1
|
Chen L, Guo P, Li W, Jiang X, Zhao Q, Li D, Wang Q, Xiao Y, Xing X, Pang Y, Aschner M, Zhang L, Chen W. Protein phosphatase 2A regulates cytotoxicity and drug resistance by dephosphorylating xenobiotic metabolism enzymes AHR and MDR1. J Biol Chem 2022; 298:101918. [PMID: 35405096 PMCID: PMC9118923 DOI: 10.1016/j.jbc.2022.101918] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2022] [Revised: 03/26/2022] [Accepted: 03/28/2022] [Indexed: 11/20/2022] Open
Abstract
Protein phosphatase 2A (PP2A) is a serine/threonine dephosphorylating enzyme complex that plays numerous roles in biological processes, including cell growth and metabolism. However, its specific actions in many of these critical pathways are unclear. To explore mechanisms underlying metabolic enzyme regulation in the liver, we investigated the key pathways involved in regulation of xenobiotic-metabolizing enzymes in a mouse model with hepatocyte-specific deletion of Ppp2r1a, encoding the Aα subunit of PP2A. We performed transcriptome and phosphoproteome analysis in mouse livers at the age of 3 months and identified 2695 differentially expressed genes and 549 upregulated phosphoproteins in homozygous knockout mouse livers compared with WT littermates. In particular, the expression of metabolic enzymes Cyp2e1, Cyp1a1, Cyp1a2, Mdr1a, and Abcg2 was dramatically altered in homozygous knockout mouse livers. We also demonstrated that activation of PP2A reversed the decline of metabolic enzyme expression in primary mouse hepatocytes. We found that specific PP2A holoenzymes were involved in metabolic enzyme induction through dephosphorylation of transcription factors, nuclear receptors, or the target enzymes themselves, leading to dysregulation of xenobiotic metabolism or drug-induced hepatotoxicity. Notably, we confirmed that a regulatory axis, PP2A B56α–aryl hydrocarbon receptor–Cyp1a1, was involved in benzo(a)pyrene-induced cytotoxicity through dephosphorylation of the metabolic nuclear receptor, aryl hydrocarbon receptor, at serine 36. In addition, we showed that PP2A B56δ complexes directly dephosphorylated the multidrug efflux pump MDR1 (encoded by multi-drug resistance gene 1), contributing to drug resistance against the chemotherapeutic 5-fluorouracil. Taken together, these novel findings demonstrate the involvement of PP2A in the regulation of liver metabolism.
Collapse
Affiliation(s)
- Liping Chen
- Guangdong Provincial Key Laboratory of Food, Nutrition and Health, Department of Toxicology, School of Public Health, Sun Yat-sen University, Guangzhou 510080, China
| | - Ping Guo
- Guangdong Provincial Key Laboratory of Food, Nutrition and Health, Department of Toxicology, School of Public Health, Sun Yat-sen University, Guangzhou 510080, China
| | - Wenxue Li
- Department of Toxicology, Guangzhou Center for Disease Control and Prevention, Guangzhou 510440, China
| | - Xinhang Jiang
- Guangdong Provincial Key Laboratory of Food, Nutrition and Health, Department of Toxicology, School of Public Health, Sun Yat-sen University, Guangzhou 510080, China
| | - Qun Zhao
- Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Science, National Chromatographic Research and Analysis Center, Dalian 116023, China
| | - Daochuan Li
- Guangdong Provincial Key Laboratory of Food, Nutrition and Health, Department of Toxicology, School of Public Health, Sun Yat-sen University, Guangzhou 510080, China
| | - Qing Wang
- Guangdong Provincial Key Laboratory of Food, Nutrition and Health, Department of Toxicology, School of Public Health, Sun Yat-sen University, Guangzhou 510080, China
| | - Yongmei Xiao
- Guangdong Provincial Key Laboratory of Food, Nutrition and Health, Department of Toxicology, School of Public Health, Sun Yat-sen University, Guangzhou 510080, China
| | - Xiumei Xing
- Guangdong Provincial Key Laboratory of Food, Nutrition and Health, Department of Toxicology, School of Public Health, Sun Yat-sen University, Guangzhou 510080, China
| | - Yaqin Pang
- Faculty of Toxicology, School of Public Health, Youjiang Medical College for Nationalities, Guangxi 533000, China
| | - Michael Aschner
- Department of Molecular Pharmacology, Albert Einstein College of Medicine, Forchheimer 209, 1300 Morris Park Avenue, Bronx, NY 10461, USA
| | - Lihua Zhang
- Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Science, National Chromatographic Research and Analysis Center, Dalian 116023, China.
| | - Wen Chen
- Guangdong Provincial Key Laboratory of Food, Nutrition and Health, Department of Toxicology, School of Public Health, Sun Yat-sen University, Guangzhou 510080, China.
| |
Collapse
|
2
|
Shoemaker JT, Zhang W, Atlas SI, Bryan RA, Inman SW, Vukasinovic J. A 3D Cell Culture Organ-on-a-Chip Platform With a Breathable Hemoglobin Analogue Augments and Extends Primary Human Hepatocyte Functions in vitro. Front Mol Biosci 2020; 7:568777. [PMID: 33195413 PMCID: PMC7645268 DOI: 10.3389/fmolb.2020.568777] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2020] [Accepted: 09/22/2020] [Indexed: 12/17/2022] Open
Abstract
Remarkable advances in three-dimensional (3D) cell cultures and organ-on-a-chip technologies have opened the door to recapitulate complex aspects of human physiology, pathology, and drug responses in vitro. The challenges regarding oxygen delivery, throughput, assay multiplexing, and experimental complexity are addressed to ensure that perfused 3D cell culture organ-on-a-chip models become a routine research tool adopted by academic and industrial stakeholders. To move the field forward, we present a throughput-scalable organ-on-a-chip insert system that requires a single tube to operate 48 statistically independent 3D cell culture organ models. Then, we introduce in-well perfusion to circumvent the loss of cell signaling and drug metabolites in otherwise one-way flow of perfusate. Further, to augment the relevancy of 3D cell culture models in vitro, we tackle the problem of oxygen transport by blood using, for the first time, a breathable hemoglobin analog to improve delivery of respiratory gases to cells, because in vivo approximately 98% of oxygen delivery to cells takes place via reversible binding to hemoglobin. Next, we show that improved oxygenation shifts cellular metabolic pathways toward oxidative phosphorylation that contributes to the maintenance of differentiated liver phenotypes in vitro. Lastly, we demonstrate that the activity of cytochrome P450 family of drug metabolizing enzymes is increased and prolonged in primary human hepatocytes cultured in 3D compared to two-dimensional (2D) cell culture gold standard with important ramifications for drug metabolism, drug-drug interactions and pharmacokinetic studies in vitro.
Collapse
Affiliation(s)
| | - Wanrui Zhang
- Lena Biosciences, Inc., Atlanta, GA, United States
| | | | | | | | | |
Collapse
|
3
|
Kammerer S, Küpper JH. Human hepatocyte systems for in vitro toxicology analysis. ACTA ACUST UNITED AC 2018. [DOI: 10.3233/jcb-179012] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Affiliation(s)
- Sarah Kammerer
- Institute of Biotechnology, Brandenburg University of Technology, Cottbus-Senftenberg, Germany
| | - Jan-Heiner Küpper
- Institute of Biotechnology, Brandenburg University of Technology, Cottbus-Senftenberg, Germany
| |
Collapse
|
4
|
Vilas-Boas V, Cooreman A, Gijbels E, Van Campenhout R, Gustafson E, Ballet S, Annaert P, Cogliati B, Vinken M. Primary hepatocytes and their cultures for the testing of drug-induced liver injury. ADVANCES IN PHARMACOLOGY (SAN DIEGO, CALIF.) 2018; 85:1-30. [PMID: 31307583 DOI: 10.1016/bs.apha.2018.08.001] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
Drug-induced liver injury is a major reason for discontinuation of drug development and withdrawal of drugs from the market. Intensive efforts in the last decades have focused on the establishment and finetuning of liver-based in vitro models for reliable prediction of hepatotoxicity triggered by drug candidates. Of those, primary hepatocytes and their cultures still are considered the gold standard, as they provide an acceptable reflection of the hepatic in vivo situation. Nevertheless, these in vitro systems cope with gradual deterioration of the differentiated morphological and functional phenotype. The present paper gives an overview of traditional and more recently introduced strategies to counteract this dedifferentiation process in an attempt to set up culture models that can be used for long-term testing purposes. The relevance and applicability of such optimized cultures of primary hepatocytes for the testing of drug-induced cholestatic liver injury is demonstrated.
Collapse
Affiliation(s)
- Vânia Vilas-Boas
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Brussels, Belgium
| | - Axelle Cooreman
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Brussels, Belgium
| | - Eva Gijbels
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Brussels, Belgium
| | - Raf Van Campenhout
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Brussels, Belgium
| | - Emma Gustafson
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Brussels, Belgium
| | - Steven Ballet
- Research Group of Organic Chemistry, Departments of Chemistry and Bioengineering Sciences, Vrije Universiteit Brussel, Brussels, Belgium
| | - Pieter Annaert
- Drug Delivery and Disposition, KU Leuven Department of Pharmaceutical and Pharmacological Sciences, Leuven, Belgium
| | - Bruno Cogliati
- Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil
| | - Mathieu Vinken
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Brussels, Belgium.
| |
Collapse
|
5
|
Chen C, Soto-Gutierrez A, Baptista PM, Spee B. Biotechnology Challenges to In Vitro Maturation of Hepatic Stem Cells. Gastroenterology 2018; 154:1258-1272. [PMID: 29428334 PMCID: PMC6237283 DOI: 10.1053/j.gastro.2018.01.066] [Citation(s) in RCA: 59] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/07/2017] [Revised: 01/05/2018] [Accepted: 01/10/2018] [Indexed: 12/16/2022]
Abstract
The incidence of liver disease is increasing globally. The only curative therapy for severe end-stage liver disease, liver transplantation, is limited by the shortage of organ donors. In vitro models of liver physiology have been developed and new technologies and approaches are progressing rapidly. Stem cells might be used as a source of liver tissue for development of models, therapies, and tissue-engineering applications. However, we have been unable to generate and maintain stable and mature adult liver cells ex vivo. We review factors that promote hepatocyte differentiation and maturation, including growth factors, transcription factors, microRNAs, small molecules, and the microenvironment. We discuss how the hepatic circulation, microbiome, and nutrition affect liver function, and the criteria for considering cells derived from stem cells to be fully mature hepatocytes. We explain the challenges to cell transplantation and consider future technologies for use in hepatic stem cell maturation, including 3-dimensional biofabrication and genome modification.
