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Ma R, Gong L, Dong C, Utsumi T, Qi J, Zhuang ZW, Zhang X, Yang Y, McConnell MJ, Huang HC, Iwakiri Y. Hepatic Arterial Flow-Induced Portal Tract Fibrosis in Portal Hypertension: The Role of VCAM-1 and Osteopontin-Expressing Macrophages. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.01.31.634947. [PMID: 39975181 PMCID: PMC11838461 DOI: 10.1101/2025.01.31.634947] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/21/2025]
Abstract
Background The liver undergoes significant hemodynamic changes during surgery, transplantation, or cirrhosis with portal hypertension(PH). The hepatic artery buffer response(HABR), which compensates for reduced portal venous flow by increasing hepatic artery(HA) flow, is hypothesized to induce pathological portal tract remodeling. This study investigates the molecular mechanisms underlying this process. Methods PH was induced in Sprague-Dawley rats via partial portal vein ligation(PPVL). Structural evaluation(microCT), immune cell profiling, hemodynamic measurements, and transcriptomic analysis in macrophages(Mϕ) from sham or PPVL rats were conducted. Results MicroCT revealed decreased portal vein flow and increased HA flow correlated with portal pressure(r=0.799, p<0.01). A 2-fold increase in portal tract fibrosis(p<0.001) was observed with increased α-SMA+ myofibroblasts in PPVL rats. CD68+ Mϕ peaked at 10 days post-PPVL, and their depletion significantly reduced fibrosis(p<0.001), indicating critical roles of Mϕ in portal tract remodeling. VCAM-1 was elevated in HA endothelium and portal fibroblasts (PFs); VCAM-1 neutralization reduced collagen accumulation(p<0.05), CD68+ Mϕ(46.3%, p<0.01), and CD3+ T cells(18%, p<0.05). Mϕ-conditioned medium increased VCAM-1 in PFs(8-fold, p<0.001) and enhanced PF migration, while VCAM-1 knockdown reduced this effect (p<0.01). Single-cell RNA sequencing data(GSE171904) and RNA-FISH revealed increased interactions between osteopontin (Spp1)+ Mϕ and PFs, with Spp1+ Mϕ driving fibrosis. Spp1 knockdown in Mϕ co-culture reduced PF fibrogenic markers, while recombinant Spp1 upregulated Col1a1, Fn1, and Acta2 expression in PFs. Conclusion Increased VCAM-1 in arterial endothelial cells and PFs facilitates the recruitment of Spp1+ Mϕ, which drive HA flow-mediated vascular remodeling and portal tract fibrosis. These findings highlight arterial flow-induced fibrosis as a key mechanism in PH, potentially contributing to disease progression and decompensation. Synopsis Liver hemodynamic changes in portal hypertension drive extracellular matrix accumulation and portal tract remodeling via Spp1+ macrophages. This study highlights how altered blood flow induces fibrosis, and its potential role in decompensation, and identifies therapeutic targets for advanced liver disease.
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Urushima H, Yuasa H, Matsubara T, Kuroda N, Hara Y, Inoue K, Wake K, Sato T, Friedman SL, Ikeda K. Activation of Hepatic Stellate Cells Requires Dissociation of E-Cadherin-Containing Adherens Junctions with Hepatocytes. THE AMERICAN JOURNAL OF PATHOLOGY 2020; 191:438-453. [PMID: 33345995 DOI: 10.1016/j.ajpath.2020.12.007] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/22/2020] [Revised: 12/02/2020] [Accepted: 12/03/2020] [Indexed: 12/12/2022]
Abstract
Hepatic stellate cells (HSCs) are resident mesenchymal cells in the space of Disse interposed between liver sinusoidal endothelial cells and hepatocytes. Thorn-like microprojections, or spines, project out from the cell surface of HSCs, crossing the space of Disse, to establish adherens junctions with neighboring hepatocytes. Although HSC activation is initiated largely from stimulation by adjacent cells, isolated HSCs also activate spontaneously in primary culture on plastic. Therefore, other unknown HSC-initiating factors apart from paracrine stimuli may promote activation. The dissociation of adherens junctions between HSCs and hepatocytes as an activating signal for HSCs was explored, establishing epithelial cadherin (E-cadherin) as an adhesion molecule linking hepatocytes and HSCs. In vivo, following carbon tetrachloride-induced liver injury, HSCs lost their spines and dissociated from adherens junctions in the early stages of injury, and were subsequently activated along with an increase in YAP/TAZ expression. After abrogation of liver injury, HSCs reconstructed their spines and adherens junctions. In vitro, reconstitution of E-cadherin-containing adherens junctions by forced E-cadherin expression quiesced HSCs and suppressed TAZ expression. Additionally, increase of TAZ expression leading to the activation of HSCs by autocrine stimulation of transforming growth factor-β, was revealed as a mechanism of spontaneous activation. Thus, we have uncovered a critical event required for HSC activation through enhanced TAZ-mediated mechanotransduction after the loss of adherens junctions between HSCs and hepatocytes.
