australia
| | - Sarah Calabro
- Centenary Institute of Cancer Medicine and Cell Biology, The University of Sydney, NSW, Australia
| | - Alison J. Potter
- Centenary Institute of Cancer Medicine and Cell Biology, The University of Sydney, NSW, Australia
| | - Tina L. Iemma
- Centenary Institute of Cancer Medicine and Cell Biology, The University of Sydney, NSW, Australia
| | - David G. Bowen
- Centenary Institute of Cancer Medicine and Cell Biology, The University of Sydney, NSW, Australia
- A.W. Morrow Gastroenterology and Liver Centre, Royal Prince Alfred Hospital, Camperdown, NSW, Australia
| | - Golo Ahlenstiel
- Western Sydney School of Medicine, Blacktown Hospital, Blacktown, NSW, Australia
| | - Fiona J. Warner
- Centenary Institute of Cancer Medicine and Cell Biology, The University of Sydney, NSW, Australia
| | - Geoffrey W. McCaughan
- Centenary Institute of Cancer Medicine and Cell Biology, The University of Sydney, NSW, Australia
- A.W. Morrow Gastroenterology and Liver Centre, Royal Prince Alfred Hospital, Camperdown, NSW, Australia
| | - Susan V. McLennan
- Department of Endocrinology, Department of Medicine and Bosch Institute, Royal Prince Alfred Hospital, The University of Sydney, NSW, Australia
| | - Nicholas A. Shackel
- Centenary Institute of Cancer Medicine and Cell Biology, The University of Sydney, NSW, Australia
- Gastroenterology and Liver Laboratory, Ingham Institute for Applied Medical Research, Liverpool, NSW, Australia
- A.W. Morrow Gastroenterology and Liver Centre, Royal Prince Alfred Hospital, Camperdown, NSW, Australia
- Liverpool Hospital, Liverpool, NSW, Australia
- * E-mail:
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9
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Genz B, Coleman MA, Irvine KM, Kutasovic JR, Miranda M, Gratte FD, Tirnitz-Parker JEE, Olynyk JK, Calvopina DA, Weis A, Cloonan N, Robinson H, Hill MM, Al-Ejeh F, Ramm GA. Overexpression of miRNA-25-3p inhibits Notch1 signaling and TGF-β-induced collagen expression in hepatic stellate cells. Sci Rep 2019; 9:8541. [PMID: 31189969 PMCID: PMC6561916 DOI: 10.1038/s41598-019-44865-1] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2018] [Accepted: 05/23/2019] [Indexed: 02/06/2023] Open
Abstract
During chronic liver injury hepatic stellate cells (HSCs), the principal source of extracellular matrix in the fibrotic liver, transdifferentiate into pro-fibrotic myofibroblast-like cells - a process potentially regulated by microRNAs (miRNAs). Recently, we found serum miRNA-25-3p (miR-25) levels were upregulated in children with Cystic Fibrosis (CF) without liver disease, compared to children with CF-associated liver disease and healthy individuals. Here we examine the role of miR-25 in HSC biology. MiR-25 was detected in the human HSC cell line LX-2 and in primary murine HSCs, and increased with culture-induced activation. Transient overexpression of miR-25 inhibited TGF-β and its type 1 receptor (TGFBR1) mRNA expression, TGF-β-induced Smad2 phosphorylation and subsequent collagen1α1 induction in LX-2 cells. Pull-down experiments with biotinylated miR-25 revealed Notch signaling (co-)activators ADAM-17 and FKBP14 as miR-25 targets in HSCs. NanoString analysis confirmed miR-25 regulation of Notch- and Wnt-signaling pathways. Expression of Notch signaling pathway components and endogenous Notch1 signaling was downregulated in miR-25 overexpressing LX-2 cells, as were components of Wnt signaling such as Wnt5a. We propose that miR-25 acts as a negative feedback anti-fibrotic control during HSC activation by reducing the reactivity of HSCs to TGF-β-induced collagen expression and modulating the cross-talk between Notch, Wnt and TGF-β signaling.
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Affiliation(s)
- Berit Genz
- Hepatic Fibrosis Group, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia.,Mater Research, Translational Research Institute, Brisbane, Queensland, Australia.,Faculty of Medicine, The University of Queensland, Brisbane, Queensland, Australia
| | - Miranda A Coleman
- Hepatic Fibrosis Group, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia
| | - Katharine M Irvine
- Mater Research, Translational Research Institute, Brisbane, Queensland, Australia.,Faculty of Medicine, The University of Queensland, Brisbane, Queensland, Australia
| | - Jamie R Kutasovic
- Faculty of Medicine, The University of Queensland, Brisbane, Queensland, Australia.,Personalised Medicine Team, QIMR-Berghofer Medical Research Institute, Brisbane, Queensland, Australia
| | - Mariska Miranda
- Personalised Medicine Team, QIMR-Berghofer Medical Research Institute, Brisbane, Queensland, Australia
| | - Francis D Gratte
- School of Pharmacy and Biomedical Sciences, Curtin Health Innovation Research Institute, Curtin University, Bentley, WA, Australia.,School of Veterinary and Life Sciences, Murdoch University, Perth, Western Australia, Australia
| | - Janina E E Tirnitz-Parker
- School of Pharmacy and Biomedical Sciences, Curtin Health Innovation Research Institute, Curtin University, Bentley, WA, Australia
| | - John K Olynyk
- Department of Gastroenterology & Hepatology, Fiona Stanley Fremantle Hospital Group, Murdoch, Western Australia, Australia.,School of Medical and Health Sciences, Edith Cowan University, Joondalup, Western Australia, Australia
| | - Diego A Calvopina
- Hepatic Fibrosis Group, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia
| | - Anna Weis
- Hepatic Fibrosis Group, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia
| | - Nicole Cloonan
- Genomic Biology Lab, QIMR-Berghofer Medical Research Institute, Brisbane, Queensland, Australia
| | - Harley Robinson
- Precision & Systems Biomedicine, QIMR-Berghofer Medical Research Institute, Brisbane, Queensland, Australia
| | - Michelle M Hill
- Precision & Systems Biomedicine, QIMR-Berghofer Medical Research Institute, Brisbane, Queensland, Australia
| | - Fares Al-Ejeh
- Personalised Medicine Team, QIMR-Berghofer Medical Research Institute, Brisbane, Queensland, Australia
| | - Grant A Ramm
- Hepatic Fibrosis Group, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia. .,Faculty of Medicine, The University of Queensland, Brisbane, Queensland, Australia.
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10
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Fernández-Colino A, Iop L, Ventura Ferreira MS, Mela P. Fibrosis in tissue engineering and regenerative medicine: treat or trigger? Adv Drug Deliv Rev 2019; 146:17-36. [PMID: 31295523 DOI: 10.1016/j.addr.2019.07.007] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2018] [Revised: 05/11/2019] [Accepted: 07/04/2019] [Indexed: 02/07/2023]
Abstract
Fibrosis is a life-threatening pathological condition resulting from a dysfunctional tissue repair process. There is no efficient treatment and organ transplantation is in many cases the only therapeutic option. Here we review tissue engineering and regenerative medicine (TERM) approaches to address fibrosis in the cardiovascular system, the kidney, the lung and the liver. These strategies have great potential to achieve repair or replacement of diseased organs by cell- and material-based therapies. However, paradoxically, they might also trigger fibrosis. Cases of TERM interventions with adverse outcome are also included in this review. Furthermore, we emphasize the fact that, although organ engineering is still in its infancy, the advances in the field are leading to biomedically relevant in vitro models with tremendous potential for disease recapitulation and development of therapies. These human tissue models might have increased predictive power for human drug responses thereby reducing the need for animal testing.
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11
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Marcher AB, Bendixen SM, Terkelsen MK, Hohmann SS, Hansen MH, Larsen BD, Mandrup S, Dimke H, Detlefsen S, Ravnskjaer K. Transcriptional regulation of Hepatic Stellate Cell activation in NASH. Sci Rep 2019; 9:2324. [PMID: 30787418 PMCID: PMC6382845 DOI: 10.1038/s41598-019-39112-6] [Citation(s) in RCA: 60] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2018] [Accepted: 01/17/2019] [Indexed: 12/17/2022] Open
Abstract
Non-alcoholic steatohepatitis (NASH) signified by hepatic steatosis, inflammation, hepatocellular injury, and fibrosis is a growing cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma. Hepatic fibrosis resulting from accumulation of extracellular matrix proteins secreted by hepatic myofibroblasts plays an important role in disease progression. Activated hepatic stellate cells (HSCs) have been identified as the primary source of myofibroblasts in animal models of hepatotoxic liver injury; however, so far HSC activation and plasticity have not been thoroughly investigated in the context of NASH-related fibrogenesis. Here we have determined the time-resolved changes in the HSC transcriptome during development of Western diet- and fructose-induced NASH in mice, a NASH model recapitulating human disease. Intriguingly, HSC transcriptional dynamics are highly similar across disease models pointing to HSC activation as a point of convergence in the development of fibrotic liver disease. Bioinformatic interrogation of the promoter sequences of activated genes combined with loss-of-function experiments indicates that the transcriptional regulators ETS1 and RUNX1 act as drivers of NASH-associated HSC plasticity. Taken together, our results implicate HSC activation and transcriptional plasticity as key aspects of NASH pathophysiology.
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Affiliation(s)
- Ann-Britt Marcher
- Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230, Odense M, Denmark
| | - Sofie M Bendixen
- Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230, Odense M, Denmark
| | - Mike K Terkelsen
- Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230, Odense M, Denmark
| | - Sonja S Hohmann
- Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230, Odense M, Denmark
| | - Maria H Hansen
- Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230, Odense M, Denmark
| | - Bjørk D Larsen
- Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230, Odense M, Denmark
| | - Susanne Mandrup
- Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230, Odense M, Denmark
| | - Henrik Dimke
- Department of Cardiovascular and Renal Research, Institute of Molecular Medicine, University of Southern Denmark, 5000, Odense C, Denmark
| | - Sönke Detlefsen
- Department of Pathology, Odense University Hospital, 5000, Odense C, Denmark
| | - Kim Ravnskjaer
- Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230, Odense M, Denmark.
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12
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Coll M, Perea L, Boon R, Leite SB, Vallverdú J, Mannaerts I, Smout A, El Taghdouini A, Blaya D, Rodrigo-Torres D, Graupera I, Aguilar-Bravo B, Chesne C, Najimi M, Sokal E, Lozano JJ, van Grunsven LA, Verfaillie CM, Sancho-Bru P. Generation of Hepatic Stellate Cells from Human Pluripotent Stem Cells Enables In Vitro Modeling of Liver Fibrosis. Cell Stem Cell 2018; 23:101-113.e7. [PMID: 30049452 DOI: 10.1016/j.stem.2018.05.027] [Citation(s) in RCA: 154] [Impact Index Per Article: 22.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2016] [Revised: 11/14/2017] [Accepted: 05/30/2018] [Indexed: 12/14/2022]
Abstract
The development of complex in vitro hepatic systems and artificial liver devices has been hampered by the lack of reliable sources for relevant cell types, such as hepatic stellate cells (HSCs). Here we report efficient differentiation of human pluripotent stem cells into HSC-like cells (iPSC-HSCs). iPSC-HSCs closely resemble primary human HSCs at the transcriptional, cellular, and functional levels and possess a gene expression profile intermediate between that of quiescent and activated HSCs. Functional analyses revealed that iPSC-HSCs accumulate retinyl esters in lipid droplets and are activated in response to mediators of wound healing, similar to their in vivo counterparts. When maintained as 3D spheroids with HepaRG hepatocytes, iPSC-HSCs exhibit a quiescent phenotype but mount a fibrogenic response and secrete pro-collagen in response to known stimuli and hepatocyte toxicity. Thus, this protocol provides a robust in vitro system for studying HSC development, modeling liver fibrosis, and drug toxicity screening.