Collapse
Affiliation(s)
- Chen Chen
- Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands; The Royal Netherlands Academy of Arts and Sciences, Hubrecht Institute and University Medical Center Utrecht, Utrecht, The Netherlands
| | | | - Pedro M Baptista
- Instituto de Investigación Sanitaria de Aragón, Zaragoza, Spain; Centro de Investigación Biomédica en Red en el Área Temática de Enfermedades Hepáticas, Madrid, Spain; Fundación Agencia Aragonesa para la Investigación y el Desarrollo, Zaragoza, Spain; Instituto de Investigación Sanitaria de la Fundación Jiménez Díaz, Madrid, Spain; Department of Biomedical and Aerospace Engineering, Universidad Carlos III de Madrid, Madrid, Spain
| | - Bart Spee
- Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
| |
Collapse
|
6
|
Regulation of Human Cytochrome P4501A1 (hCYP1A1): A Plausible Target for Chemoprevention? BIOMED RESEARCH INTERNATIONAL 2016; 2016:5341081. [PMID: 28105425 PMCID: PMC5220472 DOI: 10.1155/2016/5341081] [Citation(s) in RCA: 42] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 09/05/2016] [Revised: 11/09/2016] [Accepted: 11/13/2016] [Indexed: 12/13/2022]
Abstract
Human cytochrome P450 1A1 (hCYP1A1) has been an object of study due to its role in precarcinogen metabolism; for this reason it is relevant to know more in depth the mechanisms that rule out its expression and activity, which make this enzyme a target for the development of novel chemiopreventive agents. The aim of this work is to review the origin, regulation, and structural and functional characteristics of CYP1A1 letting us understand its role in the bioactivation of precarcinogen and the consequences of its modulation in other physiological processes, as well as guide us in the study of this important protein.
Collapse
|
7
|
Effect of Chromatin-Remodeling Agents in Hepatic Differentiation of Rat Bone Marrow-Derived Mesenchymal Stem Cells In Vitro and In Vivo. Stem Cells Int 2016; 2016:3038764. [PMID: 27242905 PMCID: PMC4876003 DOI: 10.1155/2016/3038764] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2015] [Revised: 03/13/2016] [Accepted: 03/29/2016] [Indexed: 02/08/2023] Open
Abstract
Epigenetic events, including covalent histone modifications and DNA methylation, play fundamental roles in the determination of lineage-specific gene expression and cell fates. The aim of this study was to determine whether the DNA methyltransferase inhibitor (DNMTi) 5-aza-2′-deoxycytidine (5-aza-dC) and the histone deacetylase inhibitor (HDACi) trichostatin A (TSA) promote the hepatic differentiation of rat bone marrow-derived mesenchymal stem cells (rBM-MSCs) and their therapeutic effect on liver damage. 1 μM TSA and 20 μM 5-aza-dC were added to standard hepatogenic medium especially at differentiation and maturation steps and their potential function on hepatic differentiation in vitro and in vivo was determined. Exposure of rBM-MSCs to 1 μM TSA at both the differentiation and maturation steps considerably improved hepatic differentiation. TSA enhanced the development of the hepatocyte shape, promoted the chronological expression of hepatocyte-specific markers, and improved hepatic functions. In contrast, treatment of rBM-MSCs with 20 μM 5-aza-dC alone or in combination with TSA was ineffective in improving hepatic differentiation in vitro. TSA and/or 5-aza-dC derived hepatocytes-like cells failed to improve the therapeutic potential in liver damage. We conclude that HDACis enhance hepatic differentiation in a time-dependent manner, while DNMTis do not induce the hepatic differentiation of rBM-MSCs in vitro. Their in vivo function needs further investigation.
Collapse
|
8
|
Ramboer E, Rogiers V, Vanhaecke T, Vinken M. Effects of Trichostatin A on drug uptake transporters in primary rat hepatocyte cultures. EXCLI JOURNAL 2015; 14:567-76. [PMID: 26648816 PMCID: PMC4669911 DOI: 10.17179/excli2015-220] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/11/2015] [Accepted: 03/30/2015] [Indexed: 11/10/2022]
Abstract
The present study was set up to investigate the effects of Trichostatin A (TSA), a prototypical epigenetic modifier, on the expression and activity of hepatic drug uptake transporters in primary cultured rat hepatocytes. To this end, the expression of the sinusoidal transporters sodium-dependent taurocholate cotransporting polypeptide (Ntcp) and organic anion transporting polypeptide 4 (Oatp4) was monitored by real-time quantitative reverse transcriptase polymerase chain reaction analysis and immunoblotting. The activity of the uptake transporters was analyzed using radiolabeled substrates and chemical inhibitors. Downregulation of the expression and activity of Oatp4 and Ntcp was observed as a function of the cultivation time and could not be counteracted by TSA. In conclusion, the epigenetic modifier TSA does not seem to exert a positive effect on the expression and activity of the investigated uptake transporters in primary rat hepatocyte cultures.
Collapse
Affiliation(s)
- Eva Ramboer
- In Vitro Toxicology and Dermato-cosmetology research group, Center for Pharmaceutical Research, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel
| | - Vera Rogiers
- In Vitro Toxicology and Dermato-cosmetology research group, Center for Pharmaceutical Research, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel
| | - Tamara Vanhaecke
- In Vitro Toxicology and Dermato-cosmetology research group, Center for Pharmaceutical Research, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel
| | - Mathieu Vinken
- In Vitro Toxicology and Dermato-cosmetology research group, Center for Pharmaceutical Research, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel
| |
Collapse
|
9
|
Alizadeh E, Eslaminejad MB, Akbarzadeh A, Sadeghi Z, Abasi M, Herizchi R, Zarghami N. Upregulation of MiR-122 via Trichostatin A Treatments in Hepatocyte-like Cells Derived from Mesenchymal Stem Cells. Chem Biol Drug Des 2015; 87:296-305. [PMID: 26360933 DOI: 10.1111/cbdd.12664] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2015] [Revised: 08/29/2015] [Accepted: 08/31/2015] [Indexed: 12/13/2022]
Abstract
The miR-122 is a tissue-specific miRNA; its expression is abundant in liver. MiR-122 upregulation is crucial for differentiation, functionality, and maintenance of differentiated phenotype in hepatocytes. The improving effects of trichostatin A (TSA) on hepatic differentiation have been reported previously. The aim of this study was to determine whether TSA can affect the expression of miR-122 in hepatocyte-like cells (HLCs) generated from human adipose tissue-derived mesenchymal stem cells (hAT-MSCs). The hepatic differentiation of hAT-MSCs induced by a mixture of growth factors and cytokines either with or without TSA treatments. The functionality of HLCs generated with or without TSA and the expression levels of miR-122 were studied. The expression levels of miR-122 in TSA-treated HLCs was significantly (p < 0.05) higher than those generated by growth factors and cytokines, only. The downregulation of a-fetoprotein (AFP) levels but enhanced albumin synthesis (p < 0.05) and upregulation of liver-enriched transcription factors (LETFs) HNF4α (hepatocyte nuclear factor 4α) and HNF6 (hepatocyte nuclear factor 6) were observed in TSA-treated HLCs (p < 0.05). In conclusion, administration of TSA in hepatogenic differentiation of hAT-MSCs resulted in higher expression levels of miR-122, facilitation of differentiation, and subsequently attenuation of AFP levels.
Collapse
Affiliation(s)
- Effat Alizadeh
- Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Golgasht Ave., Tabriz 3137851656, I.R. Iran
| | - MohamadReza Baghaban Eslaminejad
- Department of Stem Cells and Developmental Biology at Cell Sciences Research Center, Royan Institute for Stem Cell Biology and Technology, ACER, Royan Institute, Tehran, I.R. Iran
| | - Abolfazl Akbarzadeh
- Department of Medical Nanotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Golgasht Ave., Tabriz 3137851656, I.R. Iran
| | - Zohre Sadeghi
- Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Golgasht Ave., Tabriz 3137851656, I.R. Iran
| | - Mozghan Abasi
- Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Golgasht Ave., Tabriz 3137851656, I.R. Iran
| | - Roya Herizchi
- Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Golgasht Ave., Tabriz 3137851656, I.R. Iran
| | - Nosratollah Zarghami
- Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Golgasht Ave., Tabriz 3137851656, I.R. Iran.,The Umbilical Cord Stem Cell Research Center (UCSRC), Tabriz University of Medical Sciences, Golgasht Ave., Tabriz 3137851656, I.R. Iran
| |
Collapse
|
10
|
Bolleyn J, Fraczek J, Rogiers V, Vanhaecke T. Epigenetic Modifications as Antidedifferentiation Strategy for Primary Hepatocytes in Culture. Methods Mol Biol 2015; 1250:203-211. [PMID: 26272144 DOI: 10.1007/978-1-4939-2074-7_14] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/04/2023]
Abstract
A well-known problem of cultured primary hepatocytes is their rapid dedifferentiation. During the last years, several strategies to counteract this phenomenon have been developed, of which changing the in vitro environment is the most popular one. However, mimicking the in vivo setting in vitro by adding soluble media additives or the restoration of both cell-cell and cell-extracellular matrix contacts is not sufficient and only delays the dedifferentiation process instead of counteracting it. In this chapter, new strategies to prevent the deterioration of the liver-specific phenotype of primary hepatocytes in culture by targeting the (epi)genetic mechanisms that drive hepatocellular gene expression are described.
