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1
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Salas SAS, Damba T, Buist‐Homan M, Moshage H. Protective Effect of Carvedilol Against Oxidative Stress Induced by Palmitic Acid in Primary Rat Hepatocytes. Cell Biochem Funct 2025; 43:e70057. [PMID: 39924769 PMCID: PMC11808198 DOI: 10.1002/cbf.70057] [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: 07/22/2024] [Revised: 01/23/2025] [Accepted: 01/27/2025] [Indexed: 02/11/2025]
Abstract
Hepatocyte lipotoxicity (HL) is an important factor in the pathogenesis of Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD). It is defined as the detrimental effects of exposure to (excessive) amounts of toxic lipid species, leading to increased mitochondrial β-oxidation, oxidative stress (OxS), and organellar dysfunction. Carvedilol (CV) is a β-adrenergic blocker with antioxidant properties. To elucidate whether CV protects hepatocytes against lipotoxicity induced by palmitic acid (PA) by reducing OxS and endoplasmic reticulum (ER) stress. Primary rat hepatocytes (rHep) were used. Lipotoxicity was induced by PA (1 mmol/L). Cell damage was evaluated by Sytox Green staining. Mitochondrial generation of reactive oxygen species (mROS) was assessed by MitoSox. mRNA and protein expression were measured by qPCR and Western blot, respectively. Lipid accumulation was measured by Oil Red O staining and triglyceride (TG) content. PA induced cell death in > 80% of cells and increased mROS generation. PA increased mRNA expression of ER stress markers CHOP and sXBP1 and slightly increased lipid accumulation. Expression of the β-oxidation-related gene Cpt1a was increased. CV (10 µmol/L) significantly reduced PA-induced cell death to control levels (< 8% of total cells), and mROS generation and expression of the mitochondrial antioxidant enzymes Sod2 and Cat were increased by 40% by CV in the presence of PA. CV did not change the expression of ER stress markers. CV, added before PA, protects rHep against PA-induced cytotoxicity by reducing OxS and increasing the expression of antioxidant enzymes without any additional protective effect on ER stress or lipid accumulation.
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Affiliation(s)
- Sandra A. Serna Salas
- Department of Gastroenterology and Hepatology, University Medical Center GroningenUniversity of GroningenGroningenthe Netherlands
| | - Turtushikh Damba
- Department of Gastroenterology and Hepatology, University Medical Center GroningenUniversity of GroningenGroningenthe Netherlands
- School of PharmacyMongolian National University of Medical SciencesUlaanbaatarMongolia
| | - Manon Buist‐Homan
- Department of Gastroenterology and Hepatology, University Medical Center GroningenUniversity of GroningenGroningenthe Netherlands
- Department of Laboratory Medicine, University Medical Center GroningenUniversity of GroningenGroningenthe Netherlands
| | - Han Moshage
- Department of Gastroenterology and Hepatology, University Medical Center GroningenUniversity of GroningenGroningenthe Netherlands
- Department of Laboratory Medicine, University Medical Center GroningenUniversity of GroningenGroningenthe Netherlands
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2
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Arroyave-Ospina JC, Martínez M, Buist-Homan M, Palasantzas V, Arrese M, Moshage H. Coffee Compounds Protection Against Lipotoxicity Is Associated with Lipid Droplet Formation and Antioxidant Response in Primary Rat Hepatocytes. Antioxidants (Basel) 2025; 14:175. [PMID: 40002362 PMCID: PMC11851918 DOI: 10.3390/antiox14020175] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2024] [Revised: 01/16/2025] [Accepted: 01/27/2025] [Indexed: 02/27/2025] Open
Abstract
Metabolic dysfunction associated with steatotic liver disease (MASLD) is the result of disturbed lipid metabolism. In MASLD, the accumulation of free fatty acids (FFAs) in hepatocytes causes lipotoxicity mediated by oxidative stress. Coffee compounds are known for their beneficial effects in MASLD; however, the mechanisms still need to be further explored. The aim of this study was to elucidate the protective mechanisms of coffee compounds against palmitate-induced lipotoxicity in primary hepatocytes. METHODS Primary hepatocytes were isolated from male Wistar rats and treated with palmitate (1 mmol/L) in combination with caffeine (CF: 1 mmol/L) or chlorogenic acid (CGA: 5 µmol/L). Mitochondrial ROS production, palmitate-induced necrosis, antioxidant response, ER stress markers and lipid droplet (LD) formation were assessed. Monoacylglycerols 2-SG (2-Stearolylglycerol), 2-OG (2-Oleoylglycerol) and SCD-1 (Stearoyl-CoA Desaturase 1) inhibitors were used to modulate LD formation. LD formation in steatotic Zucker rat hepatocytes was also investigated. RESULTS CF and CGA prevented palmitate-induced cell death and reduced ROS production. CF and CGA induced the antioxidant response, especially HO-1 expression, but had no significant effect on ER stress markers. CF and CGA increased LD formation in palmitate-treated cells. This effect was significantly reduced by 2-SG and SCD-1 inhibitors but enhanced by 2-OG. Lipid droplets were associated with lower palmitate toxicity and reduced ROS production. CONCLUSIONS CF and CGA protect hepatocytes from lipotoxicity via modulation of the antioxidant response and enhance lipid droplet formation via an SCD-1-dependent mechanism. Oxidative stress-related toxicity in hepatocytes can be prevented by enhancing LD formation.
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Affiliation(s)
- Johanna C. Arroyave-Ospina
- Department of Fisiología y Bioquímica and Grupo de Gastrohepatología, Facultad de Medicina Universidad de Antioquia, Medellín 050010, Colombia
- Department of Gastroenterology and Hepatology, University Med ical Center of Groningen, University of Groningen, P.O. Box 30.001, 9713 GZ Groningen, The Netherlands; (M.M.); (M.B.-H.)
| | - Magnolia Martínez
- Department of Gastroenterology and Hepatology, University Med ical Center of Groningen, University of Groningen, P.O. Box 30.001, 9713 GZ Groningen, The Netherlands; (M.M.); (M.B.-H.)
| | - Manon Buist-Homan
- Department of Gastroenterology and Hepatology, University Med ical Center of Groningen, University of Groningen, P.O. Box 30.001, 9713 GZ Groningen, The Netherlands; (M.M.); (M.B.-H.)
| | - Victoria Palasantzas
- Department of Genetics and Department of Pediatrics, University Medical Center of Groningen, University of Groningen, P.O. Box 30.001, 9713 GZ Groningen, The Netherlands;
| | - Marco Arrese
- Department of Gastroenterology, Pontificia Universidad Católica de Chile, Santiago 7820436, Chile;
| | - Han Moshage
- Department of Gastroenterology and Hepatology, University Med ical Center of Groningen, University of Groningen, P.O. Box 30.001, 9713 GZ Groningen, The Netherlands; (M.M.); (M.B.-H.)
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Wang J, Zhao F, Brouwer LA, Buist-Homan M, Wolters JC, Moshage H, Harmsen MC. Collagen-rich liver-derived extracellular matrix hydrogels augment survival and function of primary rat liver sinusoidal endothelial cells and hepatocytes. Int J Biol Macromol 2024; 278:134717. [PMID: 39142477 DOI: 10.1016/j.ijbiomac.2024.134717] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2024] [Revised: 07/11/2024] [Accepted: 08/11/2024] [Indexed: 08/16/2024]
Abstract
Liver sinusoidal endothelial cells (LSECs) are key targets for addressing metabolic dysfunction-associated steatotic liver disease (MASLD). However, isolating and culturing primary LSECs is challenging due to rapid dedifferentiation, resulting in loss of function. The extracellular matrix (ECM) likely plays a crucial role in maintaining the fate and function of LSECs. In this study, we explored the influence of liver-ECM (L-ECM) on liver cells and developed culture conditions that maintain the differentiated function of liver cells in vitro for prolonged periods. Porcine liver-derived L-ECM, containing 34.9 % protein, 0.045 % glycosaminoglycans, and negligible residual DNA (41.2 ng/mg), was utilized to culture primary rat liver cells in generated hydrogels. Proteomic analyses and molecular weight distribution of proteins of solubilized L-ECM revealed the typical diverse ECM core matrisome, with abundant collagens. L-ECM hydrogels showed suitable stiffness and stress relaxation properties. Furthermore, we demonstrated that collagen-rich L-ECM hydrogels enhanced LSECs' and hepatocytes' viability, and reduced the dedifferentiation rate of LSECs. In addition, hepatocyte function was maintained longer by culture on L-ECM hydrogels compared to traditional culturing. These beneficial effects are likely attributed to the bioactive macromolecules including collagens, and mechanical and microarchitectural properties of the L-ECM hydrogels.
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Affiliation(s)
- Junyu Wang
- University of Groningen, University Medical Center Groningen, Department of Gastroenterology and Hepatology, Groningen, the Netherlands; University of Groningen, University Medical Center Groningen, Department of Pathology and Medical Biology, Groningen, the Netherlands.
| | - Fenghua Zhao
- University of Groningen, University Medical Center Groningen, Department of Pathology and Medical Biology, Groningen, the Netherlands; University of Groningen, University Medical Centre Groningen, W.J. Kolff Institute for Biomedical Engineering and Materials Science, Groningen, the Netherlands; University of Groningen, University Medical Centre Groningen, Department of Biomedical Engineering, Groningen, the Netherlands.
| | - Linda A Brouwer
- University of Groningen, University Medical Center Groningen, Department of Pathology and Medical Biology, Groningen, the Netherlands.
| | - Manon Buist-Homan
- University of Groningen, University Medical Center Groningen, Department of Gastroenterology and Hepatology, Groningen, the Netherlands; University of Groningen, University Medical Center Groningen, Department of Laboratory Medicine, Groningen, the Netherlands.
| | - Justina C Wolters
- University of Groningen, University Medical Centre Groningen, Department of Pediatrics, Groningen, the Netherlands; University of Groningen, University Medical Centre Groningen, Interfaculty Mass Spectrometry Center, Groningen, the Netherlands.
| | - Han Moshage
- University of Groningen, University Medical Center Groningen, Department of Gastroenterology and Hepatology, Groningen, the Netherlands; University of Groningen, University Medical Center Groningen, Department of Laboratory Medicine, Groningen, the Netherlands.
| | - Martin C Harmsen
- University of Groningen, University Medical Center Groningen, Department of Pathology and Medical Biology, Groningen, the Netherlands; University of Groningen, University Medical Centre Groningen, W.J. Kolff Institute for Biomedical Engineering and Materials Science, Groningen, the Netherlands; University of Groningen, University Medical Centre Groningen, Groningen Research Institute for Asthma and COPD (GRIAC), Groningen, the Netherlands.
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4
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Wu Z, Xia M, Wang J, Aguilar MM, Buist-Homan M, Moshage H. Extracellular vesicles originating from steatotic hepatocytes promote hepatic stellate cell senescence via AKT/mTOR signaling. Cell Biochem Funct 2024; 42:e4077. [PMID: 38881228 DOI: 10.1002/cbf.4077] [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: 02/16/2024] [Revised: 05/24/2024] [Accepted: 06/06/2024] [Indexed: 06/18/2024]
Abstract
The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) is increasing rapidly due to the obesity epidemic. In the inflammatory stages of MASLD (MASH), activation of hepatic stellate cells (HSCs) leads to initiation and progression of liver fibrosis. Extracellular vesicles (EVs) are released from all cell types and play an important role in intercellular communication. However, the role of EVs released from hepatocytes in the context of MASLD is largely unknown. Therefore, the present study aimed to investigate the role of EVs derived from both normal and steatotic (free fatty acid-treated) hepatocytes on the phenotype of HSCs via the senescence pathway. Primary rat hepatocytes were treated with free fatty acids (FFAs: oleic acid and palmitic acid). EVs were collected by ultracentrifugation. EVs markers and HSCs activation and senescence markers were assessed by Western blot analysis, qPCR and cytochemistry. Reactive oxygen species (ROS) production was assessed by fluorescence assay. RNA profiles of EVs were evaluated by sequencing. We found that EVs from hepatocytes treated with FFAs (FFA-EVs) inhibit collagen type 1 and α-smooth muscle actin expression, increase the production of ROS and the expression of senescence markers (IL-6, IL-1β, p21 and senescence-associated β-galactosidase activity) in early activating HSCs via the AKT-mTOR pathway. Sequencing showed differentially enriched RNA species between the EVs groups. In conclusion, EVs from FFA-treated hepatocytes inhibit HSC activation by inducing senescence via the AKT-mTOR signaling pathway. Determining the components in EVs from steatotic hepatocytes that induce HSC senescence may lead to the identification of novel targets for intervention in the treatment of MASLD in the future.
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Affiliation(s)
- Zongmei Wu
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
| | - Mengmeng Xia
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
| | - Junyu Wang
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
| | - Magnolia Martinez Aguilar
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
| | - Manon Buist-Homan
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
| | - Han Moshage
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
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Smith-Cortinez N, Heegsma J, Podunavac M, Zakarian A, Cardenas JC, Faber KN. Novel Inositol 1,4,5-Trisphosphate Receptor Inhibitor Antagonizes Hepatic Stellate Cell Activation: A Potential Drug to Treat Liver Fibrosis. Cells 2024; 13:765. [PMID: 38727301 PMCID: PMC11083487 DOI: 10.3390/cells13090765] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2024] [Revised: 04/18/2024] [Accepted: 04/23/2024] [Indexed: 05/13/2024] Open
Abstract
Liver fibrosis, characterized by excessive extracellular matrix (ECM) deposition, can progress to cirrhosis and increases the risk of liver cancer. Hepatic stellate cells (HSCs) play a pivotal role in fibrosis progression, transitioning from a quiescent to activated state upon liver injury, wherein they proliferate, migrate, and produce ECM. Calcium signaling, involving the inositol 1,4,5-trisphosphate receptor (IP3R), regulates HSC activation. This study investigated the efficacy of a novel IP3R inhibitor, desmethylxestospongin B (dmXeB), in preventing HSC activation. Freshly isolated rat HSCs were activated in vitro in the presence of varying dmXeB concentrations. The dmXeB effectively inhibited HSC proliferation, migration, and expression of fibrosis markers without toxicity to the primary rat hepatocytes or human liver organoids. Furthermore, dmXeB preserved the quiescent phenotype of HSCs marked by retained vitamin A storage. Mechanistically, dmXeB suppressed mitochondrial respiration in activated HSCs while enhancing glycolytic activity. Notably, methyl pyruvate, dimethyl α-ketoglutarate, and nucleoside supplementation all individually restored HSC proliferation despite dmXeB treatment. Overall, dmXeB demonstrates promising anti-fibrotic effects by inhibiting HSC activation via IP3R antagonism without adverse effects on other liver cells. These findings highlight dmXeB as a potential therapeutic agent for liver fibrosis treatment, offering a targeted approach to mitigate liver fibrosis progression and its associated complications.