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Affiliation(s)
- Mar Coll
- Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain; Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Spain
| | - Luis Perea
- Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain
| | - Ruben Boon
- Stem Cell Institute Leuven, Leuven, Belgium
| | - Sofia B Leite
- Liver Cell Biology Lab, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel (VUB), Brussels, Belgium
| | - Julia Vallverdú
- Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain
| | - Inge Mannaerts
- Liver Cell Biology Lab, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel (VUB), Brussels, Belgium
| | - Ayla Smout
- Liver Cell Biology Lab, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel (VUB), Brussels, Belgium
| | - Adil El Taghdouini
- Liver Cell Biology Lab, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel (VUB), Brussels, Belgium
| | - Delia Blaya
- Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain
| | - Daniel Rodrigo-Torres
- Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain
| | - Isabel Graupera
- Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain; Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Spain; Liver Unit, Hospital Clínic, Barcelona, Spain
| | - Beatriz Aguilar-Bravo
- Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain
| | | | - Mustapha Najimi
- Laboratory of Pediatric Hepatology and Cell Therapy, Institute of Experimental and Clinical Research (IREC), Université Catholique de Louvain (UCL), Leuven, Belgium
| | - Etienne Sokal
- Laboratory of Pediatric Hepatology and Cell Therapy, Institute of Experimental and Clinical Research (IREC), Université Catholique de Louvain (UCL), Leuven, Belgium
| | - Juan José Lozano
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Spain
| | - Leo A van Grunsven
- Liver Cell Biology Lab, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel (VUB), Brussels, Belgium.
| | | | - Pau Sancho-Bru
- Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain; Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Spain.
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13
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Caviglia JM, Yan J, Jang MK, Gwak GY, Affo S, Yu L, Olinga P, Friedman RA, Chen X, Schwabe RF. MicroRNA-21 and Dicer are dispensable for hepatic stellate cell activation and the development of liver fibrosis. Hepatology 2018; 67:2414-2429. [PMID: 29091291 PMCID: PMC5930143 DOI: 10.1002/hep.29627] [Citation(s) in RCA: 63] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/31/2016] [Revised: 10/20/2017] [Accepted: 10/30/2017] [Indexed: 12/21/2022]
Abstract
UNLABELLED Fibrosis and cancer represent two major complications of chronic liver disease. MicroRNAs have been implicated in the development of fibrosis and cancer, thus constituting potential therapeutic targets. Here, we investigated the role of microRNA-21 (miR-21), a microRNA that has been implicated in the development of fibrosis in multiple organs and has also been suggested to act as an "oncomir." Accordingly, miR-21 was the microRNA that showed the strongest up-regulation in activated hepatic stellate cells (HSCs) in multiple models of fibrogenesis, with an 8-fold to 24-fold induction compared to quiescent HSCs. However, miR-21 antisense inhibition did not suppress the activation of murine or human HSCs in culture or in liver slices. Moreover, genetic deletion of miR-21 in two independently generated knockout mice or miR-21 antisense inhibition did not alter HSC activation or liver fibrosis in models of toxic and biliary liver injury. Despite a strong up-regulation of miR-21 in injury-associated hepatocellular carcinoma and in cholangiocarcinoma, miR-21 deletion or antisense inhibition did not reduce the development of liver tumors. As inhibition of the most up-regulated microRNA did not affect HSC activation, liver fibrosis, or fibrosis-associated liver cancer, we additionally tested the role of microRNAs in HSCs by HSC-specific Dicer deletion. Although Dicer deletion decreased microRNA expression in HSCs and altered the expression of select genes, it only exerted negligible effects on HSC activation and liver fibrosis. CONCLUSION Genetic and pharmacologic manipulation of miR-21 does not inhibit the development of liver fibrosis and liver cancer. Moreover, suppression of microRNA synthesis does not significantly affect HSC phenotype and activation. (Hepatology 2018;67:2414-2429).
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Affiliation(s)
| | - Jun Yan
- Department of Medicine, Columbia University, New York, NY 10032, USA,Department of Pathology, Tianjin First Center Hospital, Tianjin, TJ 300192, China
| | - Myoung-Kuk Jang
- Department of Medicine, Columbia University, New York, NY 10032, USA,Department of Gastroenterology and Hepatology, Internal Medicine, Kangdong Sacred Heart Hospital of Hallym University Medical Center, Seoul, 05355, South Korea
| | - Geum-Youn Gwak
- Department of Medicine, Columbia University, New York, NY 10032, USA,Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, 06351, Korea
| | - Silvia Affo
- Department of Medicine, Columbia University, New York, NY 10032, USA
| | - Lexing Yu
- Department of Medicine, Columbia University, New York, NY 10032, USA
| | - Peter Olinga
- Division of Pharmaceutical Technology and Biopharmacy, Department of Pharmacy, University of Groningen, 9700 AB Groningen, The Netherlands
| | - Richard A. Friedman
- Biomedical Informatics Shared Resource, Herbert Irving Comprehensive Cancer Center and Department of Biomedical Informatics, Columbia University, New York, NY 10032, USA
| | - Xin Chen
- Department of Bioengineering and Therapeutic Sciences and Liver Center, University of California, San Francisco, CA, USA
| | - Robert F. Schwabe
- Department of Medicine, Columbia University, New York, NY 10032, USA
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14
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Benten D, Kluwe J, Wirth JW, Thiele ND, Follenzi A, Bhargava KK, Palestro CJ, Koepke M, Tjandra R, Volz T, Lutgehetmann M, Gupta S. A humanized mouse model of liver fibrosis following expansion of transplanted hepatic stellate cells. J Transl Med 2018; 98:525-536. [PMID: 29352225 PMCID: PMC6526950 DOI: 10.1038/s41374-017-0010-7] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2017] [Accepted: 11/06/2017] [Indexed: 02/06/2023] Open
Abstract
Hepatic stellate cells (HSCs) are major contributors to liver fibrosis, as hepatic injuries may cause their transdifferentiation into myofibroblast-like cells capable of producing excessive extracellular matrix proteins. Also, HSCs can modulate engraftment of transplanted hepatocytes and contribute to liver regeneration. Therefore, understanding the biology of human HSCs (hHSCs) is important, but effective methods have not been available to address their fate in vivo. To investigate whether HSCs could engraft and repopulate the liver, we transplanted GFP-transduced immortalized hHSCs into immunodeficient NOD/SCID mice. Biodistribution analysis with radiolabeled hHSCs showed that after intrasplenic injection, the majority of transplanted cells rapidly translocated to the liver. GFP-immunohistochemistry demonstrated that transplanted hHSCs engrafted alongside hepatic sinusoids. Prior permeabilization of the sinusoidal endothelial layer with monocrotaline enhanced engraftment of hHSCs. Transplanted hHSCs remained engrafted without relevant proliferation in the healthy liver. However, after CCl4 or bile duct ligation-induced liver damage, transplanted hHSCs expanded and contributed to extracellular matrix production, formation of bridging cell-septae and cirrhosis-like hepatic pseudolobules. CCl4-induced injury recruited hHSCs mainly to zone 3, whereas after bile duct ligation, hHSCs were mainly in zone 1 of the liver lobule. Transplanted hHSCs neither transdifferentiated into other cell types nor formed tumors in these settings. In conclusion, a humanized mouse model was generated by transplanting hHSCs, which proliferated during hepatic injury and inflammation, and contributed to liver fibrosis. The ability to repopulate the liver with transplanted hHSCs will be particularly significant for mechanistic studies of cell-cell interactions and fibrogenesis within the liver.
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Affiliation(s)
- Daniel Benten
- Departments of Medicine and Pathology, Marion Bessin Liver Research Center, Diabetes Center, Cancer Center, Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine Research, Institute for Clinical and Translational Research, Albert Einstein College of Medicine, Bronx, NY, USA. .,Department of Medicine, University Hospital Hamburg-Eppendorf, Hamburg, Germany. .,Helios Klinikum Duisburg, Duisburg, Germany.
| | - Johannes Kluwe
- Department of Medicine, University Hospital Hamburg-Eppendorf, Hamburg, Germany
| | - Jan W. Wirth
- Department of Medicine, University Hospital Hamburg-Eppendorf, Hamburg, Germany
| | - Nina D. Thiele
- Department of Medicine, University Hospital Hamburg-Eppendorf, Hamburg, Germany
| | - Antonia Follenzi
- Department of HealthSciences, Università del Piemonte Orientale “A. Avogadro”, Novara, Italy
| | - Kuldeep K. Bhargava
- Division of Nuclear Medicine and Molecular Imaging, Long Island Jewish Health Center, NorthWell Health, New Hyde Park, NY, USA
| | - Christopher J. Palestro
- Division of Nuclear Medicine and Molecular Imaging, Long Island Jewish Health Center, NorthWell Health, New Hyde Park, NY, USA
| | - Michael Koepke
- Department of Medicine, University Hospital Hamburg-Eppendorf, Hamburg, Germany
| | - Reni Tjandra
- Department of Medicine, University Hospital Hamburg-Eppendorf, Hamburg, Germany
| | - Tassilo Volz
- Department of Medicine, University Hospital Hamburg-Eppendorf, Hamburg, Germany
| | - Marc Lutgehetmann
- Department of Medicine, University Hospital Hamburg-Eppendorf, Hamburg, Germany
| | - Sanjeev Gupta
- Departments of Medicine and Pathology, Marion Bessin Liver Research Center, Diabetes Center, Cancer Center, Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine Research, Institute for Clinical and Translational Research, Albert Einstein College of Medicine, Bronx, NY, USA.
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15
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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: 89] [Impact Index Per Article: 11.1] [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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16
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Hong Y, Han YQ, Wang YZ, Gao JR, Li YX, Liu Q, Xia LZ. Paridis Rhizoma Sapoinins attenuates liver fibrosis in rats by regulating the expression of RASAL1/ERK1/2 signal pathway. JOURNAL OF ETHNOPHARMACOLOGY 2016; 192:114-122. [PMID: 27396351 DOI: 10.1016/j.jep.2016.07.010] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/21/2015] [Revised: 05/10/2016] [Accepted: 07/06/2016] [Indexed: 06/06/2023]
Abstract
ETHNO-PHARMACOLOGICAL RELEVANCE Paridis Rhizoma is a Chinese medicinal herb that has been used in liver disease treatment for thousands of years. Our previous studies found that Paridis Rhizoma saponins (PRS) are the critical components of Paridis Rhizoma which has good liver protection effect. However, the anti-hepatic fibrosis effect and the mechanism of PRS have seldom been reported. AIM OF THE STUDY To investigate the potential of PRS in the treatment of experimental liver fibrosis and the underlying mechanism. MATERIALS AND METHODS The chemical feature fingerprint of PRS was analyzed by UPLC-PDA. A total of 40 Male Sprague-Dawley (SD) rats were randomly divided into the control group, the model group, the PRS high dose group (PRS H) and the PRS low dose group (PRS L) with 10 rats in each group. The model, PRS H and L groups as liver fibrosis models were established with carbon tetrachloride (CCl4) method. PRS H and L groups were adopted PRS (300 and 150mg/kgd-1) treatment since the twelfth week of modeling till the sixteenth week. Pathological changes in hepatic tissue were examined using hematoxylin and eosin (H&E) and MASSON trichrome staining. Immunohistochemical analysis was performed to determine the protein expression of the RASAL1. RT-PCR and western blotting were used to detect the expression of ERK1/2 mRNA and protein. RESULTS Four saponins in PRS were identified from 19 detected chromatographic peaks on UPLC-PDA by comparing to the standard compounds. PRS can improve the degeneration and necrosis of hepatic tissue, reduce the extent of its fibrous hyperplasia according to H&E and MASSON staining detection. As was detected in PRS H and L groups, PRS down-regulated p-ERK1/2 mRNA and RASAL1 protein, and up-regulated the level of p-ERK1/2 mRNA and RASAL1 protein. CONCLUSION These results demonstrated that PRS can attenuate CCl4-induced liver fibrosis through the regulation of RAS/ERK1/2 signal pathway.