Collapse
Affiliation(s)
- Jennifer Bolleyn
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090, Brussels, Belgium
| | | | | | | |
Collapse
|
11
|
Yin L, Zhu Y, Yang J, Ni Y, Zhou Z, Chen Y, Wen L. Adipose tissue-derived mesenchymal stem cells differentiated into hepatocyte-like cells in vivo and in vitro. Mol Med Rep 2014; 11:1722-32. [PMID: 25395242 PMCID: PMC4270341 DOI: 10.3892/mmr.2014.2935] [Citation(s) in RCA: 67] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2013] [Accepted: 07/22/2014] [Indexed: 01/03/2023] Open
Abstract
Cell-based therapy is a potential alternative to liver transplantation. The goal of the present study was to examine the in vivo and in vitro hepatic differentiation potential of adipose tissue-derived mesenchymal stem cells (AT-MSCs) and to explore its therapeutic use. AT-MSCs were isolated and cultured with hepatic differentiation medium. Bioactivity assays were used to study the properties of AT-MSCs. The morphology of differentiated AT-MSCs in serum-free hepatic differentiation medium changed into polygonal epithelial cells, while the morphology of AT-MSCs in a similar medium containing 2% fetal bovine serum remained unchanged. The differentiated cells cultured without serum showed hepatocyte-like cell morphology and hepatocyte-specific markers, including albumin (ALB) and α-fetoprotein. The bioactivity assays revealed that hepatocyte-like cells could take up low-density lipoprotein (LDL) and store glycogen. Furthermore, trichostatin A (TSA) enhanced ALB production and LDL uptake by the hepatocyte-like cells, analogous to the functions of human liver cells. ALB was detected in the livers of the CCl4-injured mice one month post-transplantation. This suggested that transplantation of the human AT-MSCs could relieve the impairment of acute CCl4-injured livers in nude mice. This therefore implied that adipose tissue was a source of multipotent stem cells which had the potential to differentiate into mature, transplantable hepatocyte-like cells in vivo and in vitro. In addition, the present study determined that TSA was essential to promoting differentiation of human MSC towards functional hepatocyte-like cells. The relief of liver injury following treatment with AT-MSCs suggested their potential as a novel therapeutic method for liver disorders or injury.
Collapse
Affiliation(s)
- Libo Yin
- Department of Traumatic Surgery, Changzhou No. 2 People's Hospital, Nanjing Medical University, Changzhou, Jiangsu 213000, P.R. China
| | - Yuhua Zhu
- Department of Traumatic Surgery, Changzhou No. 2 People's Hospital, Nanjing Medical University, Changzhou, Jiangsu 213000, P.R. China
| | - Jiangang Yang
- Department of Traumatic Surgery, Changzhou No. 2 People's Hospital, Nanjing Medical University, Changzhou, Jiangsu 213000, P.R. China
| | - Yijiang Ni
- Department of Traumatic Surgery, Changzhou No. 2 People's Hospital, Nanjing Medical University, Changzhou, Jiangsu 213000, P.R. China
| | - Zhao Zhou
- Department of Traumatic Surgery, Changzhou No. 2 People's Hospital, Nanjing Medical University, Changzhou, Jiangsu 213000, P.R. China
| | - Yu Chen
- Department of Traumatic Surgery, Changzhou No. 2 People's Hospital, Nanjing Medical University, Changzhou, Jiangsu 213000, P.R. China
| | - Lixing Wen
- Department of Traumatic Surgery, Changzhou No. 2 People's Hospital, Nanjing Medical University, Changzhou, Jiangsu 213000, P.R. China
| |
Collapse
|
12
|
Gomez A, Ingelman-Sundberg M. Pharmacoepigenetic aspects of gene polymorphism on drug therapies: effects of DNA methylation on drug response. Expert Rev Clin Pharmacol 2014; 2:55-65. [DOI: 10.1586/17512433.2.1.55] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
|
13
|
Primary hepatocytes and their cultures in liver apoptosis research. Arch Toxicol 2013; 88:199-212. [PMID: 24013573 DOI: 10.1007/s00204-013-1123-4] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2013] [Accepted: 08/29/2013] [Indexed: 01/18/2023]
Abstract
Apoptosis not only plays a key role in physiological demise of defunct hepatocytes, but is also associated with a plethora of acute and chronic liver diseases as well as with hepatotoxicity. The present paper focuses on the modelling of this mode of programmed cell death in primary hepatocyte cultures. Particular attention is paid to the activation of spontaneous apoptosis during the isolation of hepatocytes from the liver, its progressive manifestation upon the subsequent establishment of cell cultures and simultaneously to strategies to counteract this deleterious process. In addition, currently applied approaches to experimentally induce controlled apoptosis in this in vitro setting for mechanistic research purposes and thereby its detection using relevant biomarkers are reviewed.
Collapse
|
14
|
Godoy P, Hewitt NJ, Albrecht U, Andersen ME, Ansari N, Bhattacharya S, Bode JG, Bolleyn J, Borner C, Böttger J, Braeuning A, Budinsky RA, Burkhardt B, Cameron NR, Camussi G, Cho CS, Choi YJ, Craig Rowlands J, Dahmen U, Damm G, Dirsch O, Donato MT, Dong J, Dooley S, Drasdo D, Eakins R, Ferreira KS, Fonsato V, Fraczek J, Gebhardt R, Gibson A, Glanemann M, Goldring CEP, Gómez-Lechón MJ, Groothuis GMM, Gustavsson L, Guyot C, Hallifax D, Hammad S, Hayward A, Häussinger D, Hellerbrand C, Hewitt P, Hoehme S, Holzhütter HG, Houston JB, Hrach J, Ito K, Jaeschke H, Keitel V, Kelm JM, Kevin Park B, Kordes C, Kullak-Ublick GA, LeCluyse EL, Lu P, Luebke-Wheeler J, Lutz A, Maltman DJ, Matz-Soja M, McMullen P, Merfort I, Messner S, Meyer C, Mwinyi J, Naisbitt DJ, Nussler AK, Olinga P, Pampaloni F, Pi J, Pluta L, Przyborski SA, Ramachandran A, Rogiers V, Rowe C, Schelcher C, Schmich K, Schwarz M, Singh B, Stelzer EHK, Stieger B, Stöber R, Sugiyama Y, Tetta C, Thasler WE, Vanhaecke T, Vinken M, Weiss TS, Widera A, Woods CG, Xu JJ, Yarborough KM, Hengstler JG. Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME. Arch Toxicol 2013; 87:1315-530. [PMID: 23974980 PMCID: PMC3753504 DOI: 10.1007/s00204-013-1078-5] [Citation(s) in RCA: 1074] [Impact Index Per Article: 89.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2013] [Accepted: 05/06/2013] [Indexed: 12/15/2022]
Abstract
This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell-derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.
Collapse
Affiliation(s)
- Patricio Godoy
- Leibniz Research Centre for Working Environment and Human Factors (IFADO), 44139 Dortmund, Germany
| | | | - Ute Albrecht
- Clinic for Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine-University, Moorenstrasse 5, 40225 Düsseldorf, Germany
| | - Melvin E. Andersen
- The Hamner Institutes for Health Sciences, Research Triangle Park, NC USA
| | - Nariman Ansari
- Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University Frankfurt, Max-von-Laue-Str. 15, 60438 Frankfurt am Main, Germany
| | - Sudin Bhattacharya
- The Hamner Institutes for Health Sciences, Research Triangle Park, NC USA
| | - Johannes Georg Bode
- Clinic for Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine-University, Moorenstrasse 5, 40225 Düsseldorf, Germany
| | - Jennifer Bolleyn
- Department of Toxicology, Centre for Pharmaceutical Research, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, 1090 Brussels, Belgium
| | - Christoph Borner
- Institute of Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Germany
| | - Jan Böttger
- Institute of Biochemistry, Faculty of Medicine, University of Leipzig, 04103 Leipzig, Germany
| | - Albert Braeuning
- Department of Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, Wilhelmstr. 56, 72074 Tübingen, Germany
| | - Robert A. Budinsky
- Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, MI USA
| | - Britta Burkhardt
- BG Trauma Center, Siegfried Weller Institut, Eberhard Karls University Tübingen, 72076 Tübingen, Germany
| | - Neil R. Cameron
- Department of Chemistry, Durham University, Durham, DH1 3LE UK
| | - Giovanni Camussi
- Department of Medical Sciences, University of Torino, 10126 Turin, Italy
| | - Chong-Su Cho
- Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, 151-921 Korea
| | - Yun-Jaie Choi
- Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, 151-921 Korea
| | - J. Craig Rowlands
- Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, MI USA
| | - Uta Dahmen
- Experimental Transplantation Surgery, Department of General Visceral, and Vascular Surgery, Friedrich-Schiller-University Jena, 07745 Jena, Germany
| | - Georg Damm
- Department of General-, Visceral- and Transplantation Surgery, Charité University Medicine Berlin, 13353 Berlin, Germany
| | - Olaf Dirsch
- Institute of Pathology, Friedrich-Schiller-University Jena, 07745 Jena, Germany
| | - María Teresa Donato
- Unidad de Hepatología Experimental, IIS Hospital La Fe Avda Campanar 21, 46009 Valencia, Spain
- CIBERehd, Fondo de Investigaciones Sanitarias, Barcelona, Spain
- Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Valencia, Valencia, Spain
| | - Jian Dong
- The Hamner Institutes for Health Sciences, Research Triangle Park, NC USA
| | - Steven Dooley
- Department of Medicine II, Section Molecular Hepatology, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
| | - Dirk Drasdo
- Interdisciplinary Center for Bioinformatics (IZBI), University of Leipzig, 04107 Leipzig, Germany
- INRIA (French National Institute for Research in Computer Science and Control), Domaine de Voluceau-Rocquencourt, B.P. 105, 78153 Le Chesnay Cedex, France
- UPMC University of Paris 06, CNRS UMR 7598, Laboratoire Jacques-Louis Lions, 4, pl. Jussieu, 75252 Paris cedex 05, France
| | - Rowena Eakins
- Department of Molecular and Clinical Pharmacology, Centre for Drug Safety Science, Institute of Translational Medicine, University of Liverpool, Liverpool, UK
| | - Karine Sá Ferreira
- Institute of Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Germany