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Affiliation(s)
- Natalia Smith-Cortinez
- Department of Gastroenterology and Hepatology, University of Groningen, University Medical Center Groningen (UMCG), 9713 GZ Groningen, The Netherlands
| | - Janette Heegsma
- Department of Gastroenterology and Hepatology, University of Groningen, University Medical Center Groningen (UMCG), 9713 GZ Groningen, The Netherlands
| | - Masa Podunavac
- Department of Chemistry and Biochemistry, University of California, Oakland, CA 94607, USA
| | - Armen Zakarian
- Department of Chemistry and Biochemistry, University of California, Oakland, CA 94607, USA
| | - J. César Cardenas
- Department of Chemistry and Biochemistry, University of California, Oakland, CA 94607, USA
- Center for Integrative Biology, Universidad Mayor, Santiago 7510041, Chile
- Buck Institute for Research on Aging, Novato, CA 94945, USA
| | - Klaas Nico Faber
- Department of Gastroenterology and Hepatology, University of Groningen, University Medical Center Groningen (UMCG), 9713 GZ Groningen, The Netherlands
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6
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Wang J, Wu Z, Xia M, Salas SS, Ospina JA, Buist-Homan M, Harmsen MC, Moshage H. Extracellular vesicles derived from liver sinusoidal endothelial cells inhibit the activation of hepatic stellate cells and Kupffer cells in vitro. Biochim Biophys Acta Mol Basis Dis 2024; 1870:167020. [PMID: 38244390 DOI: 10.1016/j.bbadis.2024.167020] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2023] [Revised: 12/22/2023] [Accepted: 01/05/2024] [Indexed: 01/22/2024]
Abstract
Liver sinusoidal endothelial cells (LSECs) play a crucial role in maintaining liver microcirculation and exchange of nutrients in the liver and are thought to be involved in the pathogenesis of metabolic dysfunction-associated steatotic liver disease (MASLD). The activation of hepatic stellate cells (HSCs) and Kupffer cells (KCs) has been considered to be responsible for the onset of liver fibrosis and the aggravation of liver injury. However, the paracrine regulatory effects of LSECs in the development of MASLD, in particular the role of LSEC-derived extracellular vesicles (EVs) remains unclear. Therefore, the aim of the present study was to investigate the influence of LSEC-derived EVs on HSCs and KCs. Primary rat LSECs, HSCs and KCs were isolated from male Wistar rats. LSEC-derived EVs were isolated from conditioned medium by ultracentrifugation and analyzed by nanoparticle tracking analysis, and expression of specific markers. LSEC-derived EVs reduced the expression of activation markers in activated HSCs but did not affect quiescent HSCs. Also, LSEC-derived EVs suppressed proliferation of activated HSCs activation, as assessed by Xcelligence and BrdU assay. LSEC-derived EVs also increased the expression of inflammatory genes in HSCs that normally are lowly expression during their activation. In contrast, EVs decreased the expression of inflammatory genes in activated KCs. In summary, our results suggest that LSEC-derived EVs may attenuate the fibrogenic phenotype of activated HSCs and the inflammatory phenotype of KCs. Our results show promise for LSEC-derived EVs as therapeutic moieties to treat MASLD. In addition, these EVs might prove of diagnostic value.
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Affiliation(s)
- Junyu Wang
- University Medical Center Groningen, University of Groningen, Department of Gastroenterology and Hepatology, Groningen, the Netherlands
| | - Zongmei Wu
- University Medical Center Groningen, University of Groningen, Department of Gastroenterology and Hepatology, Groningen, the Netherlands
| | - Mengmeng Xia
- University Medical Center Groningen, University of Groningen, Department of Gastroenterology and Hepatology, Groningen, the Netherlands
| | - Sandra Serna Salas
- University Medical Center Groningen, University of Groningen, Department of Gastroenterology and Hepatology, Groningen, the Netherlands
| | - Johanna Arroyave Ospina
- University Medical Center Groningen, University of Groningen, Department of Gastroenterology and Hepatology, Groningen, the Netherlands
| | - Manon Buist-Homan
- University Medical Center Groningen, University of Groningen, Department of Gastroenterology and Hepatology, Groningen, the Netherlands; University Medical Center Groningen, University of Groningen, Department of Laboratory Medicine, Groningen, the Netherlands
| | - Martin C Harmsen
- University Medical Center Groningen, University of Groningen, Department of Gastroenterology and Hepatology, Groningen, the Netherlands; University Medical Center Groningen, University of Groningen, Department of Pathology and Medical Biology, Groningen, the Netherlands
| | - Han Moshage
- University Medical Center Groningen, University of Groningen, Department of Gastroenterology and Hepatology, Groningen, the Netherlands; University Medical Center Groningen, University of Groningen, Department of Laboratory Medicine, Groningen, the Netherlands.
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7
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Crawford JM, Bioulac-Sage P, Hytiroglou P. Structure, Function and Responses to Injury. MACSWEEN'S PATHOLOGY OF THE LIVER 2024:1-95. [DOI: 10.1016/b978-0-7020-8228-3.00001-6] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/03/2025]
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8
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Xia M, Wu Z, Wang J, Buist-Homan M, Moshage H. The Coumarin-Derivative Esculetin Protects against Lipotoxicity in Primary Rat Hepatocytes via Attenuating JNK-Mediated Oxidative Stress and Attenuates Free Fatty Acid-Induced Lipid Accumulation. Antioxidants (Basel) 2023; 12:1922. [PMID: 38001774 PMCID: PMC10669015 DOI: 10.3390/antiox12111922] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2023] [Revised: 10/22/2023] [Accepted: 10/25/2023] [Indexed: 11/26/2023] Open
Abstract
Coumarin derivates have been proposed as a potential treatment for metabolic-dysfunction-associated fatty liver disease (MAFLD). However, the mechanisms underlying their beneficial effects remain unclear. In the present study, we explored the potential of the coumarin derivate esculetin in MAFLD, focusing on hepatocyte lipotoxicity and lipid accumulation. Primary cultures of rat hepatocytes were exposed to palmitic acid (PA) and palmitic acid plus oleic acid (OA/PA) as models of lipotoxicity and lipid accumulation, respectively. Esculetin significantly reduced oxidative stress in PA-treated hepatocytes, as shown by decreased total reactive oxygen species (ROS) and mitochondrial superoxide production and elevated expression of antioxidant genes, including Nrf2 and Gpx1. In addition, esculetin protects against PA-induced necrosis. Esculetin also improved lipid metabolism in primary hepatocytes exposed to nonlipotoxic OA/PA by decreasing the expression of the lipogenesis-related gene Srebp1c and increasing the expression of the fatty acid β-oxidation-related gene Ppar-α. Moreover, esculetin attenuated lipid accumulation in OA/PA-treated hepatocytes. The protective effects of esculetin against lipotoxicity and lipid accumulation were shown to be dependent on the inhibition of JNK and the activation of AMPK, respectively. We conclude that esculetin is a promising compound to target lipotoxicity and lipid accumulation in the treatment of MAFLD.
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Affiliation(s)
- Mengmeng Xia
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, 9713 GZ Groningen, The Netherlands; (M.X.); (Z.W.); (J.W.); (M.B.-H.)
| | - Zongmei Wu
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, 9713 GZ Groningen, The Netherlands; (M.X.); (Z.W.); (J.W.); (M.B.-H.)
| | - Junyu Wang
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, 9713 GZ Groningen, The Netherlands; (M.X.); (Z.W.); (J.W.); (M.B.-H.)
| | - Manon Buist-Homan
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, 9713 GZ Groningen, The Netherlands; (M.X.); (Z.W.); (J.W.); (M.B.-H.)
- Department of Laboratory Medicine, University Medical Center Groningen, University of Groningen, 9713 GZ Groningen, The Netherlands
| | - Han Moshage
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, 9713 GZ Groningen, The Netherlands; (M.X.); (Z.W.); (J.W.); (M.B.-H.)
- Department of Laboratory Medicine, University Medical Center Groningen, University of Groningen, 9713 GZ Groningen, The Netherlands
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9
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Geng Y, Wang J, Serna-Salas SA, Villanueva AH, Buist-Homan M, Arrese M, Olinga P, Blokzijl H, Moshage H. Hepatic stellate cells induce an inflammatory phenotype in Kupffer cells via the release of extracellular vesicles. J Cell Physiol 2023; 238:2293-2303. [PMID: 37555553 DOI: 10.1002/jcp.31086] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2023] [Revised: 06/15/2023] [Accepted: 07/12/2023] [Indexed: 08/10/2023]
Abstract
Liver fibrosis is the response of the liver to chronic liver inflammation. The communication between the resident liver macrophages (Kupffer cells [KCs]) and hepatic stellate cells (HSCs) has been mainly viewed as one-directional: from KCs to HSCs with KCs promoting fibrogenesis. However, recent studies indicated that HSCs may function as a hub of intercellular communications. Therefore, the aim of the present study was to investigate the role of HSCs on the inflammatory phenotype of KCs. Primary rat HSCs and KCs were isolated from male Wistar rats. HSCs-derived conditioned medium (CM) was harvested from different time intervals (Day 0-2: CM-D2 and Day 5-7: CM-D7) during the activation of HSCs. Extracellular vesicles (EVs) were isolated from CM by ultracentrifugation and evaluated by nanoparticle tracking analysis and western blot analysis. M1 and M2 markers of inflammation were measured by quantitative PCR and macrophage function by assessing phagocytic capacity. CM-D2 significantly induced the inflammatory phenotype in KCs, but not CM-D7. Neither CM-D2 nor CM-D7 affected the phagocytosis of KCs. Importantly, the proinflammatory effect of HSCs-derived CM is mediated via EVs released from HSCs since EVs isolated from CM mimicked the effect of CM, whereas EV-depleted CM lost its ability to induce a proinflammatory phenotype in KCs. In addition, when the activation of HSCs was inhibited, HSCs produced less EVs. Furthermore, the proinflammatory effects of CM and EVs are related to activating Toll-like receptor 4 (TLR4) in KCs. In conclusion, HSCs at an early stage of activation induce a proinflammatory phenotype in KCs via the release of EVs. This effect is absent in CM derived from HSCs at a later stage of activation and is dependent on the activation of TLR4 signaling pathway.
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Affiliation(s)
- Yana Geng
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
- Department of Pharmaceutical Technology and Biopharmacy, Groningen Research Institute of Pharmacy, University of Groningen, Groningen, The Netherlands
| | - Junyu Wang
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
| | - Sandra Alejandra Serna-Salas
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
| | - Alejandra Hernández Villanueva
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
- Department of Gastroenterology, Pontificia Universidad Católica de Chile, Santiago de Chile, Chile
| | - Manon Buist-Homan
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
- Department of Laboratory Medicine, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
| | - Marco Arrese
- Department of Gastroenterology, Pontificia Universidad Católica de Chile, Santiago de Chile, Chile
| | - Peter Olinga
- Department of Pharmaceutical Technology and Biopharmacy, Groningen Research Institute of Pharmacy, University of Groningen, Groningen, The Netherlands
| | - Hans Blokzijl
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
| | - Han Moshage
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
- Department of Laboratory Medicine, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
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10
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Arroyave-Ospina JC, Buist-Homan M, Schmidt M, Moshage H. Protective effects of caffeine against palmitate-induced lipid toxicity in primary rat hepatocytes is associated with modulation of adenosine receptor A1 signaling. Biomed Pharmacother 2023; 165:114884. [PMID: 37423170 DOI: 10.1016/j.biopha.2023.114884] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2022] [Revised: 05/05/2023] [Accepted: 05/12/2023] [Indexed: 07/11/2023] Open
Abstract
BACKGROUND Epidemiological evidence has shown an association between coffee consumption and reduced risk for chronic liver diseases, including metabolic-dysfunction-associated liver disease (MALFD). Lipotoxicity is a key cause of hepatocyte injury during MAFLD. The coffee component caffeine is known to modulate adenosine receptor signaling via the antagonism of adenosine receptors. The involvement of these receptors in the prevention of hepatic lipotoxicity has not yet been explored. The aim of this study was to explore whether caffeine protects against palmitate-induced lipotoxicity by modulating adenosine receptor signaling. METHODS Primary hepatocytes were isolated from male rats. Hepatocytes were treated with palmitate with or without caffeine or 1,7DMX. Lipotoxicity was verified using Sytox viability staining and mitochondrial JC-10 staining. PKA activation was verified by Western blotting. Selective (ant)agonists of A1AR (DPCPX and CPA, respectively) and A2AR (istradefyline and regadenoson, respectively), the AMPK inhibitor compound C, and the Protein Kinase A (PKA) inhibitor Rp8CTP were used. Lipid accumulation was verified by ORO and BODIPY 453/50 staining. RESULTS Caffeine and its metabolite 1,7DMX prevented palmitate-induced toxicity in hepatocytes. The A1AR antagonist DPCPX also prevented lipotoxicity, whereas both the inhibition of PKA and the A1AR agonist CPA (partially) abolished the protective effect. Caffeine and DPCPX increased lipid droplet formation only in palmitate-treated hepatocytes and decreased mitochondrial ROS production. CONCLUSIONS The protective effect of caffeine against palmitate lipotoxicity was shown to be dependent on A1AR receptor and PKA activation. Antagonism of A1AR also protects against lipotoxicity. Targeting A1AR receptor may be a potential therapeutic intervention with which to treat MAFLD.
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Affiliation(s)
- Johanna C Arroyave-Ospina
- Department of Laboratory Medicine, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands; Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands.
| | - Manon Buist-Homan
- Department of Laboratory Medicine, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands; Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands
| | - Martina Schmidt
- Department Molecular Pharmacology, Groningen Research Institute of Pharmacy, Groningen Research Institute for Asthma and COPD, GRIAC, University Medical Center Groningen University of Groningen, Groningen, the Netherlands
| | - Han Moshage
- Department of Laboratory Medicine, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands; Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands
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11
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Haijer F, Koets-Shajari S, Heegsma J, Serna-Salas S, Blokzijl T, Buist-Homan M, Moshage H, Faber KN. Hydroxyurea attenuates hepatic stellate cell proliferation in vitro and liver fibrogenesis in vivo. FASEB J 2023; 37:e23124. [PMID: 37552464 DOI: 10.1096/fj.202300920r] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2023] [Revised: 06/24/2023] [Accepted: 07/24/2023] [Indexed: 08/09/2023]
Abstract
Liver fibrosis results from excessive proliferation of, and collagen production by hepatic stellate cells (HSCs) that is caused by chronic liver injury. No drugs are available to cure liver fibrosis. Hydroxyurea is an anti-proliferative drug that is used in benign and malignant disorders. Here, we studied the effect of hydroxyurea on primary HSCs and its anti-fibrotic effect in the CCl4 mouse model of liver fibrosis. Primary rat HSCs were cultured in the absence or presence of hydroxyurea (0.1-1.0 mmol/L). CCl4 or vehicle was administered to C57BL/6/J mice for 4 weeks, with or without hydroxyurea (100 mg/kg/day) co-treatment. We used real-time cell proliferation analysis, Oil Red O (lipid droplet) staining, immunohistochemistry, Acridine Orange staining (apoptosis), Sytox green staining (necrosis), RT-qPCR, ELISA, and Western Blotting for analysis. Hydroxyurea dose-dependently suppressed lipid droplet-loss and mRNA levels of Col1α1 and Acta2 in transdifferentiating HSCs. In fully-activated HSCs, hydroxyurea dose-dependently attenuated PCNA protein levels and BrdU incorporation, but did not reverse Col1α1 and Acta2 mRNA expression. Hydroxyurea did not induce apoptosis or necrosis in HSCs or hepatocytes. Hydroxyurea suppressed accumulation of desmin-positive HSCs and hepatic collagen deposition after CCl4 treatment. CCl4 -induced regenerative hepatocyte proliferation, Col1α1 and Acta2 mRNA expression and α-SMA protein levels were not affected. This study demonstrates that hydroxyurea inhibits HSC proliferation in vitro and attenuates early development of liver fibrosis in vivo, while preserving hepatocyte regeneration after toxic insults by CCl4. Thus, hydroxyurea may have therapeutic value against liver fibrosis.