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MESH Headings
- Animals
- Blotting, Western
- Carbon Tetrachloride
- Chemical and Drug Induced Liver Injury/enzymology
- Chemical and Drug Induced Liver Injury/pathology
- Chemical and Drug Induced Liver Injury/prevention & control
- Chromatography, High Pressure Liquid
- Cytoprotection
- GTPase-Activating Proteins/genetics
- GTPase-Activating Proteins/metabolism
- Gene Expression Regulation, Enzymologic
- Hyperplasia
- Immunohistochemistry
- Liver/drug effects
- Liver/enzymology
- Liver/pathology
- Liver Cirrhosis, Experimental/chemically induced
- Liver Cirrhosis, Experimental/enzymology
- Liver Cirrhosis, Experimental/pathology
- Liver Cirrhosis, Experimental/prevention & control
- Male
- Melanthiaceae/chemistry
- Mitogen-Activated Protein Kinase 1/genetics
- Mitogen-Activated Protein Kinase 1/metabolism
- Mitogen-Activated Protein Kinase 3/genetics
- Mitogen-Activated Protein Kinase 3/metabolism
- Necrosis
- Phosphorylation
- Phytotherapy
- Plant Extracts/isolation & purification
- Plant Extracts/pharmacology
- Plants, Medicinal
- RNA, Messenger/genetics
- RNA, Messenger/metabolism
- Rats, Sprague-Dawley
- Reverse Transcriptase Polymerase Chain Reaction
- Saponins/isolation & purification
- Saponins/pharmacology
- Signal Transduction/drug effects
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Affiliation(s)
- Yan Hong
- Anhui University of Chinese Medicine, Hefei, Anhui 230031, China.
| | - Yan-Quan Han
- The First Affiliated Hospital, Anhui University of Chinese Medicine, Grade 3 Laboratory of TCM Preparation, State Administration of Anhui University of Chinese Medicine, Hefei, Anhui 230031, China.
| | - Yong-Zhong Wang
- The First Affiliated Hospital, Anhui University of Chinese Medicine, Grade 3 Laboratory of TCM Preparation, State Administration of Anhui University of Chinese Medicine, Hefei, Anhui 230031, China.
| | - Jia-Rong Gao
- The First Affiliated Hospital, Anhui University of Chinese Medicine, Grade 3 Laboratory of TCM Preparation, State Administration of Anhui University of Chinese Medicine, Hefei, Anhui 230031, China.
| | - Yu-Xin Li
- Anhui University of Chinese Medicine, Hefei, Anhui 230031, China; The First Affiliated Hospital, Anhui University of Chinese Medicine, Grade 3 Laboratory of TCM Preparation, State Administration of Anhui University of Chinese Medicine, Hefei, Anhui 230031, China.
| | - Qing Liu
- Anhui University of Chinese Medicine, Hefei, Anhui 230031, China; The First Affiliated Hospital, Anhui University of Chinese Medicine, Grade 3 Laboratory of TCM Preparation, State Administration of Anhui University of Chinese Medicine, Hefei, Anhui 230031, China.
| | - Lun-Zhu Xia
- The First Affiliated Hospital, Anhui University of Chinese Medicine, Grade 3 Laboratory of TCM Preparation, State Administration of Anhui University of Chinese Medicine, Hefei, Anhui 230031, China.
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17
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Morales-Ibanez O, Affò S, Rodrigo-Torres D, Blaya D, Millán C, Coll M, Perea L, Odena G, Knorpp T, Templin MF, Moreno M, Altamirano J, Miquel R, Arroyo V, Ginès P, Caballería J, Sancho-Bru P, Bataller R. Kinase analysis in alcoholic hepatitis identifies p90RSK as a potential mediator of liver fibrogenesis. Gut 2016; 65:840-51. [PMID: 25652085 PMCID: PMC4524790 DOI: 10.1136/gutjnl-2014-307979] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/15/2014] [Accepted: 12/30/2014] [Indexed: 12/20/2022]
Abstract
OBJECTIVE Alcoholic hepatitis (AH) is often associated with advanced fibrosis, which negatively impacts survival. We aimed at identifying kinases deregulated in livers from patients with AH and advanced fibrosis in order to discover novel molecular targets. DESIGN Extensive phosphoprotein analysis by reverse phase protein microarrays was performed in AH (n=12) and normal human livers (n=7). Ribosomal S6 kinase (p90RSK) hepatic expression was assessed by qPCR, Western blot and immunohistochemistry. Kaempferol was used as a selective pharmacological inhibitor of the p90RSK pathway to assess the regulation of experimentally-induced liver fibrosis and injury, using in vivo and in vitro approaches. RESULTS Proteomic analysis identified p90RSK as one of the most deregulated kinases in AH. Hepatic p90RSK gene and protein expression was also upregulated in livers with chronic liver disease. Immunohistochemistry studies showed increased p90RSK staining in areas of active fibrogenesis in cirrhotic livers. Therapeutic administration of kaempferol to carbon tetrachloride-treated mice resulted in decreased hepatic collagen deposition, and expression of profibrogenic and proinflammatory genes, compared to vehicle administration. In addition, kaempferol reduced the extent of hepatocellular injury and degree of apoptosis. In primary hepatic stellate cells, kaempferol and small interfering RNA decreased activation of p90RSK, which in turn regulated key profibrogenic actions. In primary hepatocytes, kaempferol attenuated proapoptotic signalling. CONCLUSIONS p90RSK is upregulated in patients with chronic liver disease and mediates liver fibrogenesis in vivo and in vitro. These results suggest that the p90RSK pathway could be a new therapeutic approach for liver diseases characterised by advanced fibrosis.
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Affiliation(s)
- Oriol Morales-Ibanez
- Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), CIBER de Enfermedades Hepáticas y Digestivas (CIBERehd), Facultat de Medicina, Universitat de Barcelona, Barcelona, Catalonia, Spain,Division of Gastroenterology and Hepatology, Departments of Medicine and Nutrition, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
| | - Silvia Affò
- Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), CIBER de Enfermedades Hepáticas y Digestivas (CIBERehd), Facultat de Medicina, Universitat de Barcelona, Barcelona, Catalonia, Spain
| | - Daniel Rodrigo-Torres
- Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), CIBER de Enfermedades Hepáticas y Digestivas (CIBERehd), Facultat de Medicina, Universitat de Barcelona, Barcelona, Catalonia, Spain
| | - Delia Blaya
- Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), CIBER de Enfermedades Hepáticas y Digestivas (CIBERehd), Facultat de Medicina, Universitat de Barcelona, Barcelona, Catalonia, Spain
| | - Cristina Millán
- Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), CIBER de Enfermedades Hepáticas y Digestivas (CIBERehd), Facultat de Medicina, Universitat de Barcelona, Barcelona, Catalonia, Spain
| | - Mar Coll
- Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), CIBER de Enfermedades Hepáticas y Digestivas (CIBERehd), Facultat de Medicina, Universitat de Barcelona, Barcelona, Catalonia, Spain
| | - Luis Perea
- Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), CIBER de Enfermedades Hepáticas y Digestivas (CIBERehd), Facultat de Medicina, Universitat de Barcelona, Barcelona, Catalonia, Spain
| | - Gemma Odena
- Division of Gastroenterology and Hepatology, Departments of Medicine and Nutrition, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA,Bowles Center for Alcohol Studies, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
| | - Thomas Knorpp
- Natural and Medical Sciences Institute at the University of Tübingen, Reutlingen, Baden-Württemberg, Germany
| | - Markus F Templin
- Natural and Medical Sciences Institute at the University of Tübingen, Reutlingen, Baden-Württemberg, Germany
| | - Montserrat Moreno
- Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), CIBER de Enfermedades Hepáticas y Digestivas (CIBERehd), Facultat de Medicina, Universitat de Barcelona, Barcelona, Catalonia, Spain
| | - José Altamirano
- Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), CIBER de Enfermedades Hepáticas y Digestivas (CIBERehd), Facultat de Medicina, Universitat de Barcelona, Barcelona, Catalonia, Spain,Liver Unit, Internal Medicine Department, Vall D’Hebron Institut de Recerca, Barcelona, Catalonia, Spain
| | - Rosa Miquel
- Pathology Unit, Hospital Clínic, Barcelona, Catalonia, Spain
| | - Vicente Arroyo
- Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), CIBER de Enfermedades Hepáticas y Digestivas (CIBERehd), Facultat de Medicina, Universitat de Barcelona, Barcelona, Catalonia, Spain,Liver Unit, Hospital Clínic, Barcelona, Catalonia, Spain
| | - Pere Ginès
- Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), CIBER de Enfermedades Hepáticas y Digestivas (CIBERehd), Facultat de Medicina, Universitat de Barcelona, Barcelona, Catalonia, Spain,Liver Unit, Hospital Clínic, Barcelona, Catalonia, Spain
| | - Juan Caballería
- Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), CIBER de Enfermedades Hepáticas y Digestivas (CIBERehd), Facultat de Medicina, Universitat de Barcelona, Barcelona, Catalonia, Spain,Liver Unit, Hospital Clínic, Barcelona, Catalonia, Spain
| | - Pau Sancho-Bru
- Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), CIBER de Enfermedades Hepáticas y Digestivas (CIBERehd), Facultat de Medicina, Universitat de Barcelona, Barcelona, Catalonia, Spain
| | - Ramon Bataller
- Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), CIBER de Enfermedades Hepáticas y Digestivas (CIBERehd), Facultat de Medicina, Universitat de Barcelona, Barcelona, Catalonia, Spain,Division of Gastroenterology and Hepatology, Departments of Medicine and Nutrition, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA,Bowles Center for Alcohol Studies, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
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18
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He YH, Li Z, Ni MM, Zhang XY, Li MF, Meng XM, Huang C, Li J. Cryptolepine derivative-6h inhibits liver fibrosis in TGF-β1-induced HSC-T6 cells by targeting the Shh pathway. Can J Physiol Pharmacol 2016; 94:987-95. [PMID: 27295431 DOI: 10.1139/cjpp-2016-0157] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Liver fibrosis is a worldwide problem with a significant morbidity and mortality. Cryptolepis sanguinolenta (family Periplocaceae) is widely used in West African countries for the treatment of malaria, as well as for some other diseases. However, the role of C. sanguinolenta in hepatic fibrosis is still unknown. It has been reported that Methyl-CpG binding protein 2 (MeCP2) had a high expression in liver fibrosis and played a central role in its pathobiology. Interestingly, we found that a cryptolepine derivative (HZ-6h) could inhibit liver fibrosis by reducing MeCP2 expression, as evidenced by the dramatic downregulation of α-smooth muscle actin (α-SMA) and type I collagen alpha-1 (Col1α1) in protein levels in vitro. Meanwhile, we also found that HZ-6h could reduce the cell viability and promote apoptosis of hepatic stellate cells (HSCs) treated with transforming growth factor beta 1(TGF-β1). Then, we investigated the potential molecular mechanisms and found that HZ-6h blocked Shh signaling in HSC-T6 cells, resulting in the decreased protein expression of Patched-1 (PTCH-1), Sonic hedgehog (Shh), and glioma-associated oncogene homolog 1 (GLI1). In short, these results indicate that HZ-6h inhibits liver fibrosis by downregulating MeCP2 through the Shh pathway in TGF-β1-induced HSC-T6 cells.