- GRK 1104 From Cells to Organs, Molecular Mechanisms of Organogenesis, Faculty of Biology, University of Freiburg, Freiburg, Germany
| | - Valentina Fonsato
- Department of Medical Sciences, University of Torino, 10126 Turin, Italy
| | - Joanna Fraczek
- Department of Toxicology, Centre for Pharmaceutical Research, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, 1090 Brussels, Belgium
| | - Rolf Gebhardt
- Institute of Biochemistry, Faculty of Medicine, University of Leipzig, 04103 Leipzig, Germany
| | - Andrew Gibson
- Department of Molecular and Clinical Pharmacology, Centre for Drug Safety Science, Institute of Translational Medicine, University of Liverpool, Liverpool, UK
| | - Matthias Glanemann
- Department of General-, Visceral- and Transplantation Surgery, Charité University Medicine Berlin, 13353 Berlin, Germany
| | - Chris E. P. Goldring
- Department of Molecular and Clinical Pharmacology, Centre for Drug Safety Science, Institute of Translational Medicine, University of Liverpool, Liverpool, UK
| | - María José Gómez-Lechón
- Unidad de Hepatología Experimental, IIS Hospital La Fe Avda Campanar 21, 46009 Valencia, Spain
- CIBERehd, Fondo de Investigaciones Sanitarias, Barcelona, Spain
| | - Geny M. M. Groothuis
- Department of Pharmacy, Pharmacokinetics Toxicology and Targeting, University of Groningen, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands
| | - Lena Gustavsson
- Department of Laboratory Medicine (Malmö), Center for Molecular Pathology, Lund University, Jan Waldenströms gata 59, 205 02 Malmö, Sweden
| | - Christelle Guyot
- Department of Clinical Pharmacology and Toxicology, University Hospital, 8091 Zurich, Switzerland
| | - David Hallifax
- Centre for Applied Pharmacokinetic Research (CAPKR), School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Oxford Road, Manchester, M13 9PT UK
| | - Seddik Hammad
- Department of Forensic Medicine and Veterinary Toxicology, Faculty of Veterinary Medicine, South Valley University, Qena, Egypt
| | - Adam Hayward
- Biological and Biomedical Sciences, Durham University, Durham, DH13LE UK
| | - Dieter Häussinger
- Clinic for Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine-University, Moorenstrasse 5, 40225 Düsseldorf, Germany
| | - Claus Hellerbrand
- Department of Medicine I, University Hospital Regensburg, 93053 Regensburg, Germany
| | | | - Stefan Hoehme
- Interdisciplinary Center for Bioinformatics (IZBI), University of Leipzig, 04107 Leipzig, Germany
| | - Hermann-Georg Holzhütter
- Institut für Biochemie Abteilung Mathematische Systembiochemie, Universitätsmedizin Berlin (Charité), Charitéplatz 1, 10117 Berlin, Germany
| | - J. Brian Houston
- Centre for Applied Pharmacokinetic Research (CAPKR), School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Oxford Road, Manchester, M13 9PT UK
| | | | - Kiyomi Ito
- Research Institute of Pharmaceutical Sciences, Musashino University, 1-1-20 Shinmachi, Nishitokyo-shi, Tokyo, 202-8585 Japan
| | - Hartmut Jaeschke
- Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160 USA
| | - Verena Keitel
- Clinic for Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine-University, Moorenstrasse 5, 40225 Düsseldorf, Germany
| | | | - B. Kevin Park
- Department of Molecular and Clinical Pharmacology, Centre for Drug Safety Science, Institute of Translational Medicine, University of Liverpool, Liverpool, UK
| | - Claus Kordes
- Clinic for Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine-University, Moorenstrasse 5, 40225 Düsseldorf, Germany
| | - Gerd A. Kullak-Ublick
- Department of Clinical Pharmacology and Toxicology, University Hospital, 8091 Zurich, Switzerland
| | - Edward L. LeCluyse
- The Hamner Institutes for Health Sciences, Research Triangle Park, NC USA
| | - Peng Lu
- The Hamner Institutes for Health Sciences, Research Triangle Park, NC USA
| | | | - Anna Lutz
- Department of Pharmaceutical Biology and Biotechnology, University of Freiburg, Freiburg, Germany
| | - Daniel J. Maltman
- Reinnervate Limited, NETPark Incubator, Thomas Wright Way, Sedgefield, TS21 3FD UK
| | - Madlen Matz-Soja
- Institute of Biochemistry, Faculty of Medicine, University of Leipzig, 04103 Leipzig, Germany
| | - Patrick McMullen
- The Hamner Institutes for Health Sciences, Research Triangle Park, NC USA
| | - Irmgard Merfort
- Department of Pharmaceutical Biology and Biotechnology, University of Freiburg, Freiburg, Germany
| | | | - Christoph Meyer
- Department of Medicine II, Section Molecular Hepatology, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
| | - Jessica Mwinyi
- Department of Clinical Pharmacology and Toxicology, University Hospital, 8091 Zurich, Switzerland
| | - Dean J. Naisbitt
- Department of Molecular and Clinical Pharmacology, Centre for Drug Safety Science, Institute of Translational Medicine, University of Liverpool, Liverpool, UK
| | - Andreas K. Nussler
- BG Trauma Center, Siegfried Weller Institut, Eberhard Karls University Tübingen, 72076 Tübingen, Germany
| | - Peter Olinga
- Division of Pharmaceutical Technology and Biopharmacy, Department of Pharmacy, University of Groningen, 9713 AV Groningen, The Netherlands
| | - Francesco Pampaloni
- Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University Frankfurt, Max-von-Laue-Str. 15, 60438 Frankfurt am Main, Germany
| | - Jingbo Pi
- The Hamner Institutes for Health Sciences, Research Triangle Park, NC USA
| | - Linda Pluta
- The Hamner Institutes for Health Sciences, Research Triangle Park, NC USA
| | - Stefan A. Przyborski
- Reinnervate Limited, NETPark Incubator, Thomas Wright Way, Sedgefield, TS21 3FD UK
- Biological and Biomedical Sciences, Durham University, Durham, DH13LE UK
| | - Anup Ramachandran
- Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160 USA
| | - Vera Rogiers
- Department of Toxicology, Centre for Pharmaceutical Research, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, 1090 Brussels, Belgium
| | - Cliff Rowe
- Department of Molecular and Clinical Pharmacology, Centre for Drug Safety Science, Institute of Translational Medicine, University of Liverpool, Liverpool, UK
| | - Celine Schelcher
- Department of Surgery, Liver Regeneration, Core Facility, Human in Vitro Models of the Liver, Ludwig Maximilians University of Munich, Munich, Germany
| | - Kathrin Schmich
- Department of Pharmaceutical Biology and Biotechnology, University of Freiburg, Freiburg, Germany
| | - Michael Schwarz
- Department of Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, Wilhelmstr. 56, 72074 Tübingen, Germany
| | - Bijay Singh
- Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, 151-921 Korea
| | - Ernst H. K. Stelzer
- Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University Frankfurt, Max-von-Laue-Str. 15, 60438 Frankfurt am Main, Germany
| | - Bruno Stieger
- Department of Clinical Pharmacology and Toxicology, University Hospital, 8091 Zurich, Switzerland
| | - Regina Stöber
- Leibniz Research Centre for Working Environment and Human Factors (IFADO), 44139 Dortmund, Germany
| | - Yuichi Sugiyama
- Sugiyama Laboratory, RIKEN Innovation Center, RIKEN, Yokohama Biopharmaceutical R&D Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045 Japan
| | - Ciro Tetta
- Fresenius Medical Care, Bad Homburg, Germany
| | - Wolfgang E. Thasler
- Department of Surgery, Ludwig-Maximilians-University of Munich Hospital Grosshadern, Munich, Germany
| | - Tamara Vanhaecke
- Department of Toxicology, Centre for Pharmaceutical Research, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, 1090 Brussels, Belgium
| | - Mathieu Vinken
- Department of Toxicology, Centre for Pharmaceutical Research, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, 1090 Brussels, Belgium
| | - Thomas S. Weiss
- Department of Pediatrics and Juvenile Medicine, University of Regensburg Hospital, Regensburg, Germany
| | - Agata Widera
- Leibniz Research Centre for Working Environment and Human Factors (IFADO), 44139 Dortmund, Germany
| | - Courtney G. Woods
- The Hamner Institutes for Health Sciences, Research Triangle Park, NC USA
| | | | | | - Jan G. Hengstler
- Leibniz Research Centre for Working Environment and Human Factors (IFADO), 44139 Dortmund, Germany
| |
Collapse
|
15
|
Ramboer E, Vanhaecke T, Rogiers V, Vinken M. Primary hepatocyte cultures as prominent in vitro tools to study hepatic drug transporters. Drug Metab Rev 2013; 45:196-217. [PMID: 23368091 DOI: 10.3109/03602532.2012.756010] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
Before any drug can be placed on the market, drug efficacy and safety must be ensured through rigorous testing. Animal models are used for this purpose, though currently increasing attention goes to the use of alternative in vitro systems. In particular, liver-based testing platforms that allow the prediction of pharmacokinetic (PK) and pharmacotoxicological properties during the early phase of drug development are of interest. They also enable the screening of potential effects on hepatic drug transporters. The latter are known to affect drug metabolism and disposition, thereby possibly underlying drug-drug interactions, which, in turn, may result in liver toxicity. Clearly, stable in vivo-like functional expression of drug transporters in hepatic in vitro settings is a prerequisite to be applicable in routine PK and pharmacotoxicological testing. In the first part of the article, an updated overview of hepatic drug transporters is provided, followed by a state-of-the-art review of drug-transporter production and activity in primary hepatocyte cultures (PHCs), being the gold-standard in vitro system. Specific focus is hereby put on strategies to maintain long-term functional expression, in casu of drug transporters, in these systems. In the second part, the use of PHCs to assess hepatobiliary transport and transporter-mediated interactions is outlined.