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Affiliation(s)
- Floris Haijer
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
| | - Shiva Koets-Shajari
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
| | - Janette Heegsma
- Department Laboratory Medicine, Center for Liver, Digestive, and Metabolic Diseases, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
| | - Sandra Serna-Salas
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
| | - Tjasso Blokzijl
- Department Laboratory Medicine, Center for Liver, Digestive, and Metabolic Diseases, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
| | - Manon Buist-Homan
- Department Laboratory Medicine, Center for Liver, Digestive, and Metabolic Diseases, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
| | - Han Moshage
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
- Department Laboratory Medicine, Center for Liver, Digestive, and Metabolic Diseases, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
| | - Klaas Nico Faber
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
- Department Laboratory Medicine, Center for Liver, Digestive, and Metabolic Diseases, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
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12
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Bioactive coumarin-derivative esculetin decreases hepatic stellate cell activation via induction of cellular senescence via the PI3K-Akt-GSK3β pathway. FOOD BIOSCI 2022. [DOI: 10.1016/j.fbio.2022.102164] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
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13
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Villa-Jaimes GS, Aguilar-Mora FA, González-Ponce HA, Avelar-González FJ, Martínez Saldaña MC, Buist-Homan M, Moshage H. Biocomponents from Opuntia robusta and Opuntia streptacantha fruits protect against diclofenac-induced acute liver damage in vivo and in vitro. J Funct Foods 2022. [DOI: 10.1016/j.jff.2022.104960] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
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14
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Wu Z, Geng Y, Buist-Homan M, Moshage H. Scopoletin and umbelliferone protect hepatocytes against palmitate- and bile acid-induced cell death by reducing endoplasmic reticulum stress and oxidative stress. Toxicol Appl Pharmacol 2021; 436:115858. [PMID: 34979142 DOI: 10.1016/j.taap.2021.115858] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2021] [Revised: 12/29/2021] [Accepted: 12/29/2021] [Indexed: 12/26/2022]
Abstract
BACKGROUND The number of patients with non-alcoholic fatty liver disease (NAFLD) is rapidly increasing due to the growing epidemic of obesity. Non-alcoholic steatohepatitis (NASH), the inflammatory stage of NAFLD, is characterized by lipid accumulation in hepatocytes, chronic inflammation and hepatocyte cell death. Scopoletin and umbelliferone are coumarin-like molecules and have antioxidant, anti-cancer and anti-inflammatory effects. Cytoprotective effects of these compounds have not been described in hepatocytes and the mechanisms of the beneficial effects of scopoletin and umbelliferone are unknown. AIM To investigate whether scopoletin and/or umbelliferone protect hepatocytes against palmitate-induced cell death. For comparison, we also tested the cytoprotective effect of scopoletin and umbelliferone against bile acid-induced cell death. METHODS Primary rat hepatocytes were exposed to palmitate (1 mmol/L) or the hydrophobic bile acid glycochenodeoxycholic acid (GCDCA; 50 μmol/L). Apoptosis was assessed by caspase-3 activity assay, necrosis by Sytox green assay, mRNA levels by qPCR, protein levels by Western blot and production of reactive oxygen species (ROS) by fluorescence assay. RESULTS Both scopoletin and umbelliferone protected against palmitate and GCDCA-induced cell death. Both palmitate and GCDCA induced the expression of ER stress markers. Scopoletin and umbelliferone decreased palmitate- and GCDCA-induced expression of ER stress markers, phosphorylation of the cell death signaling intermediate JNK as well as ROS production. CONCLUSION Scopoletin and umbelliferone protect against palmitate and bile acid-induced cell death of hepatocytes by inhibition of ER stress and ROS generation and decreasing phosphorylation of JNK. Scopoletin and umbelliferone may hold promise as a therapeutic modality for the treatment of NAFLD.
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Affiliation(s)
- Zongmei Wu
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands
| | - Yana Geng
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands
| | - Manon Buist-Homan
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands
| | - Han Moshage
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands.
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15
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Mora FAA, Musheshe N, Arroyave Ospina JC, Geng Y, Soto JM, Rodrigo JA, Alieva T, Buist-Homan M, Lezoualc'h F, Cheng X, Schmidt M, Moshage H. Metformin protects against diclofenac-induced toxicity in primary rat hepatocytes by preserving mitochondrial integrity via a pathway involving EPAC. Biomed Pharmacother 2021; 143:112072. [PMID: 34464747 DOI: 10.1016/j.biopha.2021.112072] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2021] [Revised: 07/23/2021] [Accepted: 08/17/2021] [Indexed: 12/24/2022] Open
Abstract
BACKGROUND AND PURPOSE It has been shown that the antidiabetic drug metformin protects hepatocytes against toxicity by various stressors. Chronic or excessive consumption of diclofenac (DF) - a pain-relieving drug, leads to drug-induced liver injury via a mechanism involving mitochondrial damage and ultimately apoptotic death of hepatocytes. However, whether metformin protects against DF-induced toxicity is unknown. Recently, it was also shown that cAMP elevation is protective against DF-induced apoptotic death in hepatocytes, a protective effect primarily involving the downstream cAMP effector EPAC and preservation of mitochondrial function. This study therefore aimed at investigating whether metformin protects against DF-induced toxicity via cAMP-EPACs. EXPERIMENTAL APPROACH Primary rat hepatocytes were exposed to 400 µmol/L DF. CE3F4 or ESI-O5 were used as EPAC-1 or 2 inhibitors respectively. Apoptosis was measured by caspase-3 activity and necrosis by Sytox green staining. Seahorse X96 assay was used to determine mitochondrial function. Mitochondrial reactive oxygen species (ROS) production was measured using MitoSox, mitochondrial MnSOD expression was determined by immunostaining and mitochondrial morphology (fusion and fission ratio) by 3D refractive index imaging. KEY RESULTS Metformin (1 mmol/L) was protective against DF-induced apoptosis in hepatocytes. This protective effect was EPAC-dependent (mainly EPAC-2). Metformin restored mitochondrial morphology in an EPAC-independent manner. DF-induced mitochondrial dysfunction which was demonstrated by decreased oxygen consumption rate, an increased ROS production and a reduced MnSOD level, were all reversed by metformin in an EPAC-dependent manner. CONCLUSION AND IMPLICATIONS Metformin protects hepatocytes against DF-induced toxicity via cAMP-dependent EPAC-2.
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Affiliation(s)
- Fabio Alejandro Aguilar Mora
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
| | - Nshunge Musheshe
- Deptartment Molecular Pharmacology, Groningen Research Institute of Pharmacy, Groningen Research Institute for Asthma and COPD, GRIAC, University Medical Center Groningen University of Groningen, Groningen, The Netherlands.
| | - Johanna C Arroyave Ospina
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
| | - Yana Geng
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
| | - Juan M Soto
- Department of Optics and Faculty of Physical Sciences, Complutense University of Madrid, Spain.
| | - José A Rodrigo
- Department of Optics and Faculty of Physical Sciences, Complutense University of Madrid, Spain.
| | - Tatiana Alieva
- Department of Optics and Faculty of Physical Sciences, Complutense University of Madrid, Spain.
| | - Manon Buist-Homan
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands; Department of Laboratory Medicine, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
| | - Frank Lezoualc'h
- Inserm UMR-1048, Institut des Maladies Metaboliques et Cardiovasculaires, Univ Toulouse Paul Sabatier, Toulouse, France.
| | - Xiaodong Cheng
- Department of Integrative Biology & Pharmacology, Texas Therapeutics Institute, University of Texas Health Science Center at Houston, Houston, TX, USA.
| | - Martina Schmidt
- Deptartment Molecular Pharmacology, Groningen Research Institute of Pharmacy, Groningen Research Institute for Asthma and COPD, GRIAC, University Medical Center Groningen University of Groningen, Groningen, The Netherlands.
| | - Han Moshage
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands; Department of Laboratory Medicine, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
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16
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Aguilar Mora FA, Musheshe N, Oun A, Buist-Homan M, Lezoualc'h F, Cheng X, Schmidt M, Moshage H. Elevated cAMP Protects against Diclofenac-Induced Toxicity in Primary Rat Hepatocytes: A Protective Effect Mediated by the Exchange Protein Directly Activated by cAMP/cAMP-Regulated Guanine Nucleotide Exchange Factors. Mol Pharmacol 2021; 99:294-307. [PMID: 33574047 PMCID: PMC11033960 DOI: 10.1124/molpharm.120.000217] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2020] [Accepted: 01/22/2021] [Indexed: 12/14/2022] Open
Abstract
Chronic consumption of the nonsteroidal anti-inflammatory drug diclofenac may induce drug-induced liver injury (DILI). The mechanism of diclofenac-induced liver injury is partially elucidated and involves mitochondrial damage. Elevated cAMP protects hepatocytes against bile acid-induced injury. However, it is unknown whether cAMP protects against DILI and, if so, which downstream targets of cAMP are implicated in the protective mechanism, including the classic protein kinase A (PKA) pathway or alternative pathways like the exchange protein directly activated by cAMP (EPAC). The aim of this study was to investigate whether cAMP and/or its downstream targets protect against diclofenac-induced injury in hepatocytes. Rat hepatocytes were exposed to 400 µmol/l diclofenac. Apoptosis and necrosis were measured by caspase-3 activity assay and Sytox green staining, respectively. Mitochondrial membrane potential (MMP) was measured by JC-10 staining. mRNA and protein expression were assessed by quantitative polymerase chain reaction (qPCR) and Western blot, respectively. The cAMP-elevating agent 7β-acetoxy-8,13-epoxy-1α,6β,9α-trihydroxylabd-14-en-11-one (forskolin), the pan-phosphodiesterase inhibitor IBMX, and EPAC inhibitors 5,7-dibromo-6-fluoro-3,4-dihydro-2-methyl-1(2H)-quinoline carboxaldehyde (CE3F4) and ESI-O5 were used to assess the role of cAMP and its effectors, PKA or EPAC. Diclofenac exposure induced apoptotic cell death and loss of MMP in hepatocytes. Both forskolin and IBMX prevented diclofenac-induced apoptosis. EPAC inhibition but not PKA inhibition abolished the protective effect of forskolin and IBMX. Forskolin and IBMX preserved the MMP, whereas both EPAC inhibitors diminished this effect. Both EPAC1 and EPAC2 were expressed in hepatocytes and localized in mitochondria. cAMP elevation protects hepatocytes against diclofenac-induced cell death, a process primarily involving EPACs. The cAMP/EPAC pathway may be a novel target for treatment of DILI. SIGNIFICANCE STATEMENT: This study shows two main highlights. First, elevated cAMP levels protect against diclofenac-induced apoptosis in primary hepatocytes via maintenance of mitochondrial integrity. In addition, this study proposes the existence of mitochondrial cAMP-EPAC microdomains in rat hepatocytes, opening new avenues for targeted therapy in drug-induced liver injury (DILI). Both EPAC1 and EPAC2, but not protein kinase A, are responsible for this protective effect. Our findings present cAMP-EPAC as a potential target for the treatment of DILI and liver injury involving mitochondrial dysfunction.
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Affiliation(s)
- Fabio Alejandro Aguilar Mora
- Dept. Gastroenterology and Hepatology (F.A.A.M., M.B.-H., H.M.), Dept. Molecular Pharmacology, Groningen Research Institute of Pharmacy, Groningen Research Institute for Asthma and COPD, GRIAC (N.M., A.O., M.S.), Dept. Laboratory Medicine (M.B.-H., H.M.), University Medical Center Groningen, University of Groningen, Groningen, The Netherlands; Inserm UMR-1048, Institut des Maladies Métaboliques et Cardiovasculaires, Univ Toulouse Paul Sabatier, Toulouse, France (F.L.); and Department of Integrative Biology and Pharmacology, Texas Therapeutics Institute, University of Texas Health Science Center at Houston, Houston, Texas (X.C.)
| | - Nshunge Musheshe
- Dept. Gastroenterology and Hepatology (F.A.A.M., M.B.-H., H.M.), Dept. Molecular Pharmacology, Groningen Research Institute of Pharmacy, Groningen Research Institute for Asthma and COPD, GRIAC (N.M., A.O., M.S.), Dept. Laboratory Medicine (M.B.-H., H.M.), University Medical Center Groningen, University of Groningen, Groningen, The Netherlands; Inserm UMR-1048, Institut des Maladies Métaboliques et Cardiovasculaires, Univ Toulouse Paul Sabatier, Toulouse, France (F.L.); and Department of Integrative Biology and Pharmacology, Texas Therapeutics Institute, University of Texas Health Science Center at Houston, Houston, Texas (X.C.)
| | - Asmaa Oun
- Dept. Gastroenterology and Hepatology (F.A.A.M., M.B.-H., H.M.), Dept. Molecular Pharmacology, Groningen Research Institute of Pharmacy, Groningen Research Institute for Asthma and COPD, GRIAC (N.M., A.O., M.S.), Dept. Laboratory Medicine (M.B.-H., H.M.), University Medical Center Groningen, University of Groningen, Groningen, The Netherlands; Inserm UMR-1048, Institut des Maladies Métaboliques et Cardiovasculaires, Univ Toulouse Paul Sabatier, Toulouse, France (F.L.); and Department of Integrative Biology and Pharmacology, Texas Therapeutics Institute, University of Texas Health Science Center at Houston, Houston, Texas (X.C.)
| | - Manon Buist-Homan
- Dept. Gastroenterology and Hepatology (F.A.A.M., M.B.-H., H.M.), Dept. Molecular Pharmacology, Groningen Research Institute of Pharmacy, Groningen Research Institute for Asthma and COPD, GRIAC (N.M., A.O., M.S.), Dept. Laboratory Medicine (M.B.-H., H.M.), University Medical Center Groningen, University of Groningen, Groningen, The Netherlands; Inserm UMR-1048, Institut des Maladies Métaboliques et Cardiovasculaires, Univ Toulouse Paul Sabatier, Toulouse, France (F.L.); and Department of Integrative Biology and Pharmacology, Texas Therapeutics Institute, University of Texas Health Science Center at Houston, Houston, Texas (X.C.)
| | - Frank Lezoualc'h
- Dept. Gastroenterology and Hepatology (F.A.A.M., M.B.-H., H.M.), Dept. Molecular Pharmacology, Groningen Research Institute of Pharmacy, Groningen Research Institute for Asthma and COPD, GRIAC (N.M., A.O., M.S.), Dept. Laboratory Medicine (M.B.-H., H.M.), University Medical Center Groningen, University of Groningen, Groningen, The Netherlands; Inserm UMR-1048, Institut des Maladies Métaboliques et Cardiovasculaires, Univ Toulouse Paul Sabatier, Toulouse, France (F.L.); and Department of Integrative Biology and Pharmacology, Texas Therapeutics Institute, University of Texas Health Science Center at Houston, Houston, Texas (X.C.)
| | - Xiaodong Cheng
- Dept. Gastroenterology and Hepatology (F.A.A.M., M.B.-H., H.M.), Dept. Molecular Pharmacology, Groningen Research Institute of Pharmacy, Groningen Research Institute for Asthma and COPD, GRIAC (N.M., A.O., M.S.), Dept. Laboratory Medicine (M.B.-H., H.M.), University Medical Center Groningen, University of Groningen, Groningen, The Netherlands; Inserm UMR-1048, Institut des Maladies Métaboliques et Cardiovasculaires, Univ Toulouse Paul Sabatier, Toulouse, France (F.L.); and Department of Integrative Biology and Pharmacology, Texas Therapeutics Institute, University of Texas Health Science Center at Houston, Houston, Texas (X.C.)
| | - Martina Schmidt
- Dept. Gastroenterology and Hepatology (F.A.A.M., M.B.-H., H.M.), Dept. Molecular Pharmacology, Groningen Research Institute of Pharmacy, Groningen Research Institute for Asthma and COPD, GRIAC (N.M., A.O., M.S.), Dept. Laboratory Medicine (M.B.-H., H.M.), University Medical Center Groningen, University of Groningen, Groningen, The Netherlands; Inserm UMR-1048, Institut des Maladies Métaboliques et Cardiovasculaires, Univ Toulouse Paul Sabatier, Toulouse, France (F.L.); and Department of Integrative Biology and Pharmacology, Texas Therapeutics Institute, University of Texas Health Science Center at Houston, Houston, Texas (X.C.)
| | - Han Moshage
- Dept. Gastroenterology and Hepatology (F.A.A.M., M.B.-H., H.M.), Dept. Molecular Pharmacology, Groningen Research Institute of Pharmacy, Groningen Research Institute for Asthma and COPD, GRIAC (N.M., A.O., M.S.), Dept. Laboratory Medicine (M.B.-H., H.M.), University Medical Center Groningen, University of Groningen, Groningen, The Netherlands; Inserm UMR-1048, Institut des Maladies Métaboliques et Cardiovasculaires, Univ Toulouse Paul Sabatier, Toulouse, France (F.L.); and Department of Integrative Biology and Pharmacology, Texas Therapeutics Institute, University of Texas Health Science Center at Houston, Houston, Texas (X.C.)