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Affiliation(s)
- Hayato Urushima
- Department of Anatomy and Regenerative Biology, Graduate School of Medicine, Osaka City University, Osaka, Japan.
| | - Hideto Yuasa
- Department of Anatomy and Regenerative Biology, Graduate School of Medicine, Osaka City University, Osaka, Japan
| | - Tsutomu Matsubara
- Department of Anatomy and Regenerative Biology, Graduate School of Medicine, Osaka City University, Osaka, Japan
| | - Noriyuki Kuroda
- Department of Anatomy, Tissue and Cell Biology, School of Dental Medicine, Tsurumi University, Yokohama, Japan
| | - Yaiko Hara
- Department of Anatomy, Tissue and Cell Biology, School of Dental Medicine, Tsurumi University, Yokohama, Japan
| | - Kouji Inoue
- Research Center of Electron Microscopy, School of Dental Medicine, Tsurumi University, Yokohama, Japan
| | - Kenjiro Wake
- Department of Anatomy and Regenerative Biology, Graduate School of Medicine, Osaka City University, Osaka, Japan; Research Center of Electron Microscopy, School of Dental Medicine, Tsurumi University, Yokohama, Japan; Liver Research Unit, Minophagen Pharmaceutical Co., Ltd., Tokyo, Japan
| | - Tetsuji Sato
- Department of Anatomy, Tissue and Cell Biology, School of Dental Medicine, Tsurumi University, Yokohama, Japan
| | - Scott L Friedman
- Division of Liver Diseases, Icahn School of Medicine at Mount Sinai, New York, New York
| | - Kazuo Ikeda
- Department of Anatomy and Regenerative Biology, Graduate School of Medicine, Osaka City University, Osaka, Japan.
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Tian X, Zhao H, Guo Z. WITHDRAWN: Effects of carvedilol on expression of TLR4 and its downstream signaling pathway in liver tissue of rats with cholestatic liver fibrosisjaundice. REVISTA ESPANOLA DE ENFERMEDADES DIGESTIVAS 2020. [PMID: 33200614 DOI: 10.17235/reed.2020.6075/2018] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Ahead of Print article withdrawn by publisher. OBJECTIVES This study was designed to investigate the effects of carvedilol on the expression of TLR4 and its downstream signaling pathway in liver tissue of rats with cholestatic liver fibrosis, and provided experimental evidence for clinical treatment of liver fibrosis with carvedilol.? METHODS A total of fifty male Sprague Dawley rats were randomly divided into five groups (10 rats per group): sham surgery control group, bile duct ligation (BDL) model group, low-dose carvedilol treatment group (0.1mgkg-1d-1), medium-dose carvedilol treatment group (1mgkg-1d-1), high-dose carvedilol treatment group (10mgkg-1d-1). Rat hepatic fibrosis model was established by applying BDL. Forty-eight hours after the operation, carvedilol was administered twice a day. The blood and liver were simultaneously collected under the aseptic condition for further detection in two weeks after operation.? RESULTS Compared with the sham group, the BDL group showed obvious liver injury, increased levels of inflammatory factors, and continued progression of liver fibrosis. Carvedilol could alleviate the above changes. The improvement effects were augmenting as dosages increasing. In addition, compared with the BDL group, carvedilol can reduce the expressions of TLR4, MyD88 and NF-?B p65 in liver tissue and increase the expression of ?-arrestin2, and the effect in the high dose group was more obvious. CONCLUSIONS Carvedilol can reduce the release of inflammatory mediators by down-regulating TLR4 expression and inhibiting its downstream signaling pathway, thus playing a therapeutic role in cholestatic liver fibrosis.