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Affiliation(s)
- Ying-Hua He
- a School of Pharmacy, Anhui Key Laboratory of Bioactivity of Natural Products, Anhui Medical University, Hefei 230032, China.,b Institute for Liver Diseases, Anhui Medical University, ILD-AMU, Hefei 230032, China.,c Anhui Institute of Innovative Drugs, Anhui Medical University, Hefei 230032, China
| | - Zeng Li
- a School of Pharmacy, Anhui Key Laboratory of Bioactivity of Natural Products, Anhui Medical University, Hefei 230032, China.,b Institute for Liver Diseases, Anhui Medical University, ILD-AMU, Hefei 230032, China.,c Anhui Institute of Innovative Drugs, Anhui Medical University, Hefei 230032, China
| | - Ming-Ming Ni
- a School of Pharmacy, Anhui Key Laboratory of Bioactivity of Natural Products, Anhui Medical University, Hefei 230032, China.,b Institute for Liver Diseases, Anhui Medical University, ILD-AMU, Hefei 230032, China.,c Anhui Institute of Innovative Drugs, Anhui Medical University, Hefei 230032, China
| | - Xing-Yan Zhang
- a School of Pharmacy, Anhui Key Laboratory of Bioactivity of Natural Products, Anhui Medical University, Hefei 230032, China.,b Institute for Liver Diseases, Anhui Medical University, ILD-AMU, Hefei 230032, China.,c Anhui Institute of Innovative Drugs, Anhui Medical University, Hefei 230032, China
| | - Ming-Fang Li
- a School of Pharmacy, Anhui Key Laboratory of Bioactivity of Natural Products, Anhui Medical University, Hefei 230032, China.,b Institute for Liver Diseases, Anhui Medical University, ILD-AMU, Hefei 230032, China.,c Anhui Institute of Innovative Drugs, Anhui Medical University, Hefei 230032, China
| | - Xiao-Ming Meng
- a School of Pharmacy, Anhui Key Laboratory of Bioactivity of Natural Products, Anhui Medical University, Hefei 230032, China.,b Institute for Liver Diseases, Anhui Medical University, ILD-AMU, Hefei 230032, China.,c Anhui Institute of Innovative Drugs, Anhui Medical University, Hefei 230032, China
| | - Cheng Huang
- a School of Pharmacy, Anhui Key Laboratory of Bioactivity of Natural Products, Anhui Medical University, Hefei 230032, China.,b Institute for Liver Diseases, Anhui Medical University, ILD-AMU, Hefei 230032, China.,c Anhui Institute of Innovative Drugs, Anhui Medical University, Hefei 230032, China
| | - Jun Li
- a School of Pharmacy, Anhui Key Laboratory of Bioactivity of Natural Products, Anhui Medical University, Hefei 230032, China.,b Institute for Liver Diseases, Anhui Medical University, ILD-AMU, Hefei 230032, China.,c Anhui Institute of Innovative Drugs, Anhui Medical University, Hefei 230032, China
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19
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Single cell analysis in native tissue: Quantification of the retinoid content of hepatic stellate cells. Sci Rep 2016; 6:24155. [PMID: 27063397 PMCID: PMC4827054 DOI: 10.1038/srep24155] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2015] [Accepted: 03/18/2016] [Indexed: 01/13/2023] Open
Abstract
Hepatic stellate cells (HSCs) are retinoid storing cells in the liver: The retinoid content of those cells changes depending on nutrition and stress level. There are also differences with regard to a HSC’s anatomical position in the liver. Up to now, retinoid levels were only accessible from bulk measurements of tissue homogenates or cell extracts. Unfortunately, they do not account for the intercellular variability. Herein, Raman spectroscopy relying on excitation by the minimally destructive wavelength 785 nm is introduced for the assessment of the retinoid state of single HSCs in freshly isolated, unprocessed murine liver lobes. A quantitative estimation of the cellular retinoid content is derived. Implications of the retinoid content on hepatic health state are reported. The Raman-based results are integrated with histological assessments of the tissue samples. This spectroscopic approach enables single cell analysis regarding an important cellular feature in unharmed tissue.
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20
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El Taghdouini A, Sørensen AL, Reiner AH, Coll M, Verhulst S, Mannaerts I, Øie CI, Smedsrød B, Najimi M, Sokal E, Luttun A, Sancho-Bru P, Collas P, van Grunsven LA. Genome-wide analysis of DNA methylation and gene expression patterns in purified, uncultured human liver cells and activated hepatic stellate cells. Oncotarget 2015; 6:26729-45. [PMID: 26353929 PMCID: PMC4694948 DOI: 10.18632/oncotarget.4925] [Citation(s) in RCA: 51] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2015] [Accepted: 08/20/2015] [Indexed: 12/15/2022] Open
Abstract
BACKGROUND & AIMS Liver fibrogenesis - scarring of the liver that can lead to cirrhosis and liver cancer - is characterized by hepatocyte impairment, capillarization of liver sinusoidal endothelial cells (LSECs) and hepatic stellate cell (HSC) activation. To date, the molecular determinants of a healthy human liver cell phenotype remain largely uncharacterized. Here, we assess the transcriptome and the genome-wide promoter methylome specific for purified, non-cultured human hepatocytes, LSECs and HSCs, and investigate the nature of epigenetic changes accompanying transcriptional changes associated with activation of HSCs. MATERIAL AND METHODS Gene expression profile and promoter methylome of purified, uncultured human liver cells and culture-activated HSCs were respectively determined using Affymetrix HG-U219 genechips and by methylated DNA immunoprecipitation coupled to promoter array hybridization. Histone modification patterns were assessed at the single-gene level by chromatin immunoprecipitation and quantitative PCR. RESULTS We unveil a DNA-methylation-based epigenetic relationship between hepatocytes, LSECs and HSCs despite their distinct ontogeny. We show that liver cell type-specific DNA methylation targets early developmental and differentiation-associated functions. Integrative analysis of promoter methylome and transcriptome reveals partial concordance between DNA methylation and transcriptional changes associated with human HSC activation. Further, we identify concordant histone methylation and acetylation changes in the promoter and putative novel enhancer elements of genes involved in liver fibrosis. CONCLUSIONS Our study provides the first epigenetic blueprint of three distinct freshly isolated, human hepatic cell types and of epigenetic changes elicited upon HSC activation.
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Affiliation(s)
- Adil El Taghdouini
- Liver Cell Biology Lab, Vrije Universiteit Brussel (VUB), Brussels, Belgium
| | - Anita L. Sørensen
- Department of Molecular medicine, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Oslo, Norway
| | - Andrew H. Reiner
- Department of Molecular medicine, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Oslo, Norway
| | - Mar Coll
- Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain
| | - Stefaan Verhulst
- Liver Cell Biology Lab, Vrije Universiteit Brussel (VUB), Brussels, Belgium
| | - Inge Mannaerts
- Liver Cell Biology Lab, Vrije Universiteit Brussel (VUB), Brussels, Belgium
| | - Cristina I. Øie
- Department of Medical Biology, Vascular Biology Research Group, UiT, The Arctic University of Norway, Tromsø, Norway
| | - Bård Smedsrød
- Department of Medical Biology, Vascular Biology Research Group, UiT, The Arctic University of Norway, Tromsø, Norway
| | - Mustapha Najimi
- Université Catholique de Louvain, Institut de Recherche Expérimentale et Clinique (IREC), Laboratory of Pediatric Hepatology and Cell Therapy, Brussels, Belgium
| | - Etienne Sokal
- Université Catholique de Louvain, Institut de Recherche Expérimentale et Clinique (IREC), Laboratory of Pediatric Hepatology and Cell Therapy, Brussels, Belgium
| | - Aernout Luttun
- Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, Katholieke Universiteit Leuven (KU Leuven), Leuven, Belgium
| | - Pau Sancho-Bru
- Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain
| | - Philippe Collas
- Department of Molecular medicine, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Oslo, Norway
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21
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Isoform-specific dynamic translocation of PKC by α1-adrenoceptor stimulation in live cells. Biochem Biophys Res Commun 2015; 465:464-70. [PMID: 26277396 DOI: 10.1016/j.bbrc.2015.08.040] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2015] [Accepted: 08/09/2015] [Indexed: 11/23/2022]
Abstract
Protein kinase C (PKC) plays key roles in the regulation of signal transduction and cellular function in various cell types. At least ten PKC isoforms have been identified and intracellular localization and trafficking of these individual isoforms are important for regulation of enzyme activity and substrate specificity. PKC can be activated downstream of Gq-protein coupled receptor (GqPCR) signaling and translocate to various cellular compartments including plasma membrane (PM). Recent reports suggested that different types of GqPCRs would activate different PKC isoforms (classic, novel and atypical PKCs) with different trafficking patterns. However, the knowledge of isoform-specific activation of PKC by each GqPCR is limited. α1-Adrenoceptor (α1-AR) is one of the GqPCRs highly expressed in the cardiovascular system. In this study, we examined the isoform-specific dynamic translocation of PKC in living HEK293T cells by α1-AR stimulation (α1-ARS). Rat PKCα, βI, βII, δ, ε and ζ fused with GFP at C-term were co-transfected with human α1A-AR into HEK293T cells. The isoform-specific dynamic translocation of PKC in living HEK293T cells by α1-ARS using phenylephrine was measured by confocal microscopy. Before stimulation, GFP-PKCs were localized at cytosolic region. α1-ARS strongly and rapidly translocated a classical PKC (cPKC), PKCα, (<30 s) to PM, with PKCα returning diffusively into the cytosol within 5 min. α1-ARS rapidly translocated other cPKCs, PKCβI and PKCβII, to the PM (<30 s), with sustained membrane localization. One novel PKC (nPKC), PKCε, but not another nPKC, PKCδ, was translocated by α1-AR stimulation to the PM (<30 s) and its membrane localization was also sustained. Finally, α1-AR stimulation did not cause a diacylglycerol-insensitive atypical PKC, PKCζ translocation. Our data suggest that PKCα, β and ε activation may underlie physiological and pathophysiological responses of α1-AR signaling for the phosphorylation of membrane-associated substrates including ion-channel and transporter proteins in the cardiovascular system.