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Smith-Cortinez N, van Eunen K, Heegsma J, Serna-Salas SA, Sydor S, Bechmann LP, Moshage H, Bakker BM, Faber KN. Simultaneous Induction of Glycolysis and Oxidative Phosphorylation during Activation of Hepatic Stellate Cells Reveals Novel Mitochondrial Targets to Treat Liver Fibrosis. Cells 2020; 9:cells9112456. [PMID: 33187083 PMCID: PMC7697161 DOI: 10.3390/cells9112456] [Citation(s) in RCA: 29] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2020] [Revised: 11/06/2020] [Accepted: 11/09/2020] [Indexed: 01/09/2023] Open
Abstract
Upon liver injury, hepatic stellate cells (HSCs) transdifferentiate to migratory, proliferative and extracellular matrix-producing myofibroblasts (e.g., activated HSCs; aHSCs) causing liver fibrosis. HSC activation is associated with increased glycolysis and glutaminolysis. Here, we compared the contribution of glycolysis, glutaminolysis and mitochondrial oxidative phosphorylation (OXPHOS) in rat and human HSC activation. Basal levels of glycolysis (extracellular acidification rate ~3-fold higher) and particularly mitochondrial respiration (oxygen consumption rate ~5-fold higher) were significantly increased in rat aHSCs, when compared to quiescent rat HSC. This was accompanied by extensive mitochondrial fusion in rat and human aHSCs, which occurred without increasing mitochondrial DNA content and electron transport chain (ETC) components. Inhibition of glycolysis (by 2-deoxy-D-glucose) and glutaminolysis (by CB-839) did not inhibit rat aHSC proliferation, but did reduce Acta2 (encoding α-SMA) expression slightly. In contrast, inhibiting mitochondrial OXPHOS (by rotenone) significantly suppressed rat aHSC proliferation, as well as Col1a1 and Acta2 expression. Other than that observed for rat aHSCs, human aHSC proliferation and expression of fibrosis markers were significantly suppressed by inhibiting either glycolysis, glutaminolysis or mitochondrial OXPHOS (by metformin). Activation of HSCs is marked by simultaneous induction of glycolysis and mitochondrial metabolism, extending the possibilities to suppress hepatic fibrogenesis by interfering with HSC metabolism.
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Affiliation(s)
- Natalia Smith-Cortinez
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, 9712 CP Groningen, The Netherlands; (N.S.-C.); (J.H.); (S.A.S.-S.); (H.M.)
| | - Karen van Eunen
- Department of Pediatrics, University Medical Center Groningen, University of Groningen, 9712 CP Groningen, The Netherlands; (K.v.E.); (B.M.B.)
| | - Janette Heegsma
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, 9712 CP Groningen, The Netherlands; (N.S.-C.); (J.H.); (S.A.S.-S.); (H.M.)
| | - Sandra Alejandra Serna-Salas
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, 9712 CP Groningen, The Netherlands; (N.S.-C.); (J.H.); (S.A.S.-S.); (H.M.)
| | - Svenja Sydor
- Department of Internal Medicine, University Hospital Knappschaftskrankenhaus, Ruhr-University, 44892 Bochum, Germany; (S.S.); (L.P.B.)
| | - Lars P. Bechmann
- Department of Internal Medicine, University Hospital Knappschaftskrankenhaus, Ruhr-University, 44892 Bochum, Germany; (S.S.); (L.P.B.)
| | - Han Moshage
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, 9712 CP Groningen, The Netherlands; (N.S.-C.); (J.H.); (S.A.S.-S.); (H.M.)
| | - Barbara M. Bakker
- Department of Pediatrics, University Medical Center Groningen, University of Groningen, 9712 CP Groningen, The Netherlands; (K.v.E.); (B.M.B.)
| | - Klaas Nico Faber
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, 9712 CP Groningen, The Netherlands; (N.S.-C.); (J.H.); (S.A.S.-S.); (H.M.)
- Correspondence: ; Tel.: +31-(0)-50-361-2364
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Geng Y, Wu Z, Buist-Homan M, Blokzijl H, Moshage H. Hesperetin protects against palmitate-induced cellular toxicity via induction of GRP78 in hepatocytes. Toxicol Appl Pharmacol 2020; 404:115183. [PMID: 32763355 DOI: 10.1016/j.taap.2020.115183] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2020] [Revised: 07/29/2020] [Accepted: 08/01/2020] [Indexed: 12/19/2022]
Abstract
Lipotoxicity plays a critical role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Hesperetin, a flavonoid derivative, has anti-oxidant, anti-inflammatory and cytoprotective properties. In the present study, we aim to examine whether hesperetin protects against palmitate-induced lipotoxic cell death and to investigate the underlying mechanisms in hepatocytes. Primary rat hepatocytes and HepG2 cells were pretreated with hesperetin for 30 min and then exposed to palmitate (1.0 mmol/L in primary rat hepatocytes; 0.5 mmol/L in HepG2 cells) in the presence or absence of hesperetin. Necrotic cell death was measured via Sytox green nuclei staining and quantified by LDH release assay. Apoptotic cell death was determined by caspase 3/7 activity and the protein level of cleaved-PARP. The unfolded protein response (UPR) was assessed by measuring the expression of GRP78, sXBP1, ATF4 and CHOP. Results show that hesperetin (50 μmol/L and 100 μmol/L) protected against palmitate-induced cell death and inhibited palmitate-induced endoplasmic reticulum (ER) stress in both primary rat hepatocytes and HepG2 cells. Hesperetin (100 μmol/L) significantly activated sXBP1/GRP78 signaling, whereas a high concentration of hesperetin (200 μmol/L) activated p-eIF2α and caused hepatic cell death. Importantly, GRP78 knockdown via siRNA abolished the protective effects of hesperetin in HepG2 cells. In conclusion, hesperetin protected against palmitate-induced hepatic cell death via activation of the sXBP1/GRP78 signaling pathway, thus inhibiting palmitate-induced ER stress. Moreover, high concentrations of hesperetin induce ER stress and subsequently cause cell death in hepatocytes.
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Affiliation(s)
- Yana Geng
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands.
| | - Zongmei Wu
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands.
| | - Manon Buist-Homan
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands; Department of Laboratory Medicine, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands.
| | - Hans Blokzijl
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands.
| | - Han Moshage
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands; Department of Laboratory Medicine, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands.
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Cao Q, Lu X, Azad BB, Pomper M, Smith M, He J, Pi L, Ren B, Ying Z, Sichani BS, Morris M, Dilsizian V. cis-4-[ 18F]fluoro-L-proline Molecular Imaging Experimental Liver Fibrosis. Front Mol Biosci 2020; 7:90. [PMID: 32500081 PMCID: PMC7243806 DOI: 10.3389/fmolb.2020.00090] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2020] [Accepted: 04/20/2020] [Indexed: 12/28/2022] Open
Abstract
Introduction: Early-stage liver fibrosis is potentially reversible, but difficult to diagnose. Clinical management would be enhanced by the development of a non-invasive imaging technique able to identify hepatic injury early, before end-stage fibrosis ensues. The analog of the amino acid proline, cis-4-[18F]fluoro-L-proline ([18F]fluoro-proline), which targets collagenogenesis in hepatic stellate cells (HSC), was used to detect fibrosis. Methods: Acute steatohepatitis was induced in experimental animals by liquid ethanol diet for 8 weeks, intra-gastric binge feedings every 10th day along with lipopolysaccharide (LPS) injection. The control animals received control diet for 8 weeks and an equivalent volume of saline on the same schedule as the acute steatohepatitis model. First, in vitro cellular experiments were carried out to assess [3H]proline uptake by HSC, hepatocytes and Kupffer cells derived from rats with acute steatohepatitis (n = 14) and controls (n = 14). Next, ex vivo liver experiments were done to investigate unlabeled proline-mediated collagen synthesis and its associated proline transporter expression in acute steatohepatitis (n = 5) and controls (n = 5). Last, in vivo dynamic and static [18F]fluoro-proline micro-PET/CT imaging was performed in animal models of acute steatohepatitis (n = 7) and control (n = 7) mice. Results: [3H]proline uptake was 5-fold higher in the HSCs of steatohepatitis rats than controls after incubation of up to 60 min. There was an excellent correlation between [3H]proline uptake and liver collagen expression (r-value > 0.90, p < 0.05). Subsequent liver tissue studies demonstrated 2–3-fold higher proline transporter expression in acute steatohepatitis animals than in controls, and proline-related collagen synthesis was blocked by this transporter inhibitor. In vivo micro-PET/CT studies with [18F]fluoro-proline showed 2–3-fold higher uptake in the livers of acute steatohepatitis mice than in controls. There was an excellent correlation between [18F]fluoro-proline uptake and liver collagen expression in the livers of acute steatohepatitis mice (r-value = 0.97, p < 0.001). Conclusion: [18F]fluoro-proline localizes in the liver and correlates with collagenogenesis in acute steatohepatitis with a signal intensity that is sufficiently high to allow imaging with micro-PET/CT. Thus, [18F]fluoro-proline could serve as a PET imaging biomarker for detecting early-stage liver fibrosis.
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Affiliation(s)
- Qi Cao
- The Department of Diagnostic Radiology and Nuclear Medicine, University of Maryland School of Medicine, Baltimore, MD, United States
| | - Xin Lu
- The Department of Diagnostic Radiology and Nuclear Medicine, University of Maryland School of Medicine, Baltimore, MD, United States
| | - Babak Behnam Azad
- Division of Nuclear Medicine and Molecular Imaging, The Johns Hopkins PET Center, Baltimore, MD, United States
| | - Martin Pomper
- The Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins School of Medicine, Baltimore, MD, United States
| | - Mark Smith
- The Department of Diagnostic Radiology and Nuclear Medicine, University of Maryland School of Medicine, Baltimore, MD, United States
| | - Jiang He
- Department of Radiology and Medical Imaging, University of Virginia, Charlottesville, VA, United States
| | - Liya Pi
- The Department of Pediatrics in the College of Medicine, University of Florida, Gainesville, FL, United States
| | - Bin Ren
- The Department of Surgery, University of Alabama at Birmingham School of Medicine, Birmingham, AL, United States
| | - Zhekang Ying
- The Department of Medicine, University of Maryland School of Medicine, Baltimore, MD, United States
| | - Babak Saboury Sichani
- The Department of Diagnostic Radiology and Nuclear Medicine, University of Maryland School of Medicine, Baltimore, MD, United States
| | - Michael Morris
- The Department of Diagnostic Radiology and Nuclear Medicine, University of Maryland School of Medicine, Baltimore, MD, United States
| | - Vasken Dilsizian
- The Department of Diagnostic Radiology and Nuclear Medicine, University of Maryland School of Medicine, Baltimore, MD, United States
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20
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Ríos-Ocampo WA, Navas MC, Buist-Homan M, Faber KN, Daemen T, Moshage H. Hepatitis C Virus Proteins Core and NS5A Are Highly Sensitive to Oxidative Stress-Induced Degradation after eIF2α/ATF4 Pathway Activation. Viruses 2020; 12:v12040425. [PMID: 32283772 PMCID: PMC7232227 DOI: 10.3390/v12040425] [Citation(s) in RCA: 11] [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: 03/19/2020] [Accepted: 04/07/2020] [Indexed: 12/11/2022] Open
Abstract
Hepatitis C virus (HCV) infection is accompanied by increased oxidative stress and endoplasmic reticulum stress as a consequence of viral replication, production of viral proteins, and pro-inflammatory signals. To overcome the cellular stress, hepatocytes have developed several adaptive mechanisms like anti-oxidant response, activation of Unfolded Protein Response and autophagy to achieve cell survival. These adaptive mechanisms could both improve or inhibit viral replication, however, little is known in this regard. In this study, we investigate the mechanisms by which hepatocyte-like (Huh7) cells adapt to cellular stress in the context of HCV protein overexpression and oxidative stress. Huh7 cells stably expressing individual HCV (Core, NS3/4A and NS5A) proteins were treated with the superoxide anion donor menadione to induce oxidative stress. Production of reactive oxygen species and activation of caspase 3 were quantified. The activation of the eIF2α/ATF4 pathway and changes in the steady state levels of the autophagy-related proteins LC3 and p62 were determined either by quantitative polymerase chain reaction (qPCR) or Western blotting. Huh7 cells expressing Core or NS5A demonstrated reduced oxidative stress and apoptosis. In addition, phosphorylation of eIF2α and increased ATF4 and CHOP expression was observed with subsequent HCV Core and NS5A protein degradation. In line with these results, in liver biopsies from patients with hepatitis C, the expression of ATF4 and CHOP was confirmed. HCV Core and NS5A protein degradation was reversed by antioxidant treatment or silencing of the autophagy adaptor protein p62. We demonstrated that hepatocyte-like cells expressing HCV proteins and additionally exposed to oxidative stress adapt to cellular stress through eIF2a/ATF4 activation and selective degradation of HCV pro-oxidant proteins Core and NS5A. This selective degradation is dependent on p62 and results in increased resistance to apoptotic cell death induced by oxidative stress. This mechanism may provide a new key for the study of HCV pathology and lead to novel clinically applicable therapeutic interventions.
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Affiliation(s)
- W. Alfredo Ríos-Ocampo
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, 9713 GZ Groningen, The Netherlands; (M.B.-H.); (K.N.F.); (H.M.)
- Department of Medical Microbiology, University Medical Center Groningen, University of Groningen, 9713 GZ Groningen, The Netherlands;
- Gastrohepatology Group, Medicine School, University of Antioquia, Medellin 050010, Colombia;
- Correspondence: ; Tel.: +31-50-361-2364 or +31-638-955-716
| | - María-Cristina Navas
- Gastrohepatology Group, Medicine School, University of Antioquia, Medellin 050010, Colombia;
| | - Manon Buist-Homan
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, 9713 GZ Groningen, The Netherlands; (M.B.-H.); (K.N.F.); (H.M.)
| | - Klaas Nico Faber
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, 9713 GZ Groningen, The Netherlands; (M.B.-H.); (K.N.F.); (H.M.)
| | - Toos Daemen
- Department of Medical Microbiology, University Medical Center Groningen, University of Groningen, 9713 GZ Groningen, The Netherlands;
| | - Han Moshage
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, 9713 GZ Groningen, The Netherlands; (M.B.-H.); (K.N.F.); (H.M.)