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Hassan SM, Taha AM, Eldahshan OA, Sayed AA, Salem AM. Modulatory effect of Prosopis juliflora leaves on hepatic fibrogenic and fibrolytic alterations induced in rats by thioacetamide. Biomed Pharmacother 2019; 115:108788. [PMID: 31035010 DOI: 10.1016/j.biopha.2019.108788] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2018] [Revised: 02/23/2019] [Accepted: 03/13/2019] [Indexed: 01/15/2023] Open
Abstract
This study investigated the antifibrotic effect of Prosopis juliflora leaves crude methanolic extract (PJEL) against thioacetamide (TAA)-induced liver fibrosis. The phytochemical analysis of PJEL was performed via HPLC/MS in association with evaluating its free radical scavenging and cytotoxic activities. The antifibrotic activity of PJEL was assessed by dividing Wistar rats into 8 groups: normal control, PJEL1-administered rats (2 mg/ Kg b.w.), PJEL2-administered rats (4 mg/ Kg b.w.), PJEL3-administered rats (8 mg/Kg b.w.), TAA-induced hepatic fibrosis, TTA + PJEL1, TAA + PJEL2, and TAA + PJEL3. Results indicated that PJEL crude methanolic extract is rich in polyphenolic compounds and alkaloids. PJEL exerted free radical scavenging activity with IC50 of 123.5 μg/mL and cytotoxic activity against a well-differentiated hepatocellular cell line (IC50 = 11.1 μg/mL). PJEL at a dose of 4 mg/Kg b.w. ameliorated serum ALT activity and improved serum albumin level and hepatic hydroxyproline content in association with a reduction in the fibrosis stage. PJEL elevated hepatic tumor necrosis factor-α and interleukin-6 contents with less necrosis grade. PJEL post-therapy ameliorated the relative expression of Bcl-2, Col1A1, Mmp-9, and Mmp-2 genes in liver. CONCLUSION: PJEL possesses a good therapeutic activity against TAA-induced liver fibrosis via enhancing extracellular matrix removal and stimulating hepatic regeneration to decrease hepatic necrosis.
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Affiliation(s)
- Salah M Hassan
- Department of Biochemistry, Faculty of Science, Ain Shams University, Cairo, Egypt
| | - AlShaimaa M Taha
- Department of Biochemistry, Faculty of Science, Ain Shams University, Cairo, Egypt.
| | - Omayma A Eldahshan
- Department of Pharmacognosy, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt.
| | - Ahmed A Sayed
- Department of Biochemistry, Faculty of Science, Ain Shams University, Cairo, Egypt; Children's Cancer Hospital, 57357, Egypt
| | - Ahmed M Salem
- Department of Biochemistry, Faculty of Science, Ain Shams University, Cairo, Egypt
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Lv J, Zhang H, Wang L, Gao J, Fan Y. Effects of A94T and P84L Polymorphisms Within theTNF-αGene on Proliferation and Activation of Hepatic Stellate Cells. DNA Cell Biol 2019; 38:162-169. [PMID: 30526018 DOI: 10.1089/dna.2018.4452] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Affiliation(s)
- Jian Lv
- Department of Pharmacy, Renmin Hospital of Wuhan University, Wuhan, Hubei, P.R. China
| | - Hong Zhang
- Department of Pharmacy, Renmin Hospital of Wuhan University, Wuhan, Hubei, P.R. China
| | - Li Wang
- Department of Pharmacy, Renmin Hospital of Wuhan University, Wuhan, Hubei, P.R. China
| | - Jiefang Gao
- Department of Pharmacy, Renmin Hospital of Wuhan University, Wuhan, Hubei, P.R. China
| | - Yueying Fan
- Department of Pharmacy, Renmin Hospital of Wuhan University, Wuhan, Hubei, P.R. China
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Emerging role and therapeutic implication of Wnt signaling pathways in liver fibrosis. Gene 2018; 674:57-69. [PMID: 29944952 DOI: 10.1016/j.gene.2018.06.053] [Citation(s) in RCA: 51] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2018] [Revised: 06/14/2018] [Accepted: 06/16/2018] [Indexed: 02/08/2023]
Abstract
Activation of hepatic stellate cells (HSCs) is a pivotal cellular event in liver fibrosis. Therefore, improving our understanding of the molecular pathways that are involved in these processes is essential to generate new therapies for liver fibrosis. Greater knowledge of the role of the Wnt signaling pathway in liver fibrosis could improve understanding of the liver fibrosis pathogenesis. The aim of this review is to describe the present knowledge about the Wnt signaling pathway, which significantly participates in liver fibrosis and HSC activation, and look ahead on new perspectives of Wnt signaling pathway research. Moreover, we will discuss the different interactions with Wnt signaling pathway-regulated liver fibrosis. The Wnt signaling pathway modulates several important aspects of function, including cell proliferation, activation and differentiation. Targeting the Wnt signaling pathway can be a promising direction in liver fibrosis treatment. We discuss new perspectives of Wnt signaling pathway activation in liver fibrosis. For example, antagonist to Wnt and Wnt ligands could inhibit liver fibrosis by regulating Wnt/β-catenin signaling pathway. These findings identify the Wnt signaling pathway as a potentially important for therapeutic targets in liver fibrosis. Future studies are needed in order to find safer and more effective Wnt-based drugs.