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22
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Integrative miRNA and Gene Expression Profiling Analysis of Human Quiescent Hepatic Stellate Cells. Sci Rep 2015; 5:11549. [PMID: 26096707 PMCID: PMC4476106 DOI: 10.1038/srep11549] [Citation(s) in RCA: 74] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2014] [Accepted: 05/22/2015] [Indexed: 12/13/2022] Open
Abstract
Unveiling the regulatory pathways maintaining hepatic stellate cells (HSC) in a quiescent (q) phenotype is essential to develop new therapeutic strategies to treat fibrogenic diseases. To uncover the miRNA-mRNA regulatory interactions in qHSCs, HSCs were FACS-sorted from healthy livers and activated HSCs (aHSCs) were generated in vitro. MiRNA Taqman array analysis showed HSCs expressed a low number of miRNAs (n = 259), from which 47 were down-regulated and 212 up-regulated upon activation. Computational integration of miRNA and gene expression profiles revealed that 66% of qHSC-associated miRNAs correlated with more than 6 altered target mRNAs (17,28 ± 10,7 targets/miRNA) whereas aHSC-associated miRNAs had an average of 1,49 targeted genes. Interestingly, interaction networks generated by miRNA-targeted genes in qHSCs were associated with key HSC activation processes. Next, selected miRNAs were validated in healthy and cirrhotic human livers and miR-192 was chosen for functional analysis. Down-regulation of miR-192 in HSCs was found to be an early event during fibrosis progression in mouse models of liver injury. Moreover, mimic assays for miR-192 in HSCs revealed its role in HSC activation, proliferation and migration. Together, these results uncover the importance of miRNAs in the maintenance of the qHSC phenotype and form the basis for understanding the regulatory networks in HSCs.
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23
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Delire B, Stärkel P, Leclercq I. Animal Models for Fibrotic Liver Diseases: What We Have, What We Need, and What Is under Development. J Clin Transl Hepatol 2015; 3:53-66. [PMID: 26357635 PMCID: PMC4542084 DOI: 10.14218/jcth.2014.00035] [Citation(s) in RCA: 119] [Impact Index Per Article: 11.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/05/2014] [Revised: 12/10/2014] [Accepted: 12/12/2014] [Indexed: 02/06/2023] Open
Abstract
Liver fibrosis is part of the wound-healing response to liver damage of various origins and represents a major health problem. Although our understanding of the pathogenesis of liver fibrosis has grown considerably over the last 20 years, effective antifibrotic therapies are still lacking. The use of animal models is crucial for determining mechanisms underlying initiation, progression, and resolution of fibrosis and for developing novel therapies. To date, no animal model can recapitulate all the hepatic and extra-hepatic features of liver disease. In this review, we will discuss the current rodent models of liver injuries. We will then focus on the available ways to target specifically particular compounds of fibrogenesis and on the new models of liver diseases like the humanized liver mouse model.
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Affiliation(s)
- Bénédicte Delire
- Laboratory of Hepato-Gastroenterology, Institut de Recherche Expérimentale et Clinique (IREC), Catholic University of Louvain (UCL), Brussels, Belgium
| | - Peter Stärkel
- Laboratory of Hepato-Gastroenterology, Institut de Recherche Expérimentale et Clinique (IREC), Catholic University of Louvain (UCL), Brussels, Belgium
- Department of Gastroenterology, Saint-Luc Academic Hospital and Institute of Clinical Research, Catholic University of Louvain, Brussels, Belgium
| | - Isabelle Leclercq
- Laboratory of Hepato-Gastroenterology, Institut de Recherche Expérimentale et Clinique (IREC), Catholic University of Louvain (UCL), Brussels, Belgium
- Correspondence to: Isabelle Leclercq, Laboratoire d'Hépato-Gastro-Entérologie, Institut de Recherche Expérimentale et Clinique, Université catholique de Louvain, Avenue E Mounier 53, Box B1.52.01, Brussels 1200, Belgium. Tel: +32-27645379, Fax: +32-27645346. E-mail:
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24
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High-yield and high-purity isolation of hepatic stellate cells from normal and fibrotic mouse livers. Nat Protoc 2015; 10:305-15. [PMID: 25612230 DOI: 10.1038/nprot.2015.017] [Citation(s) in RCA: 432] [Impact Index Per Article: 43.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Hepatic stellate cells (HSCs) have been identified as the main fibrogenic cell type in the liver. Hence, efforts to understand hepatic fibrogenesis and to develop treatment strategies have focused on this cell type. HSC isolation, originally developed in rats, has subsequently been adapted to mice, thus allowing the study of fibrogenesis by genetic approaches in transgenic mice. However, mouse HSC isolation is commonly hampered by low yield and purity. Here we present an easy-to-perform protocol for high-purity and high-yield isolation of quiescent and activated HSCs in mice, based on retrograde pronase-collagenase perfusion of the liver and subsequent density-gradient centrifugation. We describe an optional add-on protocol for ultrapure HSC isolation from normal and fibrotic livers via subsequent flow cytometric sorting, thus providing a validated method to determine gene expression changes during HSC activation devoid of cell culture artifacts or contamination with other cells. The described isolation procedure takes ∼4 h to complete.
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25
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Berardis S, Sattwika PD, Najimi M, Sokal EM. Use of mesenchymal stem cells to treat liver fibrosis: Current situation and future prospects. World J Gastroenterol 2015; 21:742-758. [PMID: 25624709 PMCID: PMC4299328 DOI: 10.3748/wjg.v21.i3.742] [Citation(s) in RCA: 99] [Impact Index Per Article: 9.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/29/2014] [Revised: 09/05/2014] [Accepted: 11/19/2014] [Indexed: 02/06/2023] Open
Abstract
Progressive liver fibrosis is a major health issue for which no effective treatment is available, leading to cirrhosis and orthotopic liver transplantation. However, organ shortage is a reality. Hence, there is an urgent need to find alternative therapeutic strategies. Cell-based therapy using mesenchymal stem cells (MSCs) may represent an attractive therapeutic option, based on their immunomodulatory properties, their potential to differentiate into hepatocytes, allowing the replacement of damaged hepatocytes, their potential to promote residual hepatocytes regeneration and their capacity to inhibit hepatic stellate cell activation or induce their apoptosis, particularly via paracrine mechanisms. The current review will highlight recent findings regarding the input of MSC-based therapy for the treatment of liver fibrosis, from in vitro studies to pre-clinical and clinical trials. Several studies have shown the ability of MSCs to reduce liver fibrosis and improve liver function. However, despite these promising results, some limitations need to be considered. Future prospects will also be discussed in this review.
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26
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Weiskirchen R, Tacke F. Cellular and molecular functions of hepatic stellate cells in inflammatory responses and liver immunology. Hepatobiliary Surg Nutr 2015; 3:344-63. [PMID: 25568859 DOI: 10.3978/j.issn.2304-3881.2014.11.03] [Citation(s) in RCA: 105] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/12/2014] [Accepted: 10/17/2014] [Indexed: 12/11/2022]
Abstract
The liver is a central immunological organ. Liver resident macrophages, Kupffer cells (KC), but also sinusoidal endothelial cells, dendritic cells (DC) and other immune cells are involved in balancing immunity and tolerance against pathogens, commensals or food antigens. Hepatic stellate cells (HSCs) have been primarily characterized as the main effector cells in liver fibrosis, due to their capacity to transdifferentiate into collagen-producing myofibroblasts (MFB). More recent studies elucidated the fundamental role of HSC in liver immunology. HSC are not only the major storage site for dietary vitamin A (Vit A) (retinol, retinoic acid), which is essential for proper function of the immune system. This pericyte further represents a versatile source of many soluble immunological active factors including cytokines [e.g., interleukin 17 (IL-17)] and chemokines [C-C motif chemokine (ligand) 2 (CCL2)], may act as an antigen presenting cell (APC), and has autophagy activity. Additionally, it responds to many immunological triggers via toll-like receptors (TLR) (e.g., TLR4, TLR9) and transduces signals through pathways and mediators traditionally found in immune cells, including the Hedgehog (Hh) pathway or inflammasome activation. Overall, HSC promote rather immune-suppressive responses in homeostasis, like induction of regulatory T cells (Treg), T cell apoptosis (via B7-H1, PDL-1) or inhibition of cytotoxic CD8 T cells. In conditions of liver injury, HSC are important sensors of altered tissue integrity and initiators of innate immune cell activation. Vice versa, several immune cell subtypes interact directly or via soluble mediators with HSC. Such interactions include the mutual activation of HSC (towards MFB) and macrophages or pro-apoptotic signals from natural killer (NK), natural killer T (NKT) and gamma-delta T cells (γδ T-cells) on activated HSC. Current directions of research investigate the immune-modulating functions of HSC in the environment of liver tumors, cellular heterogeneity or interactions promoting HSC deactivation during resolution of liver fibrosis. Understanding the role of HSC as central regulators of liver immunology may lead to novel therapeutic strategies for chronic liver diseases.
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Affiliation(s)
- Ralf Weiskirchen
- 1 Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry, 2 Department of Internal Medicine III, RWTH University Hospital Aachen, Aachen, Germany
| | - Frank Tacke
- 1 Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry, 2 Department of Internal Medicine III, RWTH University Hospital Aachen, Aachen, Germany
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27
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Abstract
In vitro systems are required to evaluate potential liver fibrogenic effects of drugs and compounds during drug development and toxicity screening, respectively. Upon liver injury or toxicity, hepatic stellate cells are activated, thereby acquiring a myofibroblastic phenotype and participating in extracellular matrix deposition and liver fibrosis. The most widely used in vitro models to investigate liver fibrogenesis are primary cultures of hepatic stellate cells, which can be isolated from healthy human livers. Currently, there are no effective methods to maintain hepatic stellate cells in vitro in a quiescent phenotype. Therefore, when cells are plated, they spontaneously become activated in few days. Most in vitro studies in this area have been performed with monocultures of hepatic stellate cells in order to assess the direct effects of a given factor on hepatic stellate cell activation or the induction of inflammatory and fibrogenic responses. In this chapter, focus is put on basic protocols to isolate hepatic stellate cells from human tissue and to maintain them in culture as well as on common in vitro assays to evaluate their response to profibrogenic factors.
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Affiliation(s)
- Luis Perea
- Laboratory of Liver Fibrosis, Institut d'Investigacions Biomèdiques, August Pi i Sunyer (IDIBAPS), Rossello 149-153, Barcelona, 08036, Spain
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28
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Lee JI, Campbell JS. Role of desmoplasia in cholangiocarcinoma and hepatocellular carcinoma. J Hepatol 2014; 61:432-4. [PMID: 24751832 DOI: 10.1016/j.jhep.2014.04.014] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/06/2014] [Revised: 04/08/2014] [Accepted: 04/13/2014] [Indexed: 02/07/2023]
Affiliation(s)
- Jung Il Lee
- Department of Internal Medicine, Yonsei University College of Medicine, Seoul, South Korea
| | - Jean S Campbell
- Department of Pathology, University of Washington, Seattle, WA, USA.
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29
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Fate tracing reveals hepatic stellate cells as dominant contributors to liver fibrosis independent of its aetiology. Nat Commun 2014; 4:2823. [PMID: 24264436 PMCID: PMC4059406 DOI: 10.1038/ncomms3823] [Citation(s) in RCA: 1025] [Impact Index Per Article: 93.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2013] [Accepted: 10/25/2013] [Indexed: 02/07/2023] Open
Abstract
Although organ fibrosis causes significant morbidity and mortality in chronic diseases, the lack of detailed knowledge about specific cellular contributors mediating fibrogenesis hampers the design of effective antifibrotic therapies. Different cellular sources, including tissue-resident and bone marrow-derived fibroblasts, pericytes and epithelial cells, have been suggested to give rise to myofibroblasts, but their relative contributions remain controversial, with profound differences between organs and different diseases. Here we employ a novel Cre-transgenic mouse that marks 99% of hepatic stellate cells (HSCs), a liver-specific pericyte population, to demonstrate that HSCs give rise to 82-96% of myofibroblasts in models of toxic, cholestatic and fatty liver disease. Moreover, we exclude that HSCs function as facultative epithelial progenitor cells in the injured liver. On the basis these findings, HSCs should be considered the primary cellular target for antifibrotic therapies across all types of liver disease.