Collapse
Affiliation(s)
- Eva Ramboer
- Department of Toxicology, Center for Pharmaceutical Research, Vrije Universiteit Brussel, Brussels, Belgium.
| | | | | | | |
Collapse
|
16
|
Fraczek J, Bolleyn J, Vanhaecke T, Rogiers V, Vinken M. Primary hepatocyte cultures for pharmaco-toxicological studies: at the busy crossroad of various anti-dedifferentiation strategies. Arch Toxicol 2012; 87:577-610. [PMID: 23242478 DOI: 10.1007/s00204-012-0983-3] [Citation(s) in RCA: 91] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2012] [Accepted: 11/19/2012] [Indexed: 01/24/2023]
Abstract
Continuously increasing understanding of the molecular triggers responsible for the onset of diseases, paralleled by an equally dynamic evolution of chemical synthesis and screening methods, offers an abundance of pharmacological agents with a potential to become new successful drugs. However, before patients can benefit of newly developed pharmaceuticals, stringent safety filters need to be applied to weed out unfavourable drug candidates. Cost effectiveness and the need to identify compound liabilities, without exposing humans to unnecessary risks, has stimulated the shift of the safety studies to the earliest stages of drug discovery and development. In this regard, in vivo relevant organotypic in vitro models have high potential to revolutionize the preclinical safety testing. They can enable automation of the process, to match the requirements of high-throughput screening approaches, while satisfying ethical considerations. Cultures of primary hepatocytes became already an inherent part of the preclinical pharmaco-toxicological testing battery, yet their routine use, particularly for long-term assays, is limited by the progressive deterioration of liver-specific features. The availability of suitable hepatic and other organ-specific in vitro models is, however, of paramount importance in the light of changing European legal regulations in the field of chemical compounds of different origin, which gradually restrict the use of animal studies for safety assessment, as currently witnessed in cosmetic industry. Fortunately, research groups worldwide spare no effort to establish hepatic in vitro systems. In the present review, both classical and innovative methodologies to stabilize the in vivo-like hepatocyte phenotype in culture of primary hepatocytes are presented and discussed.
Collapse
Affiliation(s)
- J Fraczek
- Department of Toxicology, Faculty of Medicine and Pharmacy, Centre for Pharmaceutical Research, Vrije Universiteit Brussel, Belgium.
| | | | | | | | | |
Collapse
|
17
|
Doktorova TY, Ellinger-Ziegelbauer H, Vinken M, Vanhaecke T, van Delft J, Kleinjans J, Ahr HJ, Rogiers V. Comparison of genotoxicant-modified transcriptomic responses in conventional and epigenetically stabilized primary rat hepatocytes with in vivo rat liver data. Arch Toxicol 2012; 86:1703-15. [PMID: 23052194 DOI: 10.1007/s00204-012-0946-8] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2011] [Accepted: 11/29/2011] [Indexed: 12/29/2022]
Abstract
The concept of mechanistic toxicogenomics implies that compound-induced changes in gene expression profiles provide valuable information about their mode of action. A growing number of research groups have presented evidence that whole-genome gene expression profiling techniques might be used as tools for in vivo and in vitro generation of gene signatures and elucidation of molecular mechanisms after exposure to toxic compounds. An important issue to be investigated is the in vivo relevance of in vitro-obtained data. In the current study, we compare the gene expression profiles generated in vitro, after exposing conventional and epigenetically stabilized primary rat hepatocytes to well-known genotoxic hepatocarcinogens (aflatoxin B1, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and 2-nitrofluorene) with those derived in vivo after oral exposure of rats to these compounds. Similar statistical tools were applied on both sets of data. The major molecular pathways affected in the in vivo setting were DNA damage, detoxification and cell survival response, as previously described. In the conventional hepatocyte cultures, two of the three genotoxicants showed quite similar responses as in vivo with respect to these pathways. The third compound (2-nitrofluorene) revealed in vitro response which was not observed in vivo. In the epigenetically stabilized hepatocytes, in contrast to what was expected, the responses were less relevant for the in vivo situation. This study highlights the importance of in vitro/in vivo comparison of data that are generated using in vitro models and shows that conventional primary rat hepatocyte cultures represent an appropriate in vitro model to retrieve mechanistic information on the exposure to genotoxicants.
Collapse
Affiliation(s)
- Tatyana Y Doktorova
- Department of Toxicology, Center for Pharmaceutical Research (CePhar), Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, Brussels, Belgium.
| | | | | | | | | | | | | | | |
Collapse
|
18
|
Vinken M, Vanhaecke T, Rogiers V. Primary hepatocyte cultures as in vitro tools for toxicity testing: quo vadis? Toxicol In Vitro 2012; 26:541-4. [PMID: 22261203 DOI: 10.1016/j.tiv.2012.01.002] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2011] [Revised: 12/20/2011] [Accepted: 01/04/2012] [Indexed: 12/29/2022]
Abstract
Cultures of primary hepatocytes are versatile tools that can serve many in vitro toxicity testing purposes. However, they cope with dedifferentiation, a process that is already initiated during the hepatocyte isolation procedure and that is manifested as the progressive loss of functionality upon subsequent cultivation. A number of strategies to prevent dedifferentiation have been introduced over the last decades, all which aim at re-establishing the in vivo hepatocyte micro-environment in vitro, but that are of merely limited success. Recent mechanistic insight into the mechanisms that underlie hepatocyte dedifferentiation has opened new avenues for the development of novel approaches that target the actual causes of this deteriorative process and thus for the generation of a long-term hepatic in vitro tool. Such experimental system is urgently needed, especially in the light of the stringent European legislative modifications that are currently encountered by the pharmaceutical, chemical and, particularly, the cosmetic industry.
Collapse
Affiliation(s)
- Mathieu Vinken
- Department of Toxicology, Center for Pharmaceutical Research, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium.
| | | | | |
Collapse
|
19
|
Vinken M. Role of connexin-related signalling in hepatic homeostasis and its relevance for liver-based in vitro modelling. World J Gastrointest Pathophysiol 2011; 2:82-7. [PMID: 22013553 PMCID: PMC3196623 DOI: 10.4291/wjgp.v2.i5.82] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/03/2011] [Revised: 09/02/2011] [Accepted: 09/09/2011] [Indexed: 02/06/2023] Open
Abstract
Direct intercellular communication mediated by gap junctions constitutes a major regulatory platform in the control of hepatic homeostasis. Hepatocellular gap junctions are composed of two hemichannels of adjacent cells which are built up by connexin proteins,
in casu Cx32. Mathieu Vinken, Pofessor at the Department of Toxicology of the Free University Brussels-Belgium, was one of the first investigators to demonstrate that hepatic connexin expression is controlled by epigenetic mechanisms. In particular, he found that inhibitors of histone deacetylase enzymes enhance Cx32 production and gap junction activity in cultures of primary hepatocytes, a finding that is of importance for liver-based in vitro modelling. Professor Dr. Mathieu Vinken’s recent work is focussed on the elucidation of the role of connexin proteins and their channels in the hepatocyte life cycle. Specific attention is paid to apoptosis in this context, whereby it has been found that Cx32 hemichannels control the termination of induced cell death in cultures of primary hepatocytes. Overall, Professor Dr. Mathieu Vinken’s research can be considered as an important contribution to the field of hepatic connexin physiology.
Collapse
|
20
|
Adler S, Basketter D, Creton S, Pelkonen O, van Benthem J, Zuang V, Andersen KE, Angers-Loustau A, Aptula A, Bal-Price A, Benfenati E, Bernauer U, Bessems J, Bois FY, Boobis A, Brandon E, Bremer S, Broschard T, Casati S, Coecke S, Corvi R, Cronin M, Daston G, Dekant W, Felter S, Grignard E, Gundert-Remy U, Heinonen T, Kimber I, Kleinjans J, Komulainen H, Kreiling R, Kreysa J, Leite SB, Loizou G, Maxwell G, Mazzatorta P, Munn S, Pfuhler S, Phrakonkham P, Piersma A, Poth A, Prieto P, Repetto G, Rogiers V, Schoeters G, Schwarz M, Serafimova R, Tähti H, Testai E, van Delft J, van Loveren H, Vinken M, Worth A, Zaldivar JM. Alternative (non-animal) methods for cosmetics testing: current status and future prospects-2010. Arch Toxicol 2011; 85:367-485. [PMID: 21533817 DOI: 10.1007/s00204-011-0693-2] [Citation(s) in RCA: 358] [Impact Index Per Article: 25.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2011] [Accepted: 03/03/2011] [Indexed: 01/09/2023]
Abstract
The 7th amendment to the EU Cosmetics Directive prohibits to put animal-tested cosmetics on the market in Europe after 2013. In that context, the European Commission invited stakeholder bodies (industry, non-governmental organisations, EU Member States, and the Commission's Scientific Committee on Consumer Safety) to identify scientific experts in five toxicological areas, i.e. toxicokinetics, repeated dose toxicity, carcinogenicity, skin sensitisation, and reproductive toxicity for which the Directive foresees that the 2013 deadline could be further extended in case alternative and validated methods would not be available in time. The selected experts were asked to analyse the status and prospects of alternative methods and to provide a scientifically sound estimate of the time necessary to achieve full replacement of animal testing. In summary, the experts confirmed that it will take at least another 7-9 years for the replacement of the current in vivo animal tests used for the safety assessment of cosmetic ingredients for skin sensitisation. However, the experts were also of the opinion that alternative methods may be able to give hazard information, i.e. to differentiate between sensitisers and non-sensitisers, ahead of 2017. This would, however, not provide the complete picture of what is a safe exposure because the relative potency of a sensitiser would not be known. For toxicokinetics, the timeframe was 5-7 years to develop the models still lacking to predict lung absorption and renal/biliary excretion, and even longer to integrate the methods to fully replace the animal toxicokinetic models. For the systemic toxicological endpoints of repeated dose toxicity, carcinogenicity and reproductive toxicity, the time horizon for full replacement could not be estimated.