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Vega-Magaña N, Delgado-Rizo V, García-Benavides L, Toro-Arreola SD, Segura-Ortega J, Morales ASMZ, Zepeda-Nuño JS, Escarra-Senmarti M, Gutiérrez-Franco J, Haramati J, Bueno-Topete MR. Bacterial Translocation Is Linked to Increased Intestinal IFN-γ, IL-4, IL-17, and mucin-2 in Cholestatic Rats. Ann Hepatol 2018. [DOI: 10.5604/01.3001.0010.8663] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 03/24/2023]
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Cheng C, Yu S, Kong R, Yuan Q, Ma Y, Yang W, Cao G, Xie L. CTRP3 attenuates hepatic stellate cell activation through transforming growth factor-β/Smad signaling pathway. Biomed Pharmacother 2017; 89:1387-1391. [DOI: 10.1016/j.biopha.2017.03.021] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2016] [Revised: 03/07/2017] [Accepted: 03/07/2017] [Indexed: 02/07/2023] Open
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Bader El Din NG, Farouk S, El-Shenawy R, Elhady MM, Ibrahim MK, Dawood RM, Salem AM, El Awady MK. The Synergistic Effect of TNFα -308 G/A and TGFβ1 -509 C/T Polymorphisms on Hepatic Fibrosis Progression in Hepatitis C Virus Genotype 4 Patients. Viral Immunol 2017; 30:127-135. [PMID: 28151059 DOI: 10.1089/vim.2016.0083] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Tumor necrosis factor-alpha (TNFα) and transforming growth factor-beta (TGFβ1) cytokines are highly implicated in liver fibrosis. Polymorphisms in these cytokines affect their expression, secretion, and activity. This study aimed to evaluate the influence of TNFα -308 G/A and TGFβ1 -509 C/T polymorphism on hepatic fibrosis progression in Egyptian patients with hepatitis C virus (HCV) genotype 4. Genotyping of TNFα -308 G/A and TGFβ1 -509 C/T was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis in 122 subjects (50 healthy controls and 72 HCV patients). Also, serum TNFα and TGFβ1 levels were detected by enzyme-linked immunosorbent assay (ELISA). The genotyping results of early (F0-F1, n = 36) and late (F2-F4, n = 36) HCV fibrosis patients showed that late fibrosis patients had higher TNFα -308 AA genotype and TGFβ1 -509 TT genotype than early fibrosis patients (p = 0.016, 0.028, respectively). Moreover, the TNFα and TGFβ1 serum levels were significantly higher in HCV patients with TNFα A containing genotypes (GA+AA) (p = 0.004) and patients with TGFβ1 T containing genotypes (CT+TT) (p = 0.001), respectively. The combined unfavorable TNFα (GA/AA) and TGFβ1 (CT/TT) genotypes were highly associated with abnormal liver function parameters and were significantly higher in high activity (A2-A3) and late fibrosis (F2-F4) HCV patients (p = 0.023, 0.029). The multivariate analysis results confirmed that the combined TNFα-308 (AA) and TGFβ1 -509 (TT) unfavorable genotypes increased the risk of hepatic fibrosis progression by 6.4-fold than combined favorable genotypes (odds ratio: 6.417, 95% confidence interval [1.490-27.641], p = 0.013). In conclusion, both TNFα -308 G/A and TGFβ1 -509 C/T polymorphisms synergistically influence the hepatic fibrosis progression and can be used as potential biomarkers to predict hepatic disease progression in chronic hepatitis C patients.