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30
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Gene expression profiling and secretome analysis differentiate adult-derived human liver stem/progenitor cells and human hepatic stellate cells. PLoS One 2014; 9:e86137. [PMID: 24516514 PMCID: PMC3906387 DOI: 10.1371/journal.pone.0086137] [Citation(s) in RCA: 49] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2013] [Accepted: 12/04/2013] [Indexed: 12/20/2022] Open
Abstract
Adult-derived human liver stem/progenitor cells (ADHLSC) are obtained after primary culture of the liver parenchymal fraction. The cells are of fibroblastic morphology and exhibit a hepato-mesenchymal phenotype. Hepatic stellate cells (HSC) derived from the liver non-parenchymal fraction, present a comparable morphology as ADHLSC. Because both ADHLSC and HSC are described as liver stem/progenitor cells, we strived to extensively compare both cell populations at different levels and to propose tools demonstrating their singularity. ADHLSC and HSC were isolated from the liver of four different donors, expanded in vitro and followed from passage 5 until passage 11. Cell characterization was performed using immunocytochemistry, western blotting, flow cytometry, and gene microarray analyses. The secretion profile of the cells was evaluated using Elisa and multiplex Luminex assays. Both cell types expressed α-smooth muscle actin, vimentin, fibronectin, CD73 and CD90 in accordance with their mesenchymal origin. Microarray analysis revealed significant differences in gene expression profiles. HSC present high expression levels of neuronal markers as well as cytokeratins. Such differences were confirmed using immunocytochemistry and western blotting assays. Furthermore, both cell types displayed distinct secretion profiles as ADHLSC highly secreted cytokines of therapeutic and immuno-modulatory importance, like HGF, interferon-γ and IL-10. Our study demonstrates that ADHLSC and HSC are distinct liver fibroblastic cell populations exhibiting significant different expression and secretion profiles.
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31
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Galler K, Schleser F, Fröhlich E, Requardt RP, Kortgen A, Bauer M, Popp J, Neugebauer U. Exploitation of the hepatic stellate cell Raman signature for their detection in native tissue samples. Integr Biol (Camb) 2014; 6:946-56. [DOI: 10.1039/c4ib00130c] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
The unique information concentrated in Raman spectra serves to differentiate hepatic stellate cells from hepatocytes, detect them in living tissue and provide insight in their activation state.
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Affiliation(s)
- Kerstin Galler
- Leibniz Institute of Photonic Technology
- Jena, Germany
- Center for Sepsis Control and Care
- Jena University Hospital
- Germany
| | - Franziska Schleser
- Center for Sepsis Control and Care
- Jena University Hospital
- Germany
- Department of Anesthesiology and Intensive Care Medicine
- Jena University Hospital
| | - Esther Fröhlich
- Center for Sepsis Control and Care
- Jena University Hospital
- Germany
- Department of Anesthesiology and Intensive Care Medicine
- Jena University Hospital
| | | | - Andreas Kortgen
- Center for Sepsis Control and Care
- Jena University Hospital
- Germany
- Department of Anesthesiology and Intensive Care Medicine
- Jena University Hospital
| | - Michael Bauer
- Center for Sepsis Control and Care
- Jena University Hospital
- Germany
- Department of Anesthesiology and Intensive Care Medicine
- Jena University Hospital
| | - Jürgen Popp
- Leibniz Institute of Photonic Technology
- Jena, Germany
- Center for Sepsis Control and Care
- Jena University Hospital
- Germany
| | - Ute Neugebauer
- Leibniz Institute of Photonic Technology
- Jena, Germany
- Center for Sepsis Control and Care
- Jena University Hospital
- Germany
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Morales-Ibanez O, Domínguez M, Ki SH, Marcos M, Chaves JF, Nguyen-Khac E, Houchi H, Affò S, Sancho-Bru P, Altamirano J, Michelena J, García-Pagán JC, Abraldes JG, Arroyo V, Caballería J, Laso FJ, Gao B, Bataller R. Human and experimental evidence supporting a role for osteopontin in alcoholic hepatitis. Hepatology 2013; 58:1742-56. [PMID: 23729174 PMCID: PMC3877722 DOI: 10.1002/hep.26521] [Citation(s) in RCA: 78] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/17/2012] [Accepted: 05/10/2013] [Indexed: 12/14/2022]
Abstract
UNLABELLED We identified, in the transcriptome analysis of patients with alcoholic hepatitis (AH), osteopontin (OPN) as one of the most up-regulated genes. Here, we used a translational approach to investigate its pathogenic role. OPN hepatic gene expression was quantified in patients with AH and other liver diseases. OPN protein expression and processing were assessed by immmunohistochemistry, western blotting and enzyme-linked immunosorbent assay. OPN gene polymorphisms were evaluated in patients with alcoholic liver disease. The role of OPN was evaluated in OPN(-/-) mice with alcohol-induced liver injury. OPN biological actions were studied in human hepatic stellate cells (HSCs) and in precision-cut liver slices. Hepatic expression and serum levels of OPN were markedly increased in AH, compared to normal livers and other types of chronic liver diseases, and correlated with short-term survival. Serum levels of OPN also correlated with hepatic expression and disease severity. OPN was mainly expressed in areas with inflammation and fibrosis. Two proteases that process OPN (thrombin and matrix metalloproteinase 7) and cleaved OPN were increased in livers with AH. Patients with AH had a tendency of a lower frequency of the CC genotype of the +1239C single-nucleotide polymorphism of the OPN gene, compared to patients with alcohol abuse without liver disease. Importantly, OPN(-/-) mice were protected against alcohol-induced liver injury and showed decreased expression of inflammatory cytokines. Finally, OPN was induced by lipopolysaccharide and stimulated inflammatory actions in HSCs. CONCLUSION Human and experimental data suggest a role for OPN in the pathogenesis of AH. Further studies should evaluate OPN as a potential therapeutic target.
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Affiliation(s)
- Oriol Morales-Ibanez
- Liver Unit, Hospital Clínic, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), CIBER de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Catalonia, Spain
| | - Marlene Domínguez
- Liver Unit, Hospital Clínic, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), CIBER de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Catalonia, Spain
| | - Sung H. Ki
- Laboratory of Liver Diseases, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD
| | - Miguel Marcos
- Alcoholism Unit, Department of Internal Medicine, IBSAL-University Hospital of Salamanca, Salamanca, Spain
| | - Javier F. Chaves
- Genotyping and Genetic Diagnosis Unit, Research Foundation of Hospital Clínico, Valencia, Spain
| | - Eric Nguyen-Khac
- Service d’Hépato-Gastroentérologie, Amiens University Hospital and Groupe de Recherche sur l’Alcool et les Pharmacodépendances (INSERM ERI 24, GRAP), Picardie University, Amiens, France
| | - Hakim Houchi
- Service d’Hépato-Gastroentérologie, Amiens University Hospital and Groupe de Recherche sur l’Alcool et les Pharmacodépendances (INSERM ERI 24, GRAP), Picardie University, Amiens, France
| | - Silvia Affò
- Liver Unit, Hospital Clínic, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), CIBER de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Catalonia, Spain
| | - Pau Sancho-Bru
- Liver Unit, Hospital Clínic, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), CIBER de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Catalonia, Spain
| | - José Altamirano
- Liver Unit, Hospital Clínic, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), CIBER de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Catalonia, Spain
| | - Javier Michelena
- Liver Unit, Hospital Clínic, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), CIBER de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Catalonia, Spain
| | - Juan Carlos García-Pagán
- Liver Unit, Hospital Clínic, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), CIBER de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Catalonia, Spain
| | - Juan G. Abraldes
- Liver Unit, Hospital Clínic, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), CIBER de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Catalonia, Spain
| | - Vicente Arroyo
- Liver Unit, Hospital Clínic, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), CIBER de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Catalonia, Spain
| | - Juan Caballería
- Liver Unit, Hospital Clínic, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), CIBER de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Catalonia, Spain
| | - Francisco-Javier Laso
- Alcoholism Unit, Department of Internal Medicine, IBSAL-University Hospital of Salamanca, Salamanca, Spain
| | - Bin Gao
- Laboratory of Liver Diseases, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD
| | - Ramón Bataller
- Liver Unit, Hospital Clínic, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), CIBER de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Catalonia, Spain
- Division of Gastroenterology and Hepatology, Departments of Medicine and Nutrition, University of North Carolina at Chapel Hill, NC
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Liao R, Wu H, Yi Y, Wang JX, Cai XY, He HW, Cheng YF, Zhou J, Fan J, Sun J, Qiu SJ. Clinical significance and gene expression study of human hepatic stellate cells in HBV related-hepatocellular carcinoma. J Exp Clin Cancer Res 2013; 32:22. [PMID: 23601182 PMCID: PMC3654985 DOI: 10.1186/1756-9966-32-22] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2013] [Accepted: 04/14/2013] [Indexed: 12/20/2022] Open
Abstract
BACKGROUND Peritumoral activated hepatic stellate cells (HSCs) are versatile myofibroblast-like cells closely related with hepatocellular carcinoma (HCC) progression. So far, comprehensive comparison of gene expression of human HSCs during hepatocarcinogenesis is scanty. Therefore, we identified the phenotypic and genomic characteristics of peritumoral HSCs to explore the valuable information on the prognosis and therapeutic targets of HBV related HCC. METHODS A tissue microarray containing 224 HBV related HCC patients was used to evaluate the expression of phenotype markers of HSCs including α-SMA, glial fibrillary acidic protein (GFAP), desmin, vinculin and vimentin. HSCs and cancer associated myofibroblasts (CAMFs) were isolated from normal, peritumoral human livers and cancer tissues, respectively. Flow cytometry and gene microarray analysis were performed to evaluate the phenotypic changes and gene expression in HCC, respectively. RESULTS Peritumoral α-SMA positive HSCs showed the prognostic value in time to recurrence (TTR) and overall survival (OS) of HCC patients, especially in early recurrence and AFP-normal HCC patients. Expression of GFAP positive HSCs cell lines LX-2 was significantly decreased after stimulation with tumor conditioned medium. Compared with quiescent HSCs, peritumoral HSCs and intratumoral CAMFs expressed considerable up- and down-regulated genes associated with biological process, cellular component, molecular function and signaling pathways involved in fibrogenesis, inflammation and progress of cancer. CONCLUSIONS Peritumoral activated HSCs displayed prognostic value in HBV related-HCC, and their genomic characteristics could present rational biomarkers for HCC risk and promising therapeutic targets.