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Abstract
Alcohol consumption has long been a part of human culture. However, alcohol consumption levels and alcohol consumption patterns are associated with chronic diseases. Overall, light and moderate alcohol consumption (up to 14 g per day for women and up to 28 g per day for men) may be associated with reduced mortality risk, mainly due to reduced risks for cardiovascular disease and type-2 diabetes. However, chronic heavy alcohol consumption and alcohol abuse lead to alcohol-use disorder, which results in physical and mental diseases such as liver disease, pancreatitis, dementia, and various types of cancer. Risk factors for alcohol-use disorder are largely unknown. Alcohol-use disorder and frequent heavy drinking have detrimental effects on personal health.
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Ríos-Ocampo WA, Daemen T, Buist-Homan M, Faber KN, Navas MC, Moshage H. Hepatitis C virus core or NS3/4A protein expression preconditions hepatocytes against oxidative stress and endoplasmic reticulum stress. Redox Rep 2020; 24:17-26. [PMID: 30909829 PMCID: PMC6748607 DOI: 10.1080/13510002.2019.1596431] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Objectives: The occurrence of oxidative stress and endoplasmic
reticulum (ER) stress in hepatitis C virus (HCV) infection has been demonstrated
and play an important role in liver injury. During viral infection, hepatocytes
must handle not only the replication of the virus, but also inflammatory signals
generating oxidative stress and damage. Although several mechanisms exist to
overcome cellular stress, little attention has been given to the adaptive
response of hepatocytes during exposure to multiple noxious triggers. Methods: In the present study, Huh-7 cells and hepatocytes
expressing HCV Core or NS3/4A proteins, both inducers of oxidative and ER
stress, were additionally challenged with the superoxide anion generator
menadione to mimic external oxidative stress. The production of reactive oxygen
species (ROS) as well as the response to oxidative stress and ER stress were
investigated. Results: We demonstrate that hepatocytes diminish oxidative stress
through a reduction in ROS production, ER-stress markers (HSPA5
[GRP78], sXBP1) and apoptosis (caspase-3 activity) despite
external oxidative stress. Interestingly, the level of the autophagy substrate
protein p62 was downregulated together with HCV Core degradation, suggesting
that hepatocytes can overcome excess oxidative stress through autophagic
degradation of one of the stressors, thereby increasing cell survival. Duscussion: In conclusion, hepatocytes exposed to direct and
indirect oxidative stress inducers are able to cope with cellular stress
associated with viral hepatitis and thus promote cell survival.
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Affiliation(s)
- W Alfredo Ríos-Ocampo
- a Department of Gastroenterology and Hepatology , University of Groningen, University Medical Center Groningen , Groningen , Netherlands.,b Department Medical Microbiology , University of Groningen, University Medical Center Groningen , Groningen , Netherlands.,c Grupo Gastrohepatología, Facultad de Medicina, Universidad de Antioquia , Medellin , Colombia
| | - Toos Daemen
- b Department Medical Microbiology , University of Groningen, University Medical Center Groningen , Groningen , Netherlands
| | - Manon Buist-Homan
- a Department of Gastroenterology and Hepatology , University of Groningen, University Medical Center Groningen , Groningen , Netherlands.,d Department of Laboratory Medicine , University of Groningen, University Medical Center Groningen , Groningen , Netherlands
| | - Klaas Nico Faber
- a Department of Gastroenterology and Hepatology , University of Groningen, University Medical Center Groningen , Groningen , Netherlands.,d Department of Laboratory Medicine , University of Groningen, University Medical Center Groningen , Groningen , Netherlands
| | - María-Cristina Navas
- c Grupo Gastrohepatología, Facultad de Medicina, Universidad de Antioquia , Medellin , Colombia
| | - Han Moshage
- a Department of Gastroenterology and Hepatology , University of Groningen, University Medical Center Groningen , Groningen , Netherlands.,d Department of Laboratory Medicine , University of Groningen, University Medical Center Groningen , Groningen , Netherlands
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Hernández A, Geng Y, Sepúlveda R, Solís N, Torres J, Arab JP, Barrera F, Cabrera D, Moshage H, Arrese M. Chemical hypoxia induces pro-inflammatory signals in fat-laden hepatocytes and contributes to cellular crosstalk with Kupffer cells through extracellular vesicles. Biochim Biophys Acta Mol Basis Dis 2020; 1866:165753. [PMID: 32126269 DOI: 10.1016/j.bbadis.2020.165753] [Citation(s) in RCA: 37] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2019] [Revised: 02/06/2020] [Accepted: 02/27/2020] [Indexed: 02/07/2023]
Abstract
BACKGROUND Obstructive sleep apnea syndrome (OSAS) is associated to intermittent hypoxia (IH) and is an aggravating factor of non-alcoholic fatty liver disease (NAFLD). We investigated the effects of hypoxia in both in vitro and in vivo models of NAFLD. METHODS Primary rat hepatocytes treated with free fatty acids (FFA) were subjected to chemically induced hypoxia (CH) using the hypoxia-inducible factor-1 alpha (HIF-1α) stabilizer cobalt chloride (CoCl2). Triglyceride (TG) content, mitochondrial superoxide production, cell death rates, cytokine and inflammasome components gene expression and protein levels of cleaved caspase-1 were assessed. Also, Kupffer cells (KC) were treated with conditioned medium (CM) and extracellular vehicles (EVs) from hypoxic fat-laden hepatic cells. The choline deficient L-amino acid defined (CDAA)-feeding model used to assess the effects of IH on experimental NAFLD in vivo. RESULTS Hypoxia induced HIF-1α in cells and animals. Hepatocytes exposed to FFA and CoCl2 exhibited increased TG content and higher cell death rates as well as increased mitochondrial superoxide production and mRNA levels of pro-inflammatory cytokines and of inflammasome-components interleukin-1β, NLRP3 and ASC. Protein levels of cleaved caspase-1 increased in CH-exposed hepatocytes. CM and EVs from hypoxic fat-laden hepatic cells evoked a pro-inflammatory phenotype in KC. Livers from CDAA-fed mice exposed to IH exhibited increased mRNA levels of pro-inflammatory and inflammasome genes and increased levels of cleaved caspase-1. CONCLUSION Hypoxia promotes inflammatory signals including inflammasome/caspase-1 activation in fat-laden hepatocytes and contributes to cellular crosstalk with KC by release of EVs. These mechanisms may underlie the aggravating effect of OSAS on NAFLD. [Abstract word count: 257].
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Affiliation(s)
- Alejandra Hernández
- Departamento de Gastroenterología, Escuela de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile; Departamento de Patología, Escuela de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile
| | - Yana Geng
- Departamento de Patología, Escuela de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile
| | - Rolando Sepúlveda
- Departamento de Gastroenterología, Escuela de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile
| | - Nancy Solís
- Departamento de Gastroenterología, Escuela de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile
| | - Javiera Torres
- Department of Gastroenterology and Hepatology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands
| | - Juan Pablo Arab
- Departamento de Gastroenterología, Escuela de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile; Centro de Envejecimiento y Regeneración (CARE), Departamento de Biología Celular y Molecular, Facultad de Ciencias Biológicas Pontificia Universidad Católica de Chile, Santiago, Chile
| | - Francisco Barrera
- Departamento de Gastroenterología, Escuela de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile
| | - Daniel Cabrera
- Departamento de Gastroenterología, Escuela de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile; Centro de Envejecimiento y Regeneración (CARE), Departamento de Biología Celular y Molecular, Facultad de Ciencias Biológicas Pontificia Universidad Católica de Chile, Santiago, Chile; Facultad de Ciencias Médicas, Universidad Bernardo O Higgins, Santiago, Chile
| | - Han Moshage
- Department of Gastroenterology and Hepatology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands
| | - Marco Arrese
- Departamento de Gastroenterología, Escuela de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile; Centro de Envejecimiento y Regeneración (CARE), Departamento de Biología Celular y Molecular, Facultad de Ciencias Biológicas Pontificia Universidad Católica de Chile, Santiago, Chile.
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Shajari S, Saeed A, Smith-Cortinez NF, Heegsma J, Sydor S, Faber KN. Hormone-sensitive lipase is a retinyl ester hydrolase in human and rat quiescent hepatic stellate cells. Biochim Biophys Acta Mol Cell Biol Lipids 2019; 1864:1258-1267. [PMID: 31150775 DOI: 10.1016/j.bbalip.2019.05.012] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2019] [Revised: 05/13/2019] [Accepted: 05/24/2019] [Indexed: 01/04/2023]
Abstract
Hepatic stellate cells (HSC) store vitamin A as retinyl esters and control circulating retinol levels. Upon liver injury, quiescent (q)HSC lose their vitamin A and transdifferentiate to myofibroblasts, e.g. activated (a)HSC, which promote fibrosis by producing excessive extracellular matrix. Adipose triglyceride lipase/patatin-like phospholipase domain-containing protein 2 (ATGL/PNPLA2) and adiponutrin (ADPN/PNPLA3) have so far been shown to mobilize retinol from retinyl esters in HSC. Here, we studied the putative role of hormone-sensitive lipase (HSL/LIPE) in HSC, as it is the major retinyl ester hydrolase (REH) in adipose tissue. Lipe/HSL expression was analyzed in rat liver and primary human and rat qHSC and culture-activated aHSC. Retinyl hydrolysis was analyzed after Isoproterenol-mediated phosphorylation/activation of HSL. Primary human HSC contain 2.5-fold higher LIPE mRNA levels compared to hepatocytes. Healthy rat liver contains significant mRNA and protein levels of HSL/Lipe, which predominates in qHSC and cells of the portal tree. Q-PCR comparison indicates that Lipe mRNA levels in qHSC are dominant over Pnpla2 and Pnpla3. HSL is mostly phosphorylated/activated in qHSC and partly colocalizes with vitamin A-containing lipid droplets. Lipe/HSL and Pnpla3 expression is rapidly lost during HSC culture-activation, while Pnpla2 expression is maintained. HSL super-activation by isoproterenol accelerates loss of lipid droplets and retinyl palmitate from HSC, which coincided with a small, but significant reduction in HSC proliferation and suppression of Collagen1A1 mRNA and protein levels. In conclusion, HSL participates in vitamin A metabolism in qHSC. Equivalent activities of ATGL and ADPN provide the healthy liver with multiple routes to control circulating retinol levels.
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Affiliation(s)
- Shiva Shajari
- Department of Gastroenterology and Hepatology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands
| | - Ali Saeed
- Department of Gastroenterology and Hepatology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands; Institute of Molecular Biology & Biotechnology, Bahauddin Zakariya University, Multan, Pakistan
| | - Natalia F Smith-Cortinez
- Department of Gastroenterology and Hepatology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands
| | - Janette Heegsma
- Department of Gastroenterology and Hepatology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands; Department of Laboratory Medicine, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands
| | - Svenja Sydor
- Department of Gastroenterology, Hepatology and Infectious Diseases, Otto von Guericke University Magdeburg, Magdeburg, Germany
| | - Klaas Nico Faber
- Department of Gastroenterology and Hepatology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands; Department of Laboratory Medicine, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands.
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Selden C, Khalil M, Hodgson H. Three Dimensional Culture Upregulates Extracellular Matrix Protein Expression in Human Liver Cell Lines - a Step towards Mimicking the Liver in Vivo? Int J Artif Organs 2018. [DOI: 10.1177/039139880002301107] [Citation(s) in RCA: 38] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
Extracellular matrix (ECM) in the liver affects the phenotype of both hepatocytes and non-parenchymal cells. To be able to mimic in vivo liver function for extracorporeal hepatic support using human cell lines, a necessary step is to upregulate the function normally seen in monolayer culture. 3-D spheroid colonies were formed by culturing single HepG2 cells encapsulated in alginate beads. ECM expression in these cultures was compared to monolayer Hep G2 cultures. The following ECM proteins were detected immunohistochemically:- collagens I, III, V and VI, the glycoproteins fibronectin, tenascin and vitronectin, and the basement membrane protein laminin. In 3-D cultures, all proteins except tenascin were strongly expressed, as compared with weak or undetectable expression in monolayer cultures, even with 10-fold increases in the antibody concentration used. In conclusion, we have demonstrated that the 3-D environment created by alginate encapsulation of cell lines leads to cell behaviour mimicking that in vivo.
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Affiliation(s)
- C. Selden
- Department of Medicine, Royal Free and University College Medical School, London - UK
| | - M. Khalil
- Department of Medicine, Royal Free and University College Medical School, London - UK
| | - H. Hodgson
- Department of Medicine, Royal Free and University College Medical School, London - UK
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26
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Crawford JM, Bioulac-Sage P, Hytiroglou P. Structure, Function, and Responses to Injury. MACSWEEN'S PATHOLOGY OF THE LIVER 2018:1-87. [DOI: 10.1016/b978-0-7020-6697-9.00001-7] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/03/2025]
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Rani V, Verma Y, Rana K, Rana SVS. Zinc oxide nanoparticles inhibit dimethylnitrosamine induced liver injury in rat. Chem Biol Interact 2017; 295:84-92. [PMID: 29024620 DOI: 10.1016/j.cbi.2017.10.009] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2017] [Accepted: 10/06/2017] [Indexed: 02/07/2023]
Abstract
Dimethylnitrosamine (DMN) is a potent hepatotoxic, carcinogenic and mutagenic compound. It induces massive liver cell necrosis and death in experimental animals. Several drugs have been tested in the past for their protective behavior against DMN toxicity. However, it is for the first time that therapeutic intervention of ZnONPs (zinc oxide nanoparticles) has been studied against its toxicity. Present results show that a post treatment of ZnONPs (50 mg/kg) to DMN (2 μl/100 g body weight) treated rats reduces lipid peroxidation, oxidative stress and fibrosis in the liver. It diminishes serum ALT (alanine transaminases), AST (aspartate transaminases) and LDH (lactate dehydrogenase) showing improvement in liver function. Reduced values of proinflammatory cytokines viz. TNF-α and IL-12 also support its protective effects. Histopathological observations also indicate improvement in liver cell morphology. It is postulated that ZnONPs offer protection through selective toxicity to proliferating tissue including adenomatous islands formed in the liver. Zinc metallothionein (Zn-MT) induced by ZnONPs may also contribute in the amelioration of DMN induced toxic effects. Diminution of oxidative stress by ZnONPs remains to be the key mechanism involved in its protective effects. However, toxicity of ZnONPs in the liver needs to be monitored simultaneously.
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Affiliation(s)
- Varsha Rani
- Toxicology Laboratory, Department of Zoology/Toxicology, Chaudhary Charan Singh University, Meerut 250004, India
| | - Yeshvandra Verma
- Toxicology Laboratory, Department of Zoology/Toxicology, Chaudhary Charan Singh University, Meerut 250004, India
| | - Kavita Rana
- Toxicology Laboratory, Department of Zoology/Toxicology, Chaudhary Charan Singh University, Meerut 250004, India
| | - Suresh Vir Singh Rana
- Toxicology Laboratory, Department of Zoology/Toxicology, Chaudhary Charan Singh University, Meerut 250004, India.