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Affiliation(s)
- Noha G Bader El Din
- 1 Department of Microbial Biotechnology, National Research Centre , Giza, Egypt
| | - Sally Farouk
- 1 Department of Microbial Biotechnology, National Research Centre , Giza, Egypt
| | - Reem El-Shenawy
- 1 Department of Microbial Biotechnology, National Research Centre , Giza, Egypt
| | - Mostafa M Elhady
- 2 Department of Biochemistry, Faculty of Science, Ain Shams University , Cairo, Egypt
| | - Marwa K Ibrahim
- 1 Department of Microbial Biotechnology, National Research Centre , Giza, Egypt
| | - Reham M Dawood
- 1 Department of Microbial Biotechnology, National Research Centre , Giza, Egypt
| | - Ahmed M Salem
- 2 Department of Biochemistry, Faculty of Science, Ain Shams University , Cairo, Egypt
| | - Mostafa K El Awady
- 1 Department of Microbial Biotechnology, National Research Centre , Giza, Egypt
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Jayawardena UA, Ratnasooriya WD, Wickramasinghe DD, Udagama PV. Heavy metal mediated innate immune responses of the Indian green frog, Euphlyctis hexadactylus (Anura: Ranidae): Cellular profiles and associated Th1 skewed cytokine response. THE SCIENCE OF THE TOTAL ENVIRONMENT 2016; 566-567:1194-1204. [PMID: 27335164 DOI: 10.1016/j.scitotenv.2016.05.171] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/26/2016] [Revised: 05/23/2016] [Accepted: 05/24/2016] [Indexed: 06/06/2023]
Abstract
Immune cell and cytokine profiles in relation to metal exposure though much studied in mammals has not been adequately investigated in amphibians, due mainly to lack of suitable reagents for cytokine profiling in non-model species. However, interspecies cross reactivity of cytokines permitted us to assay levels of IFNγ, TNFα, IL6 and IL10in a common anuran, the Indian green frog (Euphlyctis hexadactylus), exposed to heavy metals (Cd, Cr, Cu, Zn and Pb, at ~5ppm each) under field and laboratory settings in Sri Lanka. Enumeration of immune cells in blood and melanomacrophages in the liver, assay of serum and hepatic cytokines, and Th1/Th2 cytokine polarisation were investigated. Immune cell counts indicated overall immunosuppression with decreasing total WBC and splenocyte counts while neutrophil/lymphocyte ratio increased with metal exposure, indicating metal mediated stress. Serum IL6 levels of metal exposed frogs reported the highest (~9360pg/mL) of all cytokines tested. Significantly elevated IFNγ production (P<0.05) was evident in heavy metal exposed frogs. Th1/Th2 cytokine ratio in both serum and liver tissue homogenates was Th1 skewed due to significantly higher production of pro-inflammatory cytokines, IFNγ in serum and TNFα in the liver (P<0.01).Metal mediated aggregations of melanomacrophages in the liver were positively and significantly (P<0.05) correlated with the hepatic expression of TNFα, IL6 and IL10 activity. Overall, Th1 skewed response may well be due to oxidative stress mediated nuclear factor κ-light chain enhancer of activated B cells (NFκB) which enhances the transcription of pro-inflammatory cytokines. Xenobiotic stress has recently imposed an unprecedented level of threat to wildlife, particularly to sensitive species such as amphibians. Therefore, understanding the interactions between physiological stress and related immune responses is fundamental to conserve these environmental sentinels in the face of emerging eco-challenges.
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Affiliation(s)
| | | | | | - Preethi V Udagama
- Department of Zoology, University of Colombo, Colombo 03, Sri Lanka.
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Cogliati B, Crespo Yanguas S, da Silva TC, Aloia TP, Nogueira MS, Real-Lima MA, Chaible LM, Sanches DS, Willebrords J, Maes M, Pereira IV, de Castro IA, Vinken M, Dagli ML. Connexin32 deficiency exacerbates carbon tetrachloride-induced hepatocellular injury and liver fibrosis in mice. Toxicol Mech Methods 2016; 26:362-370. [PMID: 27268753 PMCID: PMC5417356 DOI: 10.1080/15376516.2016.1190991] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
OBJECTIVE Liver fibrosis results from the perpetuation of the normal wound healing response to several types of injury. Despite the wealth of knowledge regarding the involvement of intracellular and extracellular signaling pathways in liver fibrogenesis, information about the role of intercellular communication mediated by gap junctions is scarce. METHODS In this study, liver fibrosis was chemically induced by carbon tetrachloride in mice lacking connexin32, the major liver gap junction constituent. The manifestation of liver fibrosis was evaluated based on a series of read-outs, including collagen morphometric and mRNA analysis, oxidative stress, apoptotic, proliferative and inflammatory markers. RESULTS More pronounced liver damage and enhanced collagen deposition were observed in connexin32 knockout mice compared to wild-type animals in experimentally triggered induced liver fibrosis. No differences between both groups were noticed in apoptotic signaling nor in inflammation markers. However, connexin32 deficient mice displayed decreased catalase activity and increased malondialdehyde levels. CONCLUSION These findings could suggest that connexin32-based signaling mediates tissue resistance against liver damage by the modulation of the antioxidant capacity. In turn, this could point to a role for connexin32 signaling as a therapeutic target in the treatment of liver fibrosis.