Collapse
Affiliation(s)
- Sarah Adler
- Centre for Documentation and Evaluation of Alternatives to Animal Experiments (ZEBET), Federal Institute for Risk Assessment (BfR), Berlin, Germany
| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
Collapse
|
21
|
Vinci B, Duret C, Klieber S, Gerbal-Chaloin S, Sa-Cunha A, Laporte S, Suc B, Maurel P, Ahluwalia A, Daujat-Chavanieu M. Modular bioreactor for primary human hepatocyte culture: medium flow stimulates expression and activity of detoxification genes. Biotechnol J 2011; 6:554-64. [PMID: 21259441 PMCID: PMC3123466 DOI: 10.1002/biot.201000326] [Citation(s) in RCA: 86] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2010] [Revised: 12/02/2010] [Accepted: 12/05/2010] [Indexed: 01/19/2023]
Abstract
Down-regulation of detoxification genes, notably cytochrome P450 (CYPs), in primary hepatocyte cultures is a long-standing and major concern. We evaluated the influence of medium flow in this model. Hepatocytes isolated from 12 different liver donors were cultured either in a multichamber modular bioreactor (MCmB, flow rate 250-500 μL/min) or under standard/static conditions, and the expression of 32 genes, enzyme activities and biological parameters were measured 7-21 days later. mRNA expression of genes involved in xenobiotic/drug metabolism and transport, including CYP1A1, 1A2, 2B6, 2C9, 3A4 (and activities for some of them), UDP-glucuronosyltransferase (UGT) 1A1, UGT2B4, UGT2B7, glutathione S-transferase (GSTα), and multidrug resistance protein 1 (MDR1) and MRP2, were specifically up-regulated by medium flow as compared with static controls in all cultures tested. In 2-week-old cultures, expression of detoxification genes reached levels close to or higher than those measured in freshly isolated hepatocytes. In contrast, CYP2D6 and most of other tested genes were not affected by medium flow. We conclude that medium flow specifically interferes with, and up-regulates, the activity of xenosensors and/or the expression of detoxification genes in primary human hepatocytes. Down-regulation of detoxification genes in conventional (static) cultures is therefore partly a consequence of the absence of medium circulation.
Collapse
Affiliation(s)
- Bruna Vinci
- Centro Interdipartimentale di Ricerca E. Piaggio, Faculty of Engineering, University of Pisa, Pisa, Italy
| | | | | | | | | | | | | | | | | | | |
Collapse
|
22
|
Bolleyn J, Fraczek J, Vinken M, Lizarraga D, Gaj S, van Delft JHM, Rogiers V, Vanhaecke T. Effect of Trichostatin A on miRNA expression in cultures of primary rat hepatocytes. Toxicol In Vitro 2011; 25:1173-82. [PMID: 21513791 DOI: 10.1016/j.tiv.2011.04.013] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2010] [Revised: 03/01/2011] [Accepted: 04/08/2011] [Indexed: 10/18/2022]
Abstract
In the present study, the effect of Trichostatin A (TSA), a histone deacetylase inhibitor, was investigated on the microRNA (miR, miRNA) expression profile in cultured primary rat hepatocytes by means of microarray analysis. Simultaneously, albumin secretory capacity and morphological features of the hepatocytes were evaluated throughout the culture time. In total, 25 out of 348 miRNAs were found to be differentially expressed between freshly isolated hepatocytes and 7-day cultured cells. Nineteen of these miRNAs were connected with 'general metabolism'. miR-21 and miR-126 were shown to be the most up and down regulated miRs upon cultivation and could be linked to the proliferative response triggered in the hepatocytes upon their isolation from the liver. miR-379 and miR-143, on the other hand, were found to be the most up and down regulated miRs upon TSA treatment. Together with the higher expression of miR-122 observed in TSA-treated versus non-treated cultures, we hypothesize that the changes observed for miR-122, miR-143 and miR-379 could be related to the inhibitory effects of TSA on hepatocellular proliferation.
Collapse
Affiliation(s)
- Jennifer Bolleyn
- Department of Toxicology, Center for Pharmaceutical Research, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, B-1090 Brussels, Belgium.
| | | | | | | | | | | | | | | |
Collapse
|
23
|
Synergetic effects of DNA demethylation and histone deacetylase inhibition in primary rat hepatocytes. Invest New Drugs 2011; 30:1715-24. [DOI: 10.1007/s10637-011-9659-8] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2011] [Accepted: 03/15/2011] [Indexed: 12/16/2022]
|
24
|
Abstract
IMPORTANCE OF THE FIELD Reactive oxygen species (ROS) occur as natural by-products of oxygen metabolism and have important cellular functions. Normally, the cell is able to maintain an adequate balance between the formation and removal of ROS either via anti-oxidants or through the use specific enzymatic pathways. However, if this balance is disturbed, oxidative stress may occur in the cell, a situation linked to the pathogenesis of many diseases, including cancer. AREAS COVERED IN THIS REVIEW HDACs are important regulators of many oxidative stress pathways including those involved with both sensing and coordinating the cellular response to oxidative stress. In particular aberrant regulation of these pathways by histone deacetylases may play critical roles in cancer progression. WHAT THE READER WILL GAIN In this review we discuss the notion that targeting HDACs may be a useful therapeutic avenue in the treatment of oxidative stress in cancer, using chronic obstructive pulmonary disease (COPD), NSCLC and hepatocellular carcinoma (HCC) as examples to illustrate this possibility. TAKE HOME MESSAGE Epigenetic mechanisms may be an important new therapeutic avenue for targeting oxidative stress in cancer.
Collapse
Affiliation(s)
- Matthew W Lawless
- Mater Misericordiae University Hospital, University College Dublin, Centre for Liver Disease, Dublin, Ireland
| | | | | |
Collapse
|
25
|
Abstract
In vitro hepatocyte models represent very useful systems in both fundamental research and various application areas. Primary hepatocytes appear as the closest model for the liver in vivo. However, they are phenotypically unstable, have a limited life span and in addition, exhibit large interdonor variability when of human origin. Hepatoma cell lines appear as an alternative but only the HepaRG cell line exhibits various functions, including major cytochrome P450 activities, at levels close to those found in primary hepatocytes. In vitro hepatocyte models have brought a substantial contribution to the understanding of the biochemistry, physiology, and cell biology of the normal and diseased liver and in various application domains such as xenobiotic metabolism and toxicity, virology, parasitology, and more generally cell therapies. In the future, new well-differentiated hepatocyte cell lines derived from tumors or from either embryonic or adult stem cells might be expected and although hepatocytes will continue to be used in various fields, these in vitro liver models should allow marked advances, especially in cell-based therapies and predictive and mechanistic hepatotoxicity of new drugs and other chemicals. All models will benefit from new developments in throughput screening based on cell chips coupled with high-content imaging and in toxicogenomics technologies.
Collapse
|
26
|
Preservation of hepatocellular functionality in cultures of primary rat hepatocytes upon exposure to 4-Me2N-BAVAH, a hydroxamate-based HDAC-inhibitor. Toxicol In Vitro 2010; 25:100-9. [PMID: 20932894 DOI: 10.1016/j.tiv.2010.09.013] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2010] [Revised: 09/25/2010] [Accepted: 09/27/2010] [Indexed: 01/27/2023]
Abstract
Great efforts are being put in the development/optimization of reliable and highly predictive models for high-throughput screening of efficacy and toxicity of promising drug candidates. The use of primary hepatocyte cultures, however, is still limited by the occurrence of phenotypic alterations, including loss of xenobiotic biotransformation capacity. In the present study, the differentiation-stabilizing effect of a new histone deacetylase inhibitor 5-(4-dimethylaminobenzoyl)-aminovaleric acid hydroxamide (4-Me(2)N-BAVAH), a structural Trichostatin A (TSA)-analogue with a more favourable pharmaco-toxicological profile, was studied at a genome-wide scale by means of microarray analysis. Several genes coding for xenobiotic biotransformation enzymes were found to be positively regulated upon exposure to 4-Me(2)N-BAVAH. For CYP1A1/2B1/3A2, these observations were confirmed by qRT-PCR and immunoblot analysis. In addition, significantly higher 7-ethoxyresorufin-O-deethylase and 7-pentoxyresorufin-O-dealkylase activity levels were measured. These effects were accompanied by an increased expression of CCAAT/enhancer binding protein alpha and hepatic nuclear factor (HNF)4α, but not of HNF1α. Finally, 4-Me(2)N-BAVAH was found to induce histone H3 acetylation at the proximal promoter of the albumin, CYP1A1 and CYP2B1 genes, suggesting that chromatin remodelling is directly involved in the transcriptional regulation of these genes. In conclusion, histone deacetylase inhibitors prove to be efficient agents for better maintaining a differentiated hepatic phenotype in rat hepatocyte cultures.
Collapse
|
27
|
Evankovich J, Cho SW, Zhang R, Cardinal J, Dhupar R, Zhang L, Klune JR, Zlotnicki J, Billiar T, Tsung A. High mobility group box 1 release from hepatocytes during ischemia and reperfusion injury is mediated by decreased histone deacetylase activity. J Biol Chem 2010; 285:39888-97. [PMID: 20937823 DOI: 10.1074/jbc.m110.128348] [Citation(s) in RCA: 200] [Impact Index Per Article: 13.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
The mobilization and extracellular release of nuclear high mobility group box-1 (HMGB1) by ischemic cells activates inflammatory pathways following liver ischemia/reperfusion (I/R) injury. In immune cells such as macrophages, post-translational modification by acetylation appears to be critical for active HMGB1 release. Hyperacetylation shifts its equilibrium from a predominant nuclear location toward cytosolic accumulation and subsequent release. However, mechanisms governing its release by parenchymal cells such as hepatocytes are unknown. In this study, we found that serum HMGB1 released following liver I/R in vivo is acetylated, and that hepatocytes exposed to oxidative stress in vitro also released acetylated HMGB1. Histone deacetylases (HDACs) are a family of enzymes that remove acetyl groups and control the acetylation status of histones and various intracellular proteins. Levels of acetylated HMGB1 increased with a concomitant decrease in total nuclear HDAC activity, suggesting that suppression in HDAC activity contributes to the increase in acetylated HMGB1 release after oxidative stress in hepatocytes. We identified the isoforms HDAC1 and HDAC4 as critical in regulating acetylated HMGB1 release. Activation of HDAC1 was decreased in the nucleus of hepatocytes undergoing oxidative stress. In addition, HDAC1 knockdown with siRNA promoted HMGB1 translocation and release. Furthermore, we demonstrate that HDAC4 is shuttled from the nucleus to cytoplasm in response to oxidative stress, resulting in decreased HDAC activity in the nucleus. Together, these findings suggest that decreased nuclear HDAC1 and HDAC4 activities in hepatocytes following liver I/R is a mechanism that promotes the hyperacetylation and subsequent release of HMGB1.