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Arabpour M, Cool RH, Faber KN, Quax WJ, Haisma HJ. Receptor-specific TRAIL as a means to achieve targeted elimination of activated hepatic stellate cells. J Drug Target 2016; 25:360-369. [PMID: 27885847 DOI: 10.1080/1061186x.2016.1262867] [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: 02/07/2023]
Abstract
Activated hepatic stellate cells (HSCs) are known to play a central role in liver fibrosis and their elimination is a crucial step toward the resolution and reversion of liver fibrosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a molecule that may contribute to the apoptotic removal of activated HSC through binding to its dedicated receptors. In the present study, we investigated the potential application of recombinant receptor-specific TRAIL proteins in the efficient elimination of activated HSCs. Our finding revealed differential contribution of TRAIL receptors among HSCs populations with activated hepatic stellate cells expresses more TRAIL receptors DR5. In vitro treatment of activated HSCs with DR5-specific or wild-type TRAIL variants induced a significant reduction in viability and extracellular matrix production, whereas no significant decrease in viability was associated with the treatment of cells by DR4-specific TRAIL. Our analysis indicate the successful application of the DR5 receptor-specific TRAIL variant in the targeted elimination of activated HSCs via interference with collagen production and simultaneous induction of apoptosis via activation of the caspase pathway. DR5 receptor-specific TRAIL may thus represent a new therapeutic compound for the treatment of liver fibrosis.
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Affiliation(s)
- Mohammad Arabpour
- a Mivac Development , Arvid Wallgrens backe 20 , Gothenburg , Sweden.,b Department of Chemical and Pharmaceutical Biology , University of Groningen , Groningen , the Netherlands
| | - Robbert H Cool
- b Department of Chemical and Pharmaceutical Biology , University of Groningen , Groningen , the Netherlands
| | - Klaas Nico Faber
- c Department of Gastrointestinal and Liver Diseases , University Medical Center Groningen , Groningen , the Netherlands
| | - Wim J Quax
- b Department of Chemical and Pharmaceutical Biology , University of Groningen , Groningen , the Netherlands
| | - Hidde J Haisma
- b Department of Chemical and Pharmaceutical Biology , University of Groningen , Groningen , the Netherlands
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Zhang CY, Yuan WG, He P, Lei JH, Wang CX. Liver fibrosis and hepatic stellate cells: Etiology, pathological hallmarks and therapeutic targets. World J Gastroenterol 2016. [PMID: 28082803 DOI: 10.3748/wjg.v22.i48.10512.] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 09/29/2022] Open
Abstract
Liver fibrosis is a reversible wound-healing process aimed at maintaining organ integrity, and presents as the critical pre-stage of liver cirrhosis, which will eventually progress to hepatocellular carcinoma in the absence of liver transplantation. Fibrosis generally results from chronic hepatic injury caused by various factors, mainly viral infection, schistosomiasis, and alcoholism; however, the exact pathological mechanisms are still unknown. Although numerous drugs have been shown to have antifibrotic activity in vitro and in animal models, none of these drugs have been shown to be efficacious in the clinic. Importantly, hepatic stellate cells (HSCs) play a key role in the initiation, progression, and regression of liver fibrosis by secreting fibrogenic factors that encourage portal fibrocytes, fibroblasts, and bone marrow-derived myofibroblasts to produce collagen and thereby propagate fibrosis. These cells are subject to intricate cross-talk with adjacent cells, resulting in scarring and subsequent liver damage. Thus, an understanding of the molecular mechanisms of liver fibrosis and their relationships with HSCs is essential for the discovery of new therapeutic targets. This comprehensive review outlines the role of HSCs in liver fibrosis and details novel strategies to suppress HSC activity, thereby providing new insights into potential treatments for liver fibrosis.
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Affiliation(s)
- Chong-Yang Zhang
- Chong-Yang Zhang, Jia-Hui Lei, Department of Pathogenic Biology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China
| | - Wei-Gang Yuan
- Chong-Yang Zhang, Jia-Hui Lei, Department of Pathogenic Biology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China
| | - Pei He
- Chong-Yang Zhang, Jia-Hui Lei, Department of Pathogenic Biology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China
| | - Jia-Hui Lei
- Chong-Yang Zhang, Jia-Hui Lei, Department of Pathogenic Biology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China
| | - Chun-Xu Wang
- Chong-Yang Zhang, Jia-Hui Lei, Department of Pathogenic Biology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China
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Sargent G, van Zutphen T, Shatseva T, Zhang L, Di Giovanni V, Bandsma R, Kim PK. PEX2 is the E3 ubiquitin ligase required for pexophagy during starvation. J Cell Biol 2016; 214:677-90. [PMID: 27597759 PMCID: PMC5021090 DOI: 10.1083/jcb.201511034] [Citation(s) in RCA: 132] [Impact Index Per Article: 14.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2015] [Accepted: 08/01/2016] [Indexed: 12/31/2022] Open
Abstract
Sargent et al. identify the E3 ubiquitin ligase PEX2 as the causative agent of mammalian pexophagy. During amino acid starvation, PEX2 expression increases to ubiquitinate peroxisomal membrane proteins and signal peroxisome degradation by autophagy. Peroxisomes are metabolic organelles necessary for anabolic and catabolic lipid reactions whose numbers are highly dynamic based on the metabolic need of the cells. One mechanism to regulate peroxisome numbers is through an autophagic process called pexophagy. In mammalian cells, ubiquitination of peroxisomal membrane proteins signals pexophagy; however, the E3 ligase responsible for mediating ubiquitination is not known. Here, we report that the peroxisomal E3 ubiquitin ligase peroxin 2 (PEX2) is the causative agent for mammalian pexophagy. Expression of PEX2 leads to gross ubiquitination of peroxisomes and degradation of peroxisomes in an NBR1-dependent autophagic process. We identify PEX5 and PMP70 as substrates of PEX2 that are ubiquitinated during amino acid starvation. We also find that PEX2 expression is up-regulated during both amino acid starvation and rapamycin treatment, suggesting that the mTORC1 pathway regulates pexophagy by regulating PEX2 expression levels. Finally, we validate our findings in vivo using an animal model.
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Affiliation(s)
- Graeme Sargent
- Cell Biology Department, The Hospital for Sick Children, Toronto, ON M5G 1X8, Canada Biochemistry Department, University of Toronto, Toronto, ON M5S 1A8, Canada
| | - Tim van Zutphen
- Department of Pediatrics, Center for Liver, Digestive and Metabolic Diseases, University of Groningen, University Medical Center Groningen, 9700 AD Groningen, Netherlands
| | - Tatiana Shatseva
- Biochemistry Department, University of Toronto, Toronto, ON M5S 1A8, Canada
| | - Ling Zhang
- Physiology and Experimental Medicine Program, Research Institute, The Hospital for Sick Children, Toronto, ON M5G 1X8, Canada
| | - Valeria Di Giovanni
- Physiology and Experimental Medicine Program, Research Institute, The Hospital for Sick Children, Toronto, ON M5G 1X8, Canada
| | - Robert Bandsma
- Department of Paediatric Laboratory Medicine, The Hospital for Sick Children, Toronto, ON M5G 1X8, Canada Physiology and Experimental Medicine Program, Research Institute, The Hospital for Sick Children, Toronto, ON M5G 1X8, Canada Division of Gastroenterology, Hepatology and Nutrition, The Hospital for Sick Children, Toronto, ON M5G 1X8, Canada Centre for Global Child Health, The Hospital for Sick Children, Toronto, ON M5G 1X8, Canada
| | - Peter Kijun Kim
- Cell Biology Department, The Hospital for Sick Children, Toronto, ON M5G 1X8, Canada Biochemistry Department, University of Toronto, Toronto, ON M5S 1A8, Canada
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Kumar S, Wang J, Rani R, Gandhi CR. Hepatic Deficiency of Augmenter of Liver Regeneration Exacerbates Alcohol-Induced Liver Injury and Promotes Fibrosis in Mice. PLoS One 2016; 11:e0147864. [PMID: 26808690 PMCID: PMC4726524 DOI: 10.1371/journal.pone.0147864] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2015] [Accepted: 01/08/2016] [Indexed: 12/29/2022] Open
Abstract
Why only a subpopulation (about 15%) of humans develops liver cirrhosis due to alcohol is a critical as yet unanswered question. Liver-specific depletion of augmenter of liver regeneration (ALR) protein in mice causes robust steatosis and hepatocyte apoptosis by 2 weeks; these pathologies regress subsequently with return of ALR expression even at lower than control levels, but the mice develop modest steatohepatitis by 8 weeks. We aimed to investigate whether chronic alcohol ingestion promotes excessive hepatic fibrosis in these ALR-deficient mice. Liver-specific ALR-deficient and wild type (WT) female mice (8–10 weeks old) were placed on 4% alcohol-supplemented or isocaloric diet for 4 weeks. Liver sections were examined for histopathology, and parameters of steatosis and fibrosis were quantified. The mRNA expression of alcohol dehydrogenase-1, acetaldehyde dehydrogenase-1 and cytochrome P450-2E1 increased in WT mice but decreased in ALR-deficient mice upon alcohol ingestion. While alcohol induced steatosis and mild inflammation in WT mice, ALR-deficient mice showed minimal steatosis, strong hepatocellular injury and inflammation, prominent ductular proliferation, and robust fibrosis. Compared to the WT mice, alcohol feeding of ALR-deficient mice resulted in significantly greater increase in hepatic TNFα and TGFβ, and oxidative stress; there was also hepatic iron accumulation, robust lipid peroxidation and mitochondrial DNA damage. Importantly, similar to ALR-deficient mice, lower hepatic ALR levels in human alcoholic liver cirrhosis were associated with increased iron content, reduced expression of alcohol dehydrogenase and acetaldehyde dehydrogenase, and elevated fibrogenic markers. We conclude that ALR deficiency or anomaly can play a critical role in alcohol-induced hepatic fibrosis/cirrhosis, mechanisms of which may involve dysregulation of alcohol metabolism and iron homeostasis, mitochondrial damage and oxidative injury.
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Affiliation(s)
- Sudhir Kumar
- Department of Surgery, University of Cincinnati, Cincinnati, Ohio, United States of America
- Cincinnati VA Medical Center, Cincinnati, Ohio, United States of America
| | - Jiang Wang
- Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, Ohio, United States of America
| | - Richa Rani
- Department of Surgery, University of Cincinnati, Cincinnati, Ohio, United States of America
- Cincinnati VA Medical Center, Cincinnati, Ohio, United States of America
| | - Chandrashekhar R. Gandhi
- Department of Surgery, University of Cincinnati, Cincinnati, Ohio, United States of America
- Cincinnati VA Medical Center, Cincinnati, Ohio, United States of America
- Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, United states of America
- * E-mail:
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Hong-Brown LQ, Brown CR, Navaratnarajah M, Lang CH. Adamts1 mediates ethanol-induced alterations in collagen and elastin via a FoxO1-sestrin3-AMPK signaling cascade in myocytes. J Cell Biochem 2016; 116:91-101. [PMID: 25142777 DOI: 10.1002/jcb.24945] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2014] [Accepted: 08/15/2014] [Indexed: 12/11/2022]
Abstract
A variety of stressors including alcohol (EtOH) are known to induce collagen production and fibrotic diseases. Matrix metalloproteinases (MMP) play an important role in regulating fibrosis, but little is known regarding the relationship between EtOH and MMPs. In addition, the signaling cascades involved in this process have not been elucidated. We have identified the MMP Adamts1 as a target of EtOH regulation. To characterize the function of Adamts1, we examined EtOH-induced alterations in collagen I and elastin protein levels in C2C12 myocytes. Incubation of myocytes with 100 mM EtOH decreased elastin and increased collagen content, respectively, and these changes were associated with increased O-GLcNAc modification of Adamts1. Conversely, silencing of Adamts1 by siRNA blocked the adverse effects of EtOH on collagen and elastin levels. Similar results were obtained after treatment with a pharmacological inhibitor of MMP. Changes in collagen were due, at least in part, to a decreased interaction of Adamts1 with its endogenous inhibitor TIMP3. The AMPK inhibitor compound C blocked the EtOH-induced stimulation of collagen and O-GLcNAc Adamts1 protein. Changes in AMPK appear linked to FoxO1, since inhibition of FoxO1 blocked the effects of EtOH on AMPK phosphorylation and O-GLcNAc levels. These FoxO-dependent modifications were associated with an upregulation of the FoxO1 transcription target sestrin 3, as well as increased binding of sestrin 3 with AMPK. Collectively, these data indicate that EtOH regulates the collagen I and elastin content in an Adamts1-dependent manner in myocytes. Furthermore, Adamts1 appears to be controlled by the FoxO1-sestrin 3-AMPK signaling cascade.
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Affiliation(s)
- Ly Q Hong-Brown
- Department of Cellular and Molecular Physiology, Penn State College of Medicine, Hershey, Pennsylvania, 17033, USA
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Shajari S, Laliena A, Heegsma J, Tuñón MJ, Moshage H, Faber KN. Melatonin suppresses activation of hepatic stellate cells through RORα-mediated inhibition of 5-lipoxygenase. J Pineal Res 2015; 59:391-401. [PMID: 26308880 DOI: 10.1111/jpi.12271] [Citation(s) in RCA: 42] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/17/2015] [Accepted: 08/21/2015] [Indexed: 12/31/2022]
Abstract
Liver fibrosis is scar tissue resulting from an uncontrolled wound-healing process in response to chronic liver injury. Liver damage generates an inflammatory reaction that activates hepatic stellate cells (HSC) that transdifferentiate from quiescent cells that control retinol metabolism to proliferative and migratory myofibroblasts that produce excessive amounts of extracellular matrix proteins, in particular collagen 1a1 (COL1A1). Although liver fibrosis is reversible, no effective drug therapy is available to prevent or reverse HSC activation. Melatonin has potent hepatoprotective properties in a variety of acute and chronic liver injury models and suppresses liver fibrosis. However, it remains unclear whether melatonin acts indirectly or directly on HSC to prevent liver fibrosis. Here, we studied the effect of melatonin on culture-activated rat HSC. Melatonin dose-dependently suppressed the expression of HSC activation markers Col1a1 and alpha-smooth muscle actin (αSMA, Acta2), as well as HSC proliferation and loss of lipid droplets. The nuclear melatonin sensor retinoic acid receptor-related orphan receptor-alpha (RORα/Nr1f1) was expressed in quiescent and activated HSC, while the membranous melatonin receptors (Mtrn1a and Mtrn1b) were not. The synthetic RORα agonist SR1078 more potently suppressed Col1a1 and αSma expression, HSC proliferation, and lipid droplet loss, while the RORα antagonist SR1001 blocked the antifibrotic features of melatonin. Melatonin and SR1078 inhibited the expression of Alox5, encoding 5-lipoxygenase (5-LO). The pharmacological 5-LO inhibitor AA861 reduced Acta2 and Col1a1 expression in activated HSC. We conclude that melatonin directly suppresses HSC activation via RORα-mediated inhibition of Alox5 expression, which provides novel drug targets to treat liver fibrosis.