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Affiliation(s)
- Bruno Cogliati
- Department of Pathology, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Sao Paulo, Brazil
| | - Sara Crespo Yanguas
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Brussels, Belgium
| | - Tereza C. da Silva
- Department of Pathology, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Sao Paulo, Brazil
| | - Thiago P.A. Aloia
- Department of Pathology, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Sao Paulo, Brazil
| | - Marina S. Nogueira
- Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil
| | - Mirela A. Real-Lima
- Department of Pathology, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Sao Paulo, Brazil
| | - Lucas M. Chaible
- Department of Pathology, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Sao Paulo, Brazil
| | - Daniel S. Sanches
- Department of Pathology, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Sao Paulo, Brazil
| | - Joost Willebrords
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Brussels, Belgium
| | - Michaël Maes
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Brussels, Belgium
| | - Isabel V.A. Pereira
- Department of Pathology, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Sao Paulo, Brazil
| | - Inar A. de Castro
- Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil
| | - Mathieu Vinken
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Brussels, Belgium
| | - Maria L.Z. Dagli
- Department of Pathology, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Sao Paulo, Brazil
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Robert S, Gicquel T, Bodin A, Lagente V, Boichot E. Characterization of the MMP/TIMP Imbalance and Collagen Production Induced by IL-1β or TNF-α Release from Human Hepatic Stellate Cells. PLoS One 2016; 11:e0153118. [PMID: 27046197 PMCID: PMC4821480 DOI: 10.1371/journal.pone.0153118] [Citation(s) in RCA: 114] [Impact Index Per Article: 12.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/21/2016] [Accepted: 03/23/2016] [Indexed: 12/19/2022] Open
Abstract
Inflammation has an important role in the development of liver fibrosis in general and the activation of hepatic stellate cells (HSCs) in particular. It is known that HSCs are themselves able to produce cytokines and chemokines, and that this production may be a key event in the initiation of fibrogenesis. However, the direct involvement of cytokines and chemokines in HSC (self-)activation remains uncertain. In this study, the effects of pro-inflammatory cytokines IL-1α and β, TNF-α, and IL-8 on the activation state of HSCs were examined, in comparison to the pro-fibrogenic mediator TGF-β1. LX-2 cells were stimulated for 24 or 48 hours with recombinant human form of the pro-inflammatory cytokines IL-1α and β, TNF-α, and IL-8, and also the pro-fibrogenic mediator TGF-β1. Two drugs were also evaluated, the anti-TNF-α monoclonal antibody infliximab and the IL-1 receptor antagonist anakinra, regarding their inhibitory effects. In LX-2 human HSC, treatment with TGF-β1 are associated with downregulation of the metalloproteinase (MMP)-1 and MMP-3, with upregulation of tissue inhibitor of metalloproteinase (TIMP)-1, collagen type I α1, collagen type IV α1, α-SMA, endothelin-1 and PDGF-BB. Cytokines and chemokines expression were found to be downregulated, excepting IL-6. In contrast, we observed that LX-2 exposure to IL-1, TNF-α and IL-8 can reverse the phenotype of pro-fibrogenic activated cells. Indeed, MMP-1, MMP-3 and MMP-9 were found elevated, associated with downregulation of α-SMA and/or PDGF-BB, and a greater expression of IL-1β, IL-6, IL-8, CXCL1 and CCL2. Lastly, we found that infliximab and anakinra successfully inhibits effects of TNF-α and IL-1 respectively in LX-2 cells. Infliximab and anakinra may be of value in preclinical trials in chronic liver disease. Overall, our results suggest that (i) pro-inflammatory mediators exert complex effects in HSCs via an MMP/TIMP imbalance, and (ii) targeting IL-1 signaling may be a potentially valuable therapeutic strategy in chronic liver diseases.
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Affiliation(s)
- Sacha Robert
- UMR991 INSERM, Université de Rennes 1, Rennes, France
| | | | - Aude Bodin
- UMR991 INSERM, Université de