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Affiliation(s)
- Rui Liao
- Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, China
- Key Laboratory of Carcinogenesis and Cancer Invasion, the Chinese Ministry of Education, Shanghai, China
| | - Han Wu
- Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, China
- Key Laboratory of Carcinogenesis and Cancer Invasion, the Chinese Ministry of Education, Shanghai, China
| | - Yong Yi
- Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, China
- Key Laboratory of Carcinogenesis and Cancer Invasion, the Chinese Ministry of Education, Shanghai, China
| | - Jia-Xing Wang
- Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, China
- Key Laboratory of Carcinogenesis and Cancer Invasion, the Chinese Ministry of Education, Shanghai, China
| | - Xiao-Yan Cai
- Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, China
- Key Laboratory of Carcinogenesis and Cancer Invasion, the Chinese Ministry of Education, Shanghai, China
| | - Hong-Wei He
- Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, China
- Key Laboratory of Carcinogenesis and Cancer Invasion, the Chinese Ministry of Education, Shanghai, China
| | - Yun-Feng Cheng
- Biomedical Research Center, Zhongshan Hospital, Fudan University, Shanghai, China
| | - Jian Zhou
- Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, China
- Key Laboratory of Carcinogenesis and Cancer Invasion, the Chinese Ministry of Education, Shanghai, China
| | - Jia Fan
- Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, China
- Key Laboratory of Carcinogenesis and Cancer Invasion, the Chinese Ministry of Education, Shanghai, China
| | - Jian Sun
- Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, China
- Key Laboratory of Carcinogenesis and Cancer Invasion, the Chinese Ministry of Education, Shanghai, China
| | - Shuang-Jian Qiu
- Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, China
- Key Laboratory of Carcinogenesis and Cancer Invasion, the Chinese Ministry of Education, Shanghai, China
- Biomedical Research Center, Zhongshan Hospital, Fudan University, Shanghai, China
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DNA methylation and MeCP2 regulation of PTCH1 expression during rats hepatic fibrosis. Cell Signal 2013; 25:1202-11. [PMID: 23333245 DOI: 10.1016/j.cellsig.2013.01.005] [Citation(s) in RCA: 51] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2012] [Accepted: 01/08/2013] [Indexed: 12/11/2022]
Abstract
Hepatic stellate cell (HSC) activation plays an important role in liver fibrogenesis. Transdifferentiation of quiescent hepatic stellate cells into myofibroblastic-HSCs is a key event in liver fibrosis. The methyl-CpG-binding protein MeCP2 which promotes repressed chromatin structure is selectively detected in myofibroblasts of diseased liver. MeCP2 binds to methylated CpG dinucleotides, which are abundant in the promoters of many genes. Treatment of HSCs with DNA methylation inhibitor 5-aza-2'- deoxycytidine (5-azadC) prevented proliferation and activation. Treatment with 5-azadC prevented loss of Patched (PTCH1) expression that occurred during HSCs activation. In a search for underlying molecular medchanisms, we investigated whether the targeting of epigenetic silencing mechanisms could be useful in the treatment of PTCH1-associated fibrogenesis. It was indicated that hypermethylation of PTCH1 is associated with the perpetuation of fibroblast activation and fibrosis in the liver. siRNA knockdown of MeCP2 increased the expressions of PTCH1 mRNA and protein in hepatic myofibroblasts. These data suggest that DNA methylation and MeCP2 may provide molecular mechanisms for silencing of PTCH1.
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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: 391] [Impact Index Per Article: 30.1] [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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Liao R, Sun TW, Yi Y, Wu H, Li YW, Wang JX, Zhou J, Shi YH, Cheng YF, Qiu SJ, Fan J. Expression of TREM-1 in hepatic stellate cells and prognostic value in hepatitis B-related hepatocellular carcinoma. Cancer Sci 2012; 103:984-992. [PMID: 22417086 PMCID: PMC7685080 DOI: 10.1111/j.1349-7006.2012.02273.x] [Citation(s) in RCA: 66] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2011] [Revised: 03/05/2012] [Accepted: 03/07/2012] [Indexed: 12/31/2022] Open
Abstract
Hepatocellular carcinoma (HCC) is a typical inflammation-related malignancy characterized by high postoperative recurrence and metastasis. Although several inflammatory cells and inflammatory signatures have been linked to poor prognosis, the inflammation-associated molecular mechanisms of HCC development and progression are largely unknown. Here we show that triggering receptor expressed in myeloid cells (TREM)-1, a transmembrane receptor expressing in myeloid cells, was also expressed in tumor-activated hepatic stellate cells (HSCs) and associated with the aggressive behavior of HCC cells. Enzyme-linked immunosorbent assay was used to measure the expression levels of soluble TREM-1 (sTREM-1) in activated hepatic stellate cells supernatant and 92 preoperative and postoperative plasmas of patients with malignancy and/or benign liver tumor/disease, respectively. Expression levels of TREM-1 were assessed by immunohistochemistry in tissue microarray from 240 patients with HCC. As a result, increased secretion of sTREM-1 from activated HSCs was observed after co-culture with HCC cell lines (P < 0.001), and conditioned medium collected from activated HSCs/cancer associated myofibroblasts (CAMFs) with or without agonist/inhibitor of TREM-1 significantly changed the migratory ability of HCC cells. The levels of sTREM-1 were significantly higher in patients with HCC than those with benign liver tumors (P < 0.005). Peritumoral density of TREM-1 was shown to be an independent prognosis predictor according to univariate (P < 0.001 for both overall survival and time to recurrence) and multivariate analysis (P = 0.008 for overall survival; P = 0.005 for time to recurrence). Thus, these observations suggest that TREM-1 is related to the aggressive tumor behavior and has potential value as a prognostic factor for HCC.
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Affiliation(s)
- Rui Liao
- Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, China
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Xiong WJ, Hu LJ, Jian YC, Wang LJ, Jiang M, Li W, He Y. Wnt5a participates in hepatic stellate cell activation observed by gene expression profile and functional assays. World J Gastroenterol 2012; 18:1745-52. [PMID: 22553398 PMCID: PMC3332287 DOI: 10.3748/wjg.v18.i15.1745] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/09/2011] [Revised: 09/10/2011] [Accepted: 02/27/2012] [Indexed: 02/06/2023] Open
Abstract
AIM: To identify differentially expressed genes in quiescent and activated hepatic stellate cells (HSCs) and explore their functions.
METHODS: HSCs were isolated from the normal Sprague Dawley rats by in suit perfusion of collagenase and pronase and density Nycodenz gradient centrifugation. Total RNA and mRNA of quiescent HSCs, and culture-activated HSCs were extracted, quantified and reversely transcripted into cDNA. The global gene expression profile was analyzed by microarray with Affymetrix rat genechip. Differentially expressed genes were annotated with Gene Ontology (GO) and analyzed with Kyoto encyclopedia of genes and genomes (KEGG) pathway using the Database for Annotation, Visualization and Integrated Discovery. Microarray data were validated by quantitative real-time polymerase chain reaction (qRT-PCR). The function of Wnt5a on human HSCs line LX-2 was assessed with lentivirus-mediated Wnt5a RNAi. The expression of Wnt5a in fibrotic liver of a carbon tetrachloride (CCl4)-induced fibrosis rat model was also analyzed with Western blotting.
RESULTS: Of the 28 700 genes represented on this chip, 2566 genes displayed at least a 2-fold increase or decrease in expression at a P < 0.01 level with a false discovery rate. Of these, 1396 genes were upregulated, while 1170 genes were downregulated in culture-activated HSCs. These differentially expressed transcripts were grouped into 545 GO based on biological process GO terms. The most enriched GO terms included response to wounding, wound healing, regulation of cell growth, vasculature development and actin cytoskeleton organization. KEGG pathway analysis revealed that Wnt5a signaling pathway participated in the activation of HSCs. Wnt5a was significantly increased in culture-activated HSCs as compared with quiescent HSCs. qRT-PCR validated the microarray data. Lentivirus-mediated suppression of Wnt5a expression in activated LX-2 resulted in significantly impaired proliferation, downregulated expressions of type I collagen and transforming growth factor-β1. Wnt5a was upregulated in the fibrotic liver of a CCl4-induced fibrosis rat model.
CONCLUSION: Wnt5a is involved in the activation of HSCs, and it may serve as a novel therapeutic target in the treatment of liver fibrosis.
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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: 147] [Impact Index Per Article: 11.3] [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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Fan R, Shan X, Qian H, Song C, Wu G, Chen Y, Miao Y, Cha W. Protective effect of apocynin in an established alcoholic steatohepatitis rat model. Immunopharmacol Immunotoxicol 2012; 34:633-8. [DOI: 10.3109/08923973.2011.648266] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
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Ikeda N, Murata S, Maruyama T, Tamura T, Nozaki R, Kawasaki T, Fukunaga K, Oda T, Sasaki R, Homma M, Ohkohchi N. Platelet-derived adenosine 5'-triphosphate suppresses activation of human hepatic stellate cell: In vitro study. Hepatol Res 2012; 42:91-102. [PMID: 21988364 DOI: 10.1111/j.1872-034x.2011.00893.x] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
AIM Activated hepatic stellate cells (HSC) play a critical role in liver fibrosis. Suppressing abnormal function of HSC or reversion from activated to quiescent form is a hopeful treatment for liver cirrhosis. The interaction between platelets and HSC remains unknown although platelets go through hepatic sinusoids surrounded by HSC. This study aimed at clarifying the hypothesis that platelets control activation of HSC. METHODS We used human platelets, platelet extracts, and primary or immortalized human HSC. We examined the effect of platelets on the activation, DNA synthesis, type I collagen production, and fibrosis-relating gene expressions of HSC. We investigated what suppressed activation of HSC within platelets and examined the mechanism of controlling activation in vitro. RESULTS Platelets and platelet extracts suppressed activation of HSC. Platelets decreased type I collagen production without affecting DNA synthesis. Platelets increased the expression of matrix metallopeptidase 1. As platelet extracts co-cultured with an enzyme of degrading adenosine 5'-triphosphate (ATP) suppressed activation, we detected adenine nucleotides within platelets or on their surfaces and confirmed the degradation of adenine nucleotides by HSC and the production of adenosine. Adenosine and platelets increased the intracellular cyclic adenosine 5'-monophosphate (cAMP), which is important in quiescent HSC. A great amount of adenosine and ATP also suppressed activation of HSC. CONCLUSION Activation of human HSC is suppressed by human platelets or platelet-derived ATP via adenosine-cAMP signaling pathway in vitro. Therefore, platelets have the possibility to be used in the treatment of liver cirrhosis.
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Affiliation(s)
- Naoya Ikeda
- Departments of Surgery Pharmaceutical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Japan
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Autophagy and hepatic stellate cell activation - partners in crime? J Hepatol 2011; 55:1176-7. [PMID: 21856271 DOI: 10.1016/j.jhep.2011.08.001] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/10/2011] [Accepted: 08/11/2011] [Indexed: 01/18/2023]
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Tao H, Huang C, Yang JJ, Ma TT, Bian EB, Zhang L, Lv XW, Jin Y, Li J. MeCP2 controls the expression of RASAL1 in the hepatic fibrosis in rats. Toxicology 2011; 290:327-33. [PMID: 22056649 DOI: 10.1016/j.tox.2011.10.011] [Citation(s) in RCA: 73] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2011] [Revised: 10/17/2011] [Accepted: 10/19/2011] [Indexed: 12/11/2022]
Abstract
Hepatic stellate cells (HSCs) activation is an essential event during liver fibrogenesis. A major pathway is the transition of HSCs into hepatic myofibroblasts. The methyl-CpG-binding protein MeCP2 which promotes repressed chromatin structure is selectively detected in myofibroblasts of diseased liver. Overexpression of this protein results in an increase of global methylation levels. Treatment of HSCs with DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-azadC) blocks the cell proliferation. 5-azadC also prevents loss of Ras GTPase activating-like protein 1 (RASAL1) expression that occurs during HSCs proliferation. To further explore the underlying molecular mechanisms, we hypothesized that this perpetuation of fibrogenesis was caused by DNA methylation. Results demonstrated that hypermethylation of RASAL1 is associated with the perpetuation of fibroblast activation and fibrogenesis in the liver. knockdown of MeCP2 using siRNA technique increased RASAL1 in both mRNA and protein level in myofibroblasts. These studies demonstrated that MeCP2 and DNA methylation may provide molecular mechanisms for perpetuated fibroblast activation and fibrogenesis in the liver.