Collapse
Affiliation(s)
- John Evankovich
- Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA
| | | | | | | | | | | | | | | | | | | |
Collapse
|
28
|
Ellis JK, Chan PH, Doktorova T, Athersuch TJ, Cavill R, Vanhaecke T, Rogiers V, Vinken M, Nicholson JK, M D Ebbels T, Keun HC. Effect of the histone deacetylase inhibitor trichostatin a on the metabolome of cultured primary hepatocytes. J Proteome Res 2010; 9:413-9. [PMID: 19894772 DOI: 10.1021/pr9007656] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Trichostatin A (TSA) is a histone deacetylase inhibitor that has antiproliferative and differentiation-inducing effects on cancer cells, and in cultures of primary hepatocytes has been shown to maintain xenobiotic metabolic capacity. Using an NMR-based metabolic profiling approach, we evaluated if the endogenous metabolome was stabilized and the normal metabolic phenotype retained in this model. Aqueous soluble metabolites were extracted from isolated rat hepatocytes after 44 and 92 h exposure to TSA (25 muM) together with time-matched controls and measured by (1)H NMR spectroscopy. Multivariate analysis showed a clear difference in the global metabolic profile over time in control samples, while the TSA treated group was more closely clustered at both time points, suggesting that treatment reduced the time related effect on metabolism that was observed in the control. TSA treatment was associated with decreases in glycerophosphocholine, 3-hydroxybutyric acid, glycine and adenosine, an increase in glycogen, and a reduction in the decrease of inosine, hypoxanthine, and glutathione over time. Collectively, our data suggest that TSA treatment reduces the loss of a normal metabolic phenotype in cultured primary hepatocytes, improving the model as a tool to study endogenous liver metabolism, xenobiotic metabolism, and potentially affecting the accuracy of all biological assays in this system.
Collapse
Affiliation(s)
- James K Ellis
- Biomolecular Medicine, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, Sir Alexander Fleming Building, South Kensington, London, United Kingdom
| | | | | | | | | | | | | | | | | | | | | |
Collapse
|
29
|
EU research activities in alternative testing strategies: current status and future perspectives. Arch Toxicol 2009; 83:1037-42. [DOI: 10.1007/s00204-009-0484-1] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
|
30
|
Chemically distinct HDAC inhibitors prevent adipose conversion of subcutaneous human white preadipocytes at an early stage of the differentiation program. Exp Cell Res 2009; 315:3267-80. [DOI: 10.1016/j.yexcr.2009.09.012] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2009] [Revised: 09/08/2009] [Accepted: 09/11/2009] [Indexed: 12/22/2022]
|
31
|
Snykers S, De Kock J, Rogiers V, Vanhaecke T. In vitro differentiation of embryonic and adult stem cells into hepatocytes: state of the art. Stem Cells 2009; 27:577-605. [PMID: 19056906 PMCID: PMC2729674 DOI: 10.1634/stemcells.2008-0963] [Citation(s) in RCA: 193] [Impact Index Per Article: 12.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Stem cells are a unique source of self-renewing cells within the human body. Before the end of the last millennium, adult stem cells, in contrast to their embryonic counterparts, were considered to be lineage-restricted cells or incapable of crossing lineage boundaries. However, the unique breakthrough of muscle and liver regeneration by adult bone marrow stem cells at the end of the 1990s ended this long-standing paradigm. Since then, the number of articles reporting the existence of multipotent stem cells in skin, neuronal tissue, adipose tissue, and bone marrow has escalated, giving rise, both in vivo and in vitro, to cell types other than their tissue of origin. The phenomenon of fate reprogrammation and phenotypic diversification remains, though, an enigmatic and rare process. Understanding how to control both proliferation and differentiation of stem cells and their progeny is a challenge in many fields, going from preclinical drug discovery and development to clinical therapy. In this review, we focus on current strategies to differentiate embryonic, mesenchymal(-like), and liver stem/progenitor cells into hepatocytes in vitro. Special attention is paid to intracellular and extracellular signaling, genetic modification, and cell-cell and cell-matrix interactions. In addition, some recommendations are proposed to standardize, optimize, and enrich the in vitro production of hepatocyte-like cells out of stem/progenitor cells.
Collapse
Affiliation(s)
- Sarah Snykers
- Department of Toxicology, Vrije Universiteit Brussel, Belgium.
| | | | | | | |
Collapse
|
32
|
Joanna F, van Grunsven LA, Mathieu V, Sarah S, Sarah D, Karin V, Tamara V, Vera R. Histone deacetylase inhibition and the regulation of cell growth with particular reference to liver pathobiology. J Cell Mol Med 2009; 13:2990-3005. [PMID: 19583816 PMCID: PMC4516460 DOI: 10.1111/j.1582-4934.2009.00831.x] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
The transcriptional activity of genes largely depends on the accessibility of specific chromatin regions to transcriptional regulators. This process is controlled by diverse post-transcriptional modifications of the histone amino termini of which reversible acetylation plays a vital role. Histone acetyltransferases (HATs) are responsible for the addition of acetyl groups and histone deacetylases (HDACs) catalyse the reverse reaction. In general, though not exclusively, histone acetylation is associated with a positive regulation of transcription, whereas histone deacetylation is correlated with transcriptional silencing. The elucidation of unequivocal links between aberrant action of HDACs and tumorigenesis lies at the base of key scientific importance of these enzymes. In particular, the potential benefit of HDAC inhibition has been confirmed in various tumour cell lines, demonstrating antiproliferative, differentiating and pro-apoptotic effects. Consequently, the dynamic quest for HDAC inhibitors (HDIs) as a new class of anticancer drugs was set off, resulting in a number of compounds that are currently evaluated in clinical trials. Ironically, the knowledge with respect to the expression pattern and function of individual HDAC isoenzymes remains largely elusive. In the present review, we provide an update of the current knowledge on the involvement of HDACs in the regulation of fundamental cellular processes in the liver, being the main site for drug metabolism within the body. Focus lies on the involvement of HDACs in the regulation of growth of normal and transformed hepatocytes and the transdifferentiation process of stellate cells. Furthermore, extrapolation of our present knowledge on HDAC functionality towards innovative treatment of malignant and non-malignant, hyperproliferative and inflammatory disorders is discussed.
Collapse
Affiliation(s)
- Fraczek Joanna
- Department of Toxicology, Vrije Universiteit Brussel, Laarbeeklaan, Brussels, Belgium.
| | | | | | | | | | | | | | | |
Collapse
|
33
|
Snykers S, Henkens T, De Rop E, Vinken M, Fraczek J, De Kock J, De Prins E, Geerts A, Rogiers V, Vanhaecke T. Role of epigenetics in liver-specific gene transcription, hepatocyte differentiation and stem cell reprogrammation. J Hepatol 2009; 51:187-211. [PMID: 19457566 DOI: 10.1016/j.jhep.2009.03.009] [Citation(s) in RCA: 55] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
Controlling both growth and differentiation of stem cells and their differentiated somatic progeny is a challenge in numerous fields, from preclinical drug development to clinical therapy. Recently, new insights into the underlying molecular mechanisms have unveiled key regulatory roles of epigenetic marks driving cellular pluripotency, differentiation and self-renewal/proliferation. Indeed, the transcription of genes, governing cell-fate decisions during development and maintenance of a cell's differentiated status in adult life, critically depends on the chromatin accessibility of transcription factors to genomic regulatory and coding regions. In this review, we discuss the epigenetic control of (liver-specific) gene-transcription and the intricate interplay between chromatin modulation, including histone (de)acetylation and DNA (de)methylation, and liver-enriched transcription factors. Special attention is paid to their role in directing hepatic differentiation of primary hepatocytes and stem cells in vitro.
Collapse
Affiliation(s)
- Sarah Snykers
- Department of Toxicology, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium.
| | | | | | | | | | | | | | | | | | | |
Collapse
|
34
|
De Vocht C, Ranquin A, Willaert R, Van Ginderachter JA, Vanhaecke T, Rogiers V, Versées W, Van Gelder P, Steyaert J. Assessment of stability, toxicity and immunogenicity of new polymeric nanoreactors for use in enzyme replacement therapy of MNGIE. J Control Release 2009; 137:246-54. [PMID: 19371766 DOI: 10.1016/j.jconrel.2009.03.020] [Citation(s) in RCA: 58] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2008] [Revised: 03/26/2009] [Accepted: 03/30/2009] [Indexed: 11/30/2022]
Abstract
The lack of a crucial metabolic enzyme can lead to accumulating substrate concentrations in the bloodstream and severe human enzyme deficiency diseases. Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE) is such a fatal genetic disorder, caused by a thymidine phosphorylase deficiency. Enzyme replacement therapy is a strategy where the deficient enzyme is administered intravenously in order to decrease the toxic substrate concentrations. Such a therapy is however not very efficient due to the fast elimination of the native enzyme from the circulation. In this study we evaluate the potential of using polymeric enzyme-loaded nanoparticles to improve the delivery of therapeutic enzymes. We constructed new 200-nanometer PMOXA-PDMS-PMOXA polymeric nanoparticles that encapsulate the enzyme thymidine phosphorylase. These particles are permeabilised for substrates and products by the reconstitution of the nucleoside-specific porin Tsx in their polymeric wall. We show that the obtained 'nanoreactors' are enzymatically active and stable in blood serum at 37 degrees C. Moreover, they do not provoke cytotoxicity when incubated with hepatocytes for 4 days, nor do they induce a macrophage-mediated inflammatory response ex vivo and in vivo. All data highlight the potential of such nanoreactors for their application in enzyme replacement therapy of MNGIE.