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Affiliation(s)
- Shiva Shajari
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
| | - Almudena Laliena
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
- Institute of Biomedicine (IBIOMED), University of León, León, Spain
| | - Janette Heegsma
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
- Department of Laboratory Medicine, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
| | - María Jesús Tuñón
- Institute of Biomedicine (IBIOMED), University of León, León, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), León, Spain
| | - Han Moshage
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
| | - Klaas Nico Faber
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
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Conde de la Rosa L, Vrenken TE, Buist-Homan M, Faber KN, Moshage H. Metformin protects primary rat hepatocytes against oxidative stress-induced apoptosis. Pharmacol Res Perspect 2015; 3:e00125. [PMID: 26038701 PMCID: PMC4448984 DOI: 10.1002/prp2.125] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2014] [Revised: 01/05/2015] [Accepted: 01/14/2015] [Indexed: 12/21/2022] Open
Abstract
The majority of chronic liver diseases are accompanied by oxidative stress, which induces apoptosis in hepatocytes and liver injury. Recent studies suggest that oxidative stress and insulin resistance are important in the pathogenesis of nonalcoholic fatty liver disease (NAFLD) and the pathophysiology of diabetes complications. Metformin has been shown to be hepatoprotective in the insulin-resistant and leptin-deficient ob/ob mouse model of NAFLD. However, the mechanism involved in the protective effects of metformin has not been elucidated yet. Therefore, we investigated the protective effect of metformin against oxidative stress-induced apoptosis. Primary rat hepatocytes were exposed to the oxidative stress-generating compound menadione in the presence and absence of metformin. Apoptosis was determined by measuring caspase activity and poly(ADP-ribose) polymerase (PARP)-cleavage, and necrosis was measured by Sytox Green nuclear staining. We demonstrate that (1) Metformin inhibits menadione-induced caspase-9,-6,-3 activation and PARP-cleavage in a concentration-dependent manner. (2) Metformin increases menadione-induced heme oxygenase-1 (HO-1) expression and inhibits c-Jun N-terminal kinase (JNK)-phosphorylation. (3) Metformin does not induce necrosis in primary hepatocytes. Metformin protects hepatocytes against oxidative stress-induced caspase activation, PARP-cleavage and apoptosis. The anti-apoptotic effect of metformin is in part dependent on HO-1 and bcl-xl induction and inhibition of JNK activation and independent of insulin signaling. Our results elucidate novel protective mechanisms of metformin and indicate that metformin could be investigated as a novel therapeutic agent for the treatment of oxidative stress-related liver diseases.
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Affiliation(s)
- Laura Conde de la Rosa
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen Groningen, The Netherlands
| | - Titia E Vrenken
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen Groningen, The Netherlands
| | - Manon Buist-Homan
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen Groningen, The Netherlands
| | - Klaas Nico Faber
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen Groningen, The Netherlands
| | - Han Moshage
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen Groningen, The Netherlands
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Dunning S, Ur Rehman A, Tiebosch MH, Hannivoort RA, Haijer FW, Woudenberg J, van den Heuvel FAJ, Buist-Homan M, Faber KN, Moshage H. Glutathione and antioxidant enzymes serve complementary roles in protecting activated hepatic stellate cells against hydrogen peroxide-induced cell death. Biochim Biophys Acta Mol Basis Dis 2013; 1832:2027-34. [PMID: 23871839 DOI: 10.1016/j.bbadis.2013.07.008] [Citation(s) in RCA: 88] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2013] [Revised: 07/05/2013] [Accepted: 07/09/2013] [Indexed: 12/13/2022]
Abstract
BACKGROUND In chronic liver disease, hepatic stellate cells (HSCs) are activated, highly proliferative and produce excessive amounts of extracellular matrix, leading to liver fibrosis. Elevated levels of toxic reactive oxygen species (ROS) produced during chronic liver injury have been implicated in this activation process. Therefore, activated hepatic stellate cells need to harbor highly effective anti-oxidants to protect against the toxic effects of ROS. AIM To investigate the protective mechanisms of activated HSCs against ROS-induced toxicity. METHODS Culture-activated rat HSCs were exposed to hydrogen peroxide. Necrosis and apoptosis were determined by Sytox Green or acridine orange staining, respectively. The hydrogen peroxide detoxifying enzymes catalase and glutathione-peroxidase (GPx) were inhibited using 3-amino-1,2,4-triazole and mercaptosuccinic acid, respectively. The anti-oxidant glutathione was depleted by L-buthionine-sulfoximine and repleted with the GSH-analogue GSH-monoethylester (GSH-MEE). RESULTS Upon activation, HSCs increase their cellular glutathione content and GPx expression, while MnSOD (both at mRNA and protein level) and catalase (at the protein level, but not at the mRNA level) decreased. Hydrogen peroxide did not induce cell death in activated HSCs. Glutathione depletion increased the sensitivity of HSCs to hydrogen peroxide, resulting in 35% and 75% necrotic cells at 0.2 and 1mmol/L hydrogen peroxide, respectively. The sensitizing effect was abolished by GSH-MEE. Inhibition of catalase or GPx significantly increased hydrogen peroxide-induced apoptosis, which was not reversed by GSH-MEE. CONCLUSION Activated HSCs have increased ROS-detoxifying capacity compared to quiescent HSCs. Glutathione levels increase during HSC activation and protect against ROS-induced necrosis, whereas hydrogen peroxide-detoxifying enzymes protect against apoptotic cell death.
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Affiliation(s)
- Sandra Dunning
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
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Karimian G, Buist-Homan M, Mikus B, Henning RH, Faber KN, Moshage H. Angiotensin II protects primary rat hepatocytes against bile salt-induced apoptosis. PLoS One 2012; 7:e52647. [PMID: 23300732 PMCID: PMC3530435 DOI: 10.1371/journal.pone.0052647] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2012] [Accepted: 11/19/2012] [Indexed: 02/06/2023] Open
Abstract
Angiotensin II (AT-II) is a pro-fibrotic compound that acts via membrane-bound receptors (AT-1R/AT-2R) and thereby activates hepatic stellate cells (HSCs). AT-II receptor blockers (ARBs) are thus important candidates in the treatment of liver fibrosis. However, multiple case reports suggest that AT-1R blockers may induce hepatocyte injury. Therefore, we investigated the effect of AT-II and its receptor blockers on cytokine-, oxidative stress- and bile salt-induced cell death in hepatocytes. Primary rat hepatocytes were exposed to TNF-α/Actinomycin D, the ROS-generating agent menadione or the bile salts: glycochenodeoxycholic acid (GCDCA) and tauro-lithocholic acid-3 sulfate (TLCS), to induce apoptosis. AT-II (100 nmol/L) was added 10 minutes prior to the cell death-inducing agent. AT-1R antagonists (Sartans) and the AT-2R antagonist PD123319 were used at 1 µmol/L. Apoptosis (caspase-3 activity, acridine orange staining) and necrosis (Sytox green staining) were quantified. Expression of CHOP (marker for ER stress) and AT-II receptor mRNAs were quantified by Q-PCR. AT-II dose-dependently reduced GCDCA-induced apoptosis of hepatocytes (−50%, p<0.05) without inducing necrosis. In addition, AT-II reduced TLCS-induced apoptosis of hepatocytes (−50%, p<0.05). However, AT-II did not suppress TNF/Act-D and menadione-induced apoptosis. Only the AT-1R antagonists abolished the protective effect of AT-II against GCDCA-induced apoptosis. AT-II increased phosphorylation of ERK and a significant reversal of the protective effect of AT-II was observed when signaling kinases, including ERK, were inhibited. Moreover, AT-II prevented the GCDCA-induced expression of CHOP (the marker of the ER-mediated apoptosis).
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Affiliation(s)
- Golnar Karimian
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
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Penumathsa SV, Kode A, Rajagopalan R, Menon VP. Changes in Activities of MMP in Alcohol and Thermally Oxidized Sunflower Oil-Induced Liver Damage: NAC Antioxidant Therapy. Toxicol Mech Methods 2012; 16:267-74. [PMID: 20021024 DOI: 10.1080/15376520500194734] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/24/2022]
Abstract
Liver fibrosis is the result of imbalance between extracellular matrix (ECM) synthesis and breakdown. Ethanol-induced increase in redox state is a sign of major change in hepatic metabolism and this inhibits tricarboxylic acid cycle activity and, fatty acid oxidation and increases fatty acid uptake, thus predisposing fatty liver. Fibrotic changes induced by alcohol are provoked by diets rich in PUFA. Heating of oils rich in PUFA produces toxic volatile and nonvolatile compounds, which aggravate liver damage. Hepatotoxicity was induced in male Wistar rats by administering alcohol (20%) and thermally oxidized sunflower oil (Delta PUFA) (15%). When N-acetyl cyteine (NAC) (150 mg/kg body weight), an ROS scavenger, was administered, there was a reversal of liver damage, which was demonstrated biochemically. Matrix metalloproteinases (MMPs), being potential biochemical indicators of fibroproliferation, were estimated in the present study, which were found to be altered in alcohol, Delta PUFA, and alcohol + Delta PUFA. The altered activities of MMPs in these groups were effectively modulated by treatment with NAC. Thus, in this study, NAC was found to modulate the effect of alcohol and Delta PUFA-induced liver damage.
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Affiliation(s)
- Suresh Varma Penumathsa
- Department of Biochemistry, Faculty of Science, Annamalai University, Annamalainagar608 002, India
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Abstract
Over the last three decades, direct hepatotoxic effects of ethanol were established, some of which were linked to redox changes produced by NADH generated via the alcohol dehydrogenase (ADH) pathway and shown to affect the metabolism of lipids, carbohydrates, proteins, and purines. It was also determined that ethanol can be oxidized by a microsomal ethanol oxidizing system (MEOS) involving a specific cytochrome P-450; this newly discovered ethanol-inducible cytochrome P-450 (P-450 IIEi) contributes to ethanol metabolism, tolerance, energy wastage (with associated weight loss), and the selective hepatic perivenular toxicity of various xenobiotics. Their activation by P-450IIEi now provides an understanding of the increased susceptibility of the heavy drinker to the toxicity of industrial solvents, anaesthetic agents, commonly prescribed drugs, over-the-counter analgesics, and chemical carcinogens. P-450 induction also explains depletion (and toxicity) of nutritional factors such as vitamin A. As a consequence, treatment with vitamin A and other nutritional factors is beneficial, but must take into account a narrowed therapeutic window in alcoholics who have increased needs for nutrients and also display an enhanced susceptibility to some of their adverse effects. Acetaldehyde (the metabolite produced from ethanol by either ADH or MEOS) impairs hepatic oxygen utilization and forms protein adducts, resulting in antibody production, enzyme inactivation, and decreased DNA repair. It also stimulates collagen production by the vitamin A storing cells (lipocytes) and myofibroblasts, and causes glutathione depletion. Supplementation with S-adenosyl-L-methionine partly corrects the depletion and associated mitochondrial injury, whereas administration of polyunsaturated lecithin opposes the fibrosis. Thus, at the cellular level, the classic dichotomy between the nutritional and toxic effects of ethanol has now been bridged. The understanding of how the ensuing injury eventually results in irreversible scarring or cirrhosis may provide us with improved modalities for treatment and prevention.
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Affiliation(s)
- C S Lieber
- Alcohol Research & Treatment Center, Bronx VA Medical Center, 130 West Kingsbridge Road, Bronx, NY, 10468, USA
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Karimian G, Buist-Homan M, Faber KN, Moshage H. Pertussis toxin, an inhibitor of G(αi) PCR, inhibits bile acid- and cytokine-induced apoptosis in primary rat hepatocytes. PLoS One 2012; 7:e43156. [PMID: 22900098 PMCID: PMC3416748 DOI: 10.1371/journal.pone.0043156] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2012] [Accepted: 07/17/2012] [Indexed: 01/12/2023] Open
Abstract
Excessive hepatocyte apoptosis is a common event in acute and chronic liver diseases leading to loss of functional liver tissue. Approaches to prevent apoptosis have therefore high potential for the treatment of liver disease. G-protein coupled receptors (GPCR) play crucial roles in cell fate (proliferation, cell death) and act through heterotrimeric G-proteins. GαiPCRs have been shown to regulate lipoapoptosis in hepatocytes, but their role in inflammation- or bile acid-induced apoptosis is unknown. Here, we analyzed the effect of inhibiting GαiPCR function, using pertussis toxin (PT), on bile acid- and cytokine-induced apoptosis in hepatocytes. Primary rat hepatocytes, HepG2-rNtcp cells (human hepatocellular carcinoma cells) or H-4-II-E cells (rat hepatoma cells) were exposed to glycochenodeoxycholic acid (GCDCA) or tumor necrosis factor-α (TNFα)/actinomycin D (ActD). PT (50–200 nmol/L) was added 30 minutes prior to the apoptotic stimulus. Apoptosis (caspase-3 activity, acridine orange staining) and necrosis (sytox green staining) were assessed. PT significantly reduced GCDCA- and TNFα/ActD-induced apoptosis in rat hepatocytes (−60%, p<0.05) in a dose-dependent manner (with no shift to necrosis), but not in HepG2-rNtcp cells or rat H-4-II-E cells. The protective effect of pertussis toxin was independent of the activation of selected cell survival signal transduction pathways, including ERK, p38 MAPK, PI3K and PKC pathways, as specific protein kinase inhibitors did not reverse the protective effects of pertussis toxin in GCDCA-exposed hepatocytes. Conclusion: Pertussis toxin, an inhibitor of GαiPCRs, protects hepatocytes, but not hepatocellular carcinoma cells, against bile acid- and cytokine-induced apoptosis and has therapeutic potential as primary hepatoprotective drug, as well as adjuvant in anti-cancer therapy.
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Affiliation(s)
- Golnar Karimian
- Department of Gastroenterology and Hepatology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
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Crawford JM, Burt AD. Anatomy, pathophysiology and basic mechanisms of disease. MACSWEEN'S PATHOLOGY OF THE LIVER 2012:1-77. [DOI: 10.1016/b978-0-7020-3398-8.00001-5] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/06/2025]
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Comprehensive analysis of PPARalpha-dependent regulation of hepatic lipid metabolism by expression profiling. PPAR Res 2011; 2007:26839. [PMID: 18288265 PMCID: PMC2233741 DOI: 10.1155/2007/26839] [Citation(s) in RCA: 164] [Impact Index Per Article: 11.7] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2007] [Accepted: 07/25/2007] [Indexed: 01/30/2023] Open
Abstract
PPARα is a ligand-activated transcription factor involved in the regulation of nutrient metabolism and inflammation. Although much is already known about the function of PPARα in hepatic lipid metabolism, many PPARα-dependent pathways and genes have yet to be discovered. In order to obtain an overview of PPARα-regulated genes relevant to lipid metabolism, and to probe for novel candidate PPARα target genes, livers from several animal studies in which PPARα was activated and/or disabled were analyzed by Affymetrix GeneChips. Numerous novel PPARα-regulated genes relevant to lipid metabolism were identified. Out of this set of genes, eight genes were singled out for study of PPARα-dependent regulation in mouse liver and in mouse, rat, and human primary hepatocytes, including thioredoxin interacting protein (Txnip), electron-transferring-flavoprotein β polypeptide (Etfb), electron-transferring-flavoprotein dehydrogenase (Etfdh), phosphatidylcholine transfer protein (Pctp), endothelial lipase (EL, Lipg), adipose triglyceride lipase (Pnpla2), hormone-sensitive lipase (HSL, Lipe), and monoglyceride lipase (Mgll). Using an in silico screening approach, one or more PPAR response elements (PPREs) were identified in each of these genes. Regulation of Pnpla2, Lipe, and Mgll, which are involved in triglyceride hydrolysis, was studied under conditions of elevated hepatic lipids. In wild-type mice fed a high fat diet, the decrease in hepatic lipids following treatment with the PPARα agonist Wy14643 was paralleled by significant up-regulation of Pnpla2, Lipe, and Mgll, suggesting that induction of triglyceride hydrolysis may contribute to the anti-steatotic role of PPARα. Our study illustrates the power of transcriptional profiling to uncover novel PPARα-regulated genes and pathways in liver.