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Affiliation(s)
- Hui Tao
- School of pharmacy, Anhui key laboratory of bioactivity of natural products, Anhui Medical University, Hefei 230032, China
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Zhao W, Zhang L, Yin Z, Su W, Ren G, Zhou C, You J, Fan J, Wang X. Activated hepatic stellate cells promote hepatocellular carcinoma development in immunocompetent mice. Int J Cancer 2011; 129:2651-61. [PMID: 21213212 DOI: 10.1002/ijc.25920] [Citation(s) in RCA: 96] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2010] [Accepted: 12/22/2010] [Indexed: 12/29/2022]
Abstract
Activated hepatic stellate cells (HSCs) play a central role in the hepatic fibrosis and cirrhosis. Recently, HSCs were reported to have strong immune modulatory activities. However, the role of HSCs in hepatocellular carcinoma (HCC) remains unclear. In this study, we showed that HSCs could promote HCC growth both in vitro and in vivo. We examined the HSC-mediated inhibition of T-cell proliferation and the ability of conditioned medium from activated HSCs to promote the growth of murine HCC cell lines in vitro. We also assessed the immune suppression by HSCs during the development of HCC in immunocompetent mice. Cotransplantation of HSCs promoted HCC growth and progression by enhancing tumor angiogenesis and tumor cell proliferation and by creating an immunosuppressed microenvironment. Cotransplanted HSCs inhibited the lymphocyte infiltration in tumors and the spleens of mice bearing tumors, induced apoptosis of infiltrating mononuclear cells, and enhanced the expression of B7H1 and CD4(+) CD25(+) Treg cells. The immune modulation by HSCs seemed to be systemic. In conclusion, our data provide new information to support an integral role for HSCs in promoting HCC progression in part via their immune regulatory activities, and suggest that HSCs may serve as a therapeutic target in HCC.
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Affiliation(s)
- Wenxiu Zhao
- Department of Hepatobiliary Surgery, Zhongshan Hospital Xiamen University, Research Institute of Digestive Disease, Xiamen, Fujian, China
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Lemos QT, Andrade ZA. Angiogenesis and experimental hepatic fibrosis. Mem Inst Oswaldo Cruz 2011; 105:611-4. [PMID: 20835605 DOI: 10.1590/s0074-02762010000500002] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2009] [Accepted: 05/12/2010] [Indexed: 12/17/2022] Open
Abstract
Angiogenesis is a basic change occurring during repair by granulation tissue. This process seems to precede fibrosis formation in most types of chronic liver disease. To examine its presence and significance in different types of hepatic insults, this paper sought to identify the presence, evolution and peculiarities of angiogenesis in the most common experimental models of hepatic fibrosis. The characterization of cells, vessels and extracellular matrix and the identification of factors associated with endothelium (factor VIII RA), vascular basement membrane, other components of the vascular walls (actin, elastin) and the presence of the vascular-endothelial growth factor were investigated. The models examined included Capillaria hepatica septal fibrosis, whole pig serum injections, carbon tetrachloride administration, main bile duct ligation and Schistosoma mansoni infection. The first four models were performed in rats, while the last used mice. All models studied exhibited prominent angiogenesis. The most evident relationship between angiogenesis and fibrosis occurred with the C. hepatica model due to circumstances to be discussed. Special attention was paid to the presence of pericytes and to their tendency to become detached from the vascular wall and be transformed into myofibroblasts, which is a sequence of events that explains the decisive role angiogenesis plays in fibrosis.
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Affiliation(s)
- Queli Teixeira Lemos
- Laboratório de Patologia Experimental,, Centro de Pesquisa Gonçalo Moniz, Fiocruz, Salvador, BA, Brasil
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Troeger JS, Schwabe RF. Hypoxia and hypoxia-inducible factor 1α: potential links between angiogenesis and fibrogenesis in hepatic stellate cells. Liver Int 2011; 31:143-5. [PMID: 21176093 DOI: 10.1111/j.1478-3231.2010.02426.x] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
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Cao S, Yaqoob U, Das A, Shergill U, Jagavelu K, Huebert RC, Routray C, Abdelmoneim S, Vasdev M, Leof E, Charlton M, Watts RJ, Mukhopadhyay D, Shah VH. Neuropilin-1 promotes cirrhosis of the rodent and human liver by enhancing PDGF/TGF-beta signaling in hepatic stellate cells. J Clin Invest 2010; 120:2379-94. [PMID: 20577048 DOI: 10.1172/jci41203] [Citation(s) in RCA: 123] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2010] [Accepted: 05/05/2010] [Indexed: 12/13/2022] Open
Abstract
PDGF-dependent hepatic stellate cell (HSC) recruitment is an essential step in liver fibrosis and the sinusoidal vascular changes that accompany this process. However, the mechanisms that regulate PDGF signaling remain incompletely defined. Here, we found that in two rat models of liver fibrosis, the axonal guidance molecule neuropilin-1 (NRP-1) was upregulated in activated HSCs, which exhibit the highly motile myofibroblast phenotype. Additionally, NRP-1 colocalized with PDGF-receptor beta (PDGFRbeta) in HSCs both in the injury models and in human and rat HSC cell lines. In human HSCs, siRNA-mediated knockdown of NRP-1 attenuated PDGF-induced chemotaxis, while NRP-1 overexpression increased cell motility and TGF-beta-dependent collagen production. Similarly, mouse HSCs genetically modified to lack NRP-1 displayed reduced motility in response to PDGF treatment. Immunoprecipitation and biochemical binding studies revealed that NRP-1 increased PDGF binding affinity for PDGFRbeta-expressing cells and promoted downstream signaling. An NRP-1 neutralizing Ab ameliorated recruitment of HSCs, blocked liver fibrosis in a rat model of liver injury, and also attenuated VEGF responses in cultured liver endothelial cells. In addition, NRP-1 overexpression was observed in human specimens of liver cirrhosis caused by both hepatitis C and steatohepatitis. These studies reveal a role for NRP-1 as a modulator of multiple growth factor targets that regulate liver fibrosis and the vascular changes that accompany it and may have broad implications for liver cirrhosis and myofibroblast biology in a variety of other organ systems and disease conditions.
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Affiliation(s)
- Sheng Cao
- Gastroenterology Research Unit, Mayo Clinic, Rochester, Minnesota 55905, USA.
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Martínez-Rizo A, Bueno-Topete M, González-Cuevas J, Armendáriz-Borunda J. Plasmin plays a key role in the regulation of profibrogenic molecules in hepatic stellate cells. Liver Int 2010; 30:298-310. [PMID: 19889106 DOI: 10.1111/j.1478-3231.2009.02155.x] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/18/2023]
Abstract
BACKGROUND Plasmin role in transforming growth factor-beta (TGF-beta)-responsive gene regulation remains to be elucidated. Also, plasmin action on co-repressor Ski-related novel protein N (SnoN) and differential activation of matrix metalloproteinases (MMPs) are unknown. Thus, the role of plasmin on profibrogenic molecule expression, SnoN transcriptional kinetics and gelatinase activation was investigated. METHODS Hepatic stellate cells (HSC) were transduced with adenovirus-mediated human urokinase plasminogen activator (Ad-huPA) (4 x 10(9) viral particles/ml). Overexpression of urokinase plasminogen activator and therefore of plasmin, was blocked by tranexamic acid (TA) in transduced HSC. Gene expression was monitored by reverse transcriptase polymerase chain reaction. HSC-free supernatants were used to evaluate MMP-2 and MMP-9 by zymography. SnoN, TGF-beta and tissue inhibitor of metalloproteinase (TIMP)-1 were analysed by Western blot. Plasmin and SnoN expression kinetics were evaluated in bile duct-ligated (BDL) rats. RESULTS Plasmin overexpression in Ad-huPA-transduced HSC significantly decreased gene expression of profibrogenic molecules [alpha1(I)collagen 66%, TIMP-1 59%, alpha-smooth muscle actin 90% and TGF-beta 55%]. Interestingly, both SnoN gene and protein expression increased prominently. Plasmin inhibition by TA upregulated the profibrogenic genes, which respond to TGF-beta-intracellular signalling. In contrast, SnoN mRNA and protein dropped importantly. Plasmin-activated MMP-9 and MMP-2 in HSC supernatants. Taken together, these findings indicate that MMP-9 activation is totally plasmin dependent. SnoN levels significantly decreased in cholestatic-BDL rats (82%) as compared with control animals. Interestingly, hepatic plasmin levels dropped 46% in BDL rats as compared with control. CONCLUSION Plasmin plays a key role in regulating TGF-beta-responding genes. In particular, regulation of TGF-beta-co-repressor (SnoN) is greatly affected, which suggests SnoN as a cardinal player in cholestasis-induced fibrogenesis.
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Affiliation(s)
- Abril Martínez-Rizo
- Department of Molecular Biology and Genomics, CUCS, Institute for Molecular Biology in Medicine and Gene Therapy, University of Guadalajara, and OPD Hospital Civil de Guadalajara, Guadalajara, Jalisco, Mexico
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Shimada H, Ochi T, Imasato A, Morizane Y, Hori M, Ozaki H, Shinjo K. Gene expression profiling and functional assays of activated hepatic stellate cells suggest that myocardin has a role in activation. Liver Int 2010; 30:42-54. [PMID: 19793196 DOI: 10.1111/j.1478-3231.2009.02120.x] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/13/2023]
Abstract
BACKGROUND Myofibroblast-like cells derived from transdifferentiated hepatic stellate cells (HSC) play a central role in scar formation that leads to liver fibrosis. The molecular mechanisms underlying this process are not fully understood. AIM Our aim was to identify genes that are differentially regulated by HSC activation and to explore their function. METHODS Using oligonucleotide microarrays, we performed transcriptional analysis of the human HSC cell line, LI90, cultured on Matrigel. Microarray data were validated by quantitative real-time polymerase chain reaction and Western blotting. The function of myocardin was assessed by myocardin RNAi and overexpression. RESULTS Examination of Matrigel-induced deactivation of LI90 cells revealed marked downregulation of myocardin, an important transcriptional regulator in smooth and cardiac muscle development. Small interfering RNA-mediated suppression of myocardin expression in both activated LI90 and rat activated HSC resulted in loss of the phenotypic characteristics of myofibroblasts and significantly impaired the production of activated HSC markers, such as alpha-smooth muscle actin and extracellular matrix proteins like type I collagen. Overexpression of myocardin led to the upregulation of these marker genes. Myocardin was upregulated in rat primary HSC during in vitro activation and in the fibrotic liver of a dimethylnitrosamine-induced fibrosis rat model. CONCLUSIONS This study demonstrates that myocardin is involved in the activation of HSC; myocardin may serve as a novel therapeutic target in the treatment of liver fibrosis.
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Affiliation(s)
- Hideaki Shimada
- Discovery Biology Research, Pfizer Global Research and Development Nagoya Laboratories, Pfizer Japan Inc., Aichi, Japan.
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