Collapse
Affiliation(s)
- Caroline De Vocht
- Structural Biology Brussels, Vrije Universiteit Brussel (VUB), 1050 Brussels (Elsene), Belgium.
| | | | | | | | | | | | | | | | | |
Collapse
|
35
|
Screening of amide analogues of Trichostatin A in cultures of primary rat hepatocytes: search for potent and safe HDAC inhibitors. Invest New Drugs 2008; 27:338-46. [DOI: 10.1007/s10637-008-9180-x] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2008] [Accepted: 09/17/2008] [Indexed: 12/20/2022]
|
36
|
Henkens T, Vinken M, Lukaszuk A, Tourwé D, Vanhaecke T, Rogiers V. Differential effects of hydroxamate histone deacetylase inhibitors on cellular functionality and gap junctions in primary cultures of mitogen-stimulated hepatocytes. Toxicol Lett 2008; 178:37-43. [PMID: 18358644 DOI: 10.1016/j.toxlet.2008.02.002] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2007] [Revised: 02/05/2008] [Accepted: 02/06/2008] [Indexed: 02/06/2023]
Abstract
Histone deacetylase (HDAC)-inhibitors are well known to induce proliferative blocks and concomitant differentiation boosts in a plethora of tumor cells. Despite their promising potential as clinical therapeutics, however, the biological outcome of HDAC-inhibitors in non-tumorous cells has been poorly documented. We previously reported that the HDAC-inhibitor trichostatin A (TSA) and its metabolically more stable structural analogue 5-(4-dimethylaminobenzoyl)-aminovaleric acid hydroxamide (4-Me2N-BAVAH) cause cell cycle arrests in primary cultures of mitogen-stimulated hepatocytes. The present study was set up to explore whether this proliferative block in non-tumorous cells is also associated with inducing effects on the differentiated hepatocellular phenotype, a scenario that is usually observed in tumorous cells. In particular, the molecular actions of TSA and 4-Me2N-BAVAH on hepatic functionality and gap junctions, gatekeepers of liver homeostasis, in primary cultures of mitogen-stimulated hepatocytes are investigated. Both HDAC-inhibitors were found to promote albumin secretion and CYP1A1 gene transcription and functionality, whereas CYP2B1 gene transcription and activity were only slightly enhanced. The protein production of the gap junction component Cx26 was downregulated, whereas Cx32 expression was upregulated in response to HDAC-inhibition. Furthermore, TSA increased protein levels of the non-specific hepatocellular Cx43, whereas 4-Me2N-BAVAH rather diminished its expression. These data provide new insight into the biological impact of HDAC-inhibitors on the homeostatic balance in hepatocytes, being major executors of xenobiotic biotransformation and primary targets of drug-induced toxicity.
Collapse
Affiliation(s)
- Tom Henkens
- Department of Toxicology, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, B-1090 Brussels, Belgium.
| | | | | | | | | | | |
Collapse
|
37
|
Papeleu P, Wullaert A, Elaut G, Henkens T, Vinken M, Laus G, Tourwé D, Beyaert R, Rogiers V, Vanhaecke T. Inhibition of NF-kappaB activation by the histone deacetylase inhibitor 4-Me2N-BAVAH induces an early G1 cell cycle arrest in primary hepatocytes. Cell Prolif 2007; 40:640-55. [PMID: 17877607 PMCID: PMC6496027 DOI: 10.1111/j.1365-2184.2007.00466.x] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
OBJECTIVE Benzoylaminoalkanohydroxamic acids, including 5-(4-dimethylaminobenzoyl)aminovaleric acid hydroxamide (4-Me(2)N-BAVAH), are structural analogues of Trichostatin A, a naturally occurring histone deacetylase inhibitor (HDACi). 4-Me(2)N-BAVAH has been shown to induce histone hyperacetylation and to inhibit proliferation in Friend erythroleukaemia cells in vitro. However, the molecular mechanisms have remained unidentified. MATERIALS AND METHODS In this study, we evaluated the effects of 4-Me(2)N-BAVAH on proliferation in non-malignant cells, namely epidermal growth factor-stimulated primary rat hepatocytes. RESULTS AND CONCLUSION We have found that 4-Me(2)N-BAVAH inhibits HDAC activity at non-cytotoxic concentrations and prevents cells from responding to the mitogenic stimuli of epidermal growth factor. This results in an early G(1) cell cycle arrest that is independent of p21 activity, but instead can be attributed to inhibition of cyclin D1 transcription through a mechanism involving inhibition of nuclear factor-kappaB activation. In addition, 4-Me(2)N-BAVAH delays the onset of spontaneous apoptosis in primary rat hepatocyte cultures as evidenced by down-regulation of the pro-apoptotic proteins Bid and Bax, and inhibition of caspase-3 activation.
Collapse
Affiliation(s)
- P Papeleu
- Department of Toxicology, Vrije Universiteit Brussel, Brussels, Belgium
| | | | | | | | | | | | | | | | | | | |
Collapse
|
38
|
Snykers S, Vanhaecke T, De Becker A, Papeleu P, Vinken M, Van Riet I, Rogiers V. Chromatin remodeling agent trichostatin A: a key-factor in the hepatic differentiation of human mesenchymal stem cells derived of adult bone marrow. BMC DEVELOPMENTAL BIOLOGY 2007; 7:24. [PMID: 17407549 PMCID: PMC1852547 DOI: 10.1186/1471-213x-7-24] [Citation(s) in RCA: 64] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/15/2006] [Accepted: 04/02/2007] [Indexed: 02/05/2023]
Abstract
Background The capability of human mesenchymal stem cells (hMSC) derived of adult bone marrow to undergo in vitro hepatic differentiation was investigated. Results Exposure of hMSC to a cocktail of hepatogenic factors [(fibroblast growth factor-4 (FGF-4), hepatocyte growth factor (HGF), insulin-transferrin-sodium-selenite (ITS) and dexamethasone)] failed to induce hepatic differentiation. Sequential exposure to these factors (FGF-4, followed by HGF, followed by HGF+ITS+dexamethasone), however, resembling the order of secretion during liver embryogenesis, induced both glycogen-storage and cytokeratin (CK)18 expression. Additional exposure of the cells to trichostatin A (TSA) considerably improved endodermal differentiation, as evidenced by acquisition of an epithelial morphology, chronological expression of hepatic proteins, including hepatocyte-nuclear factor (HNF)-3β, alpha-fetoprotein (AFP), CK18, albumin (ALB), HNF1α, multidrug resistance-associated protein (MRP)2 and CCAAT-enhancer binding protein (C/EBP)α, and functional maturation, i.e. upregulated ALB secretion, urea production and inducible cytochrome P450 (CYP)-dependent activity. Conclusion hMSC are able to undergo mesenchymal-to-epithelial transition. TSA is hereby essential to promote differentiation of hMSC towards functional hepatocyte-like cells.
Collapse
Affiliation(s)
- Sarah Snykers
- Dept. Toxicology., Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090, Brussels, Belgium
| | - Tamara Vanhaecke
- Dept. Toxicology., Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090, Brussels, Belgium
| | - Ann De Becker
- Dept. Medical Oncology and Hematology, Stem Cell Laboratory, Academic. Hospital, Vrije Universiteit Brussel, Laarbeeklaan 101, B-1090, Brussels, Belgium
| | - Peggy Papeleu
- Dept. Toxicology., Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090, Brussels, Belgium
| | - Mathieu Vinken
- Dept. Toxicology., Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090, Brussels, Belgium
| | - Ivan Van Riet
- Dept. Medical Oncology and Hematology, Stem Cell Laboratory, Academic. Hospital, Vrije Universiteit Brussel, Laarbeeklaan 101, B-1090, Brussels, Belgium
| | - Vera Rogiers
- Dept. Toxicology., Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090, Brussels, Belgium
| |
Collapse
|
39
|
Snykers S, Vinken M, Rogiers V, Vanhaecke T. Differential role of epigenetic modulators in malignant and normal stem cells: a novel tool in preclinical in vitro toxicology and clinical therapy. Arch Toxicol 2007; 81:533-44. [PMID: 17387455 DOI: 10.1007/s00204-007-0195-4] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2007] [Accepted: 02/22/2007] [Indexed: 02/06/2023]
Abstract
Adult stem cells are primitive cells that undergo asymmetric division, thereby giving rise to one clonogenic, self-renewing cell and one cell able to undergo multipotent differentiation. Disturbance of this controlled process by epigenetic alterations, including imbalance of histone acetylation/histone deacetylation and DNA methylation/demethylation, may result in uncontrolled growth, formation of self-renewing malignant stem cells and eventually cancer. In view of this notion, several epigenetic modulators, in particular those with histone deacetylase inhibiting activity, are currently being tested in phase I and II clinical trials for their promising chemotherapeutic properties in cancer therapy. As chromatin modulation is also involved in regulation of differentiation, normal development, embryonic and adult stem cell functions and maintenance of their plasticity during embryonic organogenesis, the question can be raised whether predestined cell fate can be modified through epigenetic interference. And if so, could this strategy enforce adult stem cells to differentiate into different types of functional cells? In particular, functional hepatocytes seem important for preclinical toxicity screening of candidate drugs. This paper reviews the potential use and relevance of epigenetic modifiers, including inhibitors of histone deacetylases and DNA methyltransferases (1) to change cell fate and 'trans'differentiate normal adult stem cells into hepatocyte-like cells and (2) to cure disorders, caused by uncontrolled growth of malignant stem cells.
Collapse
Affiliation(s)
- Sarah Snykers
- Department of Toxicology, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium.
| | | | | | | |
Collapse
|