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Fujita K, Nozaki Y, Yoneda M, Wada K, Takahashi H, Kirikoshi H, Inamori M, Saito S, Iwasaki T, Terauchi Y, Maeyama S, Nakajima A. Nitric oxide plays a crucial role in the development/progression of nonalcoholic steatohepatitis in the choline-deficient, l-amino acid-defined diet-fed rat model. Alcohol Clin Exp Res 2009; 34 Suppl 1:S18-24. [PMID: 18986378 DOI: 10.1111/j.1530-0277.2008.00756.x] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
Abstract
BACKGROUND The pathogenesis of nonalcoholic steatohepatitis (NASH) is still unclear. Recently, the 2-hit hypothesis was proposed, in which nitric oxide production, representing oxidative stress, was proposed as a very important candidate for the second hit. METHODS The total study period was 10 weeks. A total of 20 rats were randomly divided into 2 groups. Group 1 was administered the Choline-Deficient, l-Amino Acid-Defined diet to produce a NASH model, and Group 2 as control received the Choline-Sufficient, l-Amino Acid-defined diet. The blood and tissue concentrations of nitrate + nitrite were measured using the Griess reagent and the expression levels of inducible nitric oxide synthase (iNOS) proteins and mRNA was determined by Western blotting. RESULTS In regard to nitric oxide (NO) and NO metabolites, there were significant differences in the blood (especially portal venous blood) as well as tissue (liver and visceral fat) concentrations between the 2 animal groups; the amounts of NO metabolites in the tissues were much higher in the NASH models. The level of nitrotyrosine was much markedly higher in the NASH models than in the controls. In regard to the tissue expression of iNOS a significant difference between the 2 groups was found in the visceral fat, especially in the mesenterium. CONCLUSIONS Based on these results, we hypothesize that the iNOS expression and NO levels in the visceral fat increase, with increased diffusion of NO and its metabolites into the liver, resulting in increased nitrotyrosine formation in the liver; this, in turn, induces inflammation, apoptosis, and fibrosis in the liver, which are one of the characteristic features of NASH.
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Affiliation(s)
- Koji Fujita
- From the Division of Gastroenterology, Yokohama City University Graduate School of Medicine, Yokohama, Japan
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Dunning S, Hannivoort RA, de Boer JF, Buist-Homan M, Faber KN, Moshage H. Superoxide anions and hydrogen peroxide inhibit proliferation of activated rat stellate cells and induce different modes of cell death. Liver Int 2009; 29:922-32. [PMID: 19386027 DOI: 10.1111/j.1478-3231.2009.02004.x] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/13/2023]
Abstract
BACKGROUND In chronic liver injury, hepatic stellate cells (HSCs) proliferate and produce excessive amounts of connective tissue causing liver fibrosis and cirrhosis. Oxidative stress has been implicated as a driving force of HSC activation and proliferation, although contradictory results have been described. AIM To determine the effects of oxidative stress on activated HSC proliferation, survival and signalling pathways. METHODS Serum-starved culture-activated rat HSCs were exposed to the superoxide anion donor menadione (5-25 micromol/L) or hydrogen peroxide (0.2-5 mmol/L). Haem oxygenase-1 mRNA expression, glutathione status, cell death, phosphorylation of mitogen-activated protein (MAP) kinases and proliferation were investigated. RESULTS Menadione induced apoptosis in a dose- and time-dependent, but caspase-independent manner. Hydrogen peroxide induced necrosis only at extremely high concentrations. Both menadione and hydrogen peroxide activated Jun N-terminal kinase (JNK) and p38. Hydrogen peroxide also activated extracellular signal-regulated protein. Menadione, but not hydrogen peroxide, reduced cellular glutathione levels. Inhibition of JNK or supplementation of glutathione reduced menadione-induced apoptosis. Non-toxic concentrations of menadione or hydrogen peroxide inhibited platelet-derived growth factor- or/and serum-induced proliferation. CONCLUSION Reactive oxygen species (ROS) inhibit HSC proliferation and promote HSC cell death in vitro. Different ROS induce different modes of cell death. Superoxide anion-induced HSC apoptosis is dependent on JNK activation and glutathione status.
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Affiliation(s)
- Sandra Dunning
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
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Abstract
Uncontrolled production of collagen I is the main feature of liver fibrosis. Following a fibrogenic stimulus such as alcohol, hepatic stellate cells (HSC) transform into an activated collagen-producing cell. In alcoholic liver disease, numerous changes in gene expression are associated with HSC activation, including the induction of several intracellular signaling cascades, which help maintain the activated phenotype and control the fibrogenic and proliferative state of the cell. Detailed analyses for understanding the molecular basis of the collagen I gene regulation have revealed a complex process involving reactive oxygen species (ROS) as key mediators. Less is known, however, about the contribution of reactive nitrogen species (RNS). In addition, a series of cytokines, growth factors, and chemokines, which activate extracellular matrix (ECM)-producing cells through paracrine and autocrine loops, contribute to the fibrogenic response.
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Affiliation(s)
- R. Urtasun
- Mount Sinai School of Medicine, Box 1123, Department of Medicine/Division of Liver Diseases, 1425 Madison Avenue, Room 11-76, New York, NY 10029, USA
| | - L. Conde de la Rosa
- Mount Sinai School of Medicine, Box 1123, Department of Medicine/Division of Liver Diseases, 1425 Madison Avenue, Room 11-76, New York, NY 10029, USA
| | - N. Nieto
- Mount Sinai School of Medicine, Box 1123, Department of Medicine/Division of Liver Diseases, 1425 Madison Avenue, Room 11-76, New York, NY 10029, USA
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Hannivoort RA, Dunning S, Vander Borght S, Schroyen B, Woudenberg J, Oakley F, Buist-Homan M, van den Heuvel FAJ, Geuken M, Geerts A, Roskams T, Faber KN, Moshage H. Multidrug resistance-associated proteins are crucial for the viability of activated rat hepatic stellate cells. Hepatology 2008; 48:624-34. [PMID: 18627004 DOI: 10.1002/hep.22346] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
UNLABELLED Hepatic stellate cells (HSCs) survive and proliferate in the chronically injured liver. ATP-binding cassette (ABC) transporters play a crucial role in cell viability by transporting toxic metabolites or xenobiotics out of the cell. ABC transporter expression in HSCs and its relevance to cell viability and/or activation have not been reported so far. The aim of this study was to investigate the expression, regulation, and function of multidrug resistance-associated protein (Mrp)-type and multidrug resistance protein (Mdr)-type ABC transporters in activated rat HSCs. Rat HSCs were exposed to cytokines or oxidative stress. ABC transporter expression was determined by quantitative polymerase chain reaction and immunohistochemistry. HSCs were exposed to the Mdr inhibitors verapamil and PSC-833 and the Mrp inhibitor MK571. Mdr and Mrp transporter function was evaluated with flow cytometry. Apoptosis was determined by activated caspase-3 and acridine orange staining, and necrosis was determined by Sytox green nuclear staining. An in vivo model of carbon tetrachloride (CCl(4))-induced liver fibrosis was used. With respect to hepatocytes, activated HSCs expressed high levels of Mrp1 and comparable levels of Mrp3, Mrp4, Mdr1a, and Mdr1b but not the hepatocyte-specific transporters bile salt export pump, Mrp2, and Mrp6. Mrp1 protein staining correlated with desmin staining in livers from CCl(4)-treated rats. Mrp1 expression increased upon activation of HSCs. Cytokines induced Mdr1b expression only. Oxidative stress was not a major regulator of Mdr and Mrp transporter expression. Activated HSCs became necrotic when exposed to the Mrp inhibitors. CONCLUSION Activated HSCs contain relatively high levels of Mrp1. Mrp-type transporters are required for the viability of activated HSCs. Mrp-dependent export of endogenous metabolites is important for the survival of activated HSCs in chronic liver diseases.
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Affiliation(s)
- Rebekka A Hannivoort
- Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands.
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Conde de la Rosa L, Vrenken TE, Hannivoort RA, Buist-Homan M, Havinga R, Slebos DJ, Kauffman HF, Faber KN, Jansen PLM, Moshage H. Carbon monoxide blocks oxidative stress-induced hepatocyte apoptosis via inhibition of the p54 JNK isoform. Free Radic Biol Med 2008; 44:1323-33. [PMID: 18206660 DOI: 10.1016/j.freeradbiomed.2007.12.011] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/27/2006] [Revised: 12/01/2007] [Accepted: 12/11/2007] [Indexed: 12/31/2022]
Abstract
Most chronic liver diseases are accompanied by oxidative stress, which may induce apoptosis in hepatocytes and liver injury. Oxidative stress induces heme oxygenase-1 (HO-1) expression. This stress-responsive cytoprotective protein is responsible for heme degradation into carbon monoxide (CO), free iron, and biliverdin. CO is an important intracellular messenger; however, the exact mechanisms responsible for its cytoprotective effect are not yet elucidated. Thus, we investigated whether HO-1 and CO protect primary hepatocytes against oxidative-stress-induced apoptosis. In vivo, bile duct ligation was used as model of chronic liver disease. In vitro, primary hepatocytes were exposed to the superoxide anion donor menadione in a normal and in a CO-- containing atmosphere. Apoptosis was determined by measuring caspase-9, -6, -3 activity and poly(ADP-ribose) polymerase cleavage, and necrosis was determined by Sytox green staining. The results showed that (1) HO-1 is induced in chronic cholestatic liver disease, (2) superoxide anions time- and dose-dependently induce HO-1 activity, (3) HO-1 overexpression inhibits superoxide-anions-induced apoptosis, and (4) CO blocks superoxide-anions-induced JNK phosphorylation and caspase-9, -6, -3 activation and abolishes apoptosis but does not increase necrosis. We conclude that HO-1 and CO protect primary hepatocytes against superoxide-anions-induced apoptosis partially via inhibition of JNK activity. CO could represent an important candidate for the treatment of liver diseases.
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Affiliation(s)
- Laura Conde de la Rosa
- Center for Liver, Digestive and Metabolic Diseases, University Medical Center Groningen, 9700 RB Groningen, The Netherlands.
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Okada Y, Tsuzuki Y, Hokari R, Miyazaki J, Matsuzaki K, Mataki N, Komoto S, Watanabe C, Kawaguchi A, Nagao S, Itoh K, Miura S. Pressure loading and ethanol exposure differentially modulate rat hepatic stellate cell activation. J Cell Physiol 2008; 215:472-80. [PMID: 18064666 DOI: 10.1002/jcp.21329] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
Ethanol may cause an increase in sinusoidal pressure accompanied by portal hypertension. Hepatic stellate cells (HSCs) located in hepatic sinusoids may therefore be frequently exposed to dual stimulations of mechanical pressure and ethanol exposure in alcoholic liver injury. In this study, the effects of pressure loading and ethanol exposure on activation of rat cultured HSCs were investigated using an in vitro pressure-inducing apparatus. HSCs were cultured in media containing ethanol (0-100 mM) under different pressures (1-40 mmHg). Morphological changes and migration index were determined. We also determined the expression levels of alpha-smooth muscle actin (alpha-SMA) and mitogen-activated protein kinases (MAPKs) by Western blot analysis and the level of collagen IV and transforming growth factor beta1 (TGF-beta1) by ELISA. Pressure loading alone induced up-regulation of alpha-SMA via the extracellular signal-regulated kinases 1 and 2 (ERK1/2) and c-jun N-terminal kinase (JNK) signaling pathways, prolonged extension of marginal length, and increased production of collagen IV. In contrast, ethanol exposure alone increased only extension of marginal length and cell migration. Dual stimulations of pressure loading and ethanol exposure enhanced the production of TGF-beta1 and migration index. The TGF-beta1-dependent p38 MAPK pathway may operate for production of extracellular matrix (ECM) or enhanced migration in the case of dual stimulations. In conclusion, static pressure loading is an important factor directly accelerating the activation of HSCs. Although increased sinusoidal pressure and ethanol exposure might differentially modulate HSC activation, both stimuli are involved in an additive manner in some situations.
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Affiliation(s)
- Yoshikiyo Okada
- Internal Medicine, National Defense Medical College, Saitama, Japan
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Lieber CS, DeCarli LM, Leo MA, Mak KM, Ponomarenko A, Ren C, Wang X. Beneficial effects versus toxicity of medium-chain triacylglycerols in rats with NASH. J Hepatol 2008; 48:318-26. [PMID: 18093684 DOI: 10.1016/j.jhep.2007.09.016] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/29/2007] [Revised: 09/04/2007] [Accepted: 09/13/2007] [Indexed: 12/04/2022]
Abstract
BACKGROUND/AIMS Replacing long-chain triacylglycerols (LCT) with medium-chain triacylglycerols (MCT) reduces alcohol-induced liver injury. Because of the similarity of the pathogenesis of alcohol-induced liver damage and non-alcoholic steatohepatitis (NASH), our aim was to assess whether MCT is also beneficial in NASH. METHODS We used a rat NASH model in which corn oil (35% of total calories) was isocalorically replaced with MCT. RESULTS Partial replacement of LCT did not ameliorate hepatic fat accumulation, 4-hydroxynonenal, collagen type I and its mRNA but it increased TNF-alpha and its mRNA (p<0.001). However, in rats given the high-fat diet restricted to 2/3 of the amount they were consuming, these adverse effects decreased, with and without MCT including less liver steatosis and lower triacylglycerols, but without beneficial effects of MCT. When 70% of the fat calories were replaced with MCT with no LCT remaining in the diet, no steatosis developed and hepatic TNF-alpha was low. When all MCT were given with carbohydrates (instead of LCT) hepatic TNF-alpha also decreased (p<0.001). CONCLUSIONS MCT are not hepatotoxic, provided the diet contains no significant amount of LCT. Total replacement of dietary LCT with MCT fed ad libitum is beneficial whereas partial replacement becomes hepatotoxic, unless the dietary intake is restricted.
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Affiliation(s)
- Charles S Lieber
- Alcohol Research Center, James J. Peters VA Medical Center, 130 West Kingsbridge Road (151-2), Bronx, NY 10468, USA.
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Schoemaker MH, Rots MG, Beljaars L, Ypma AY, Jansen PLM, Poelstra K, Moshage H, Haisma HJ. PDGF-receptor beta-targeted adenovirus redirects gene transfer from hepatocytes to activated stellate cells. Mol Pharm 2008; 5:399-406. [PMID: 18217712 DOI: 10.1021/mp700118p] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Chronic liver damage may lead to liver fibrosis. In this process, hepatic activated stellate cells are the key players. Thus, activated stellate cells are attractive targets for antifibrotic gene therapy. Recombinant adenovirus is a promising vehicle for delivering therapeutic genes to liver cells. However, this vector has considerable tropism for hepatocytes and Kupffer cells. The aim of this study is therefore to retarget the adenovirus to the activated stellate cells while reducing its affinity for hepatocytes. We constructed a fusion protein with affinity for both the adenovirus and the platelet derived growth factor-receptor beta (PDGF-Rbeta). In contrast to other cells, the PDFG-Rbeta is highly expressed on activated stellate cells. The targeting moiety, the PDGF peptide CSRNLIDC, was cloned in front of the single-chain antibody fragment (S11) directed against the adenoviral knob. This fusion protein enhanced adenoviral gene transfer in both 3T3 fibroblasts and primary isolated activated rat stellate cells by 10-60-fold. A fusion protein with a scrambled PDGF peptide (CIDNLSRC) did not accomplish this effect. Importantly, the PDGF-Rbeta-retargeted adenovirus showed a 25-fold reduced tropism for primary rat hepatocytes. Our novel approach demonstrates that therapeutic genes can be selectively directed to stellate cells. This opens new possibilities for the treatment of liver fibrosis.
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Affiliation(s)
- Marieke H Schoemaker
- Department of Gastroenterology and Hepatology, University of Groningen and University Medical Center Groningen, Groningen, The Netherlands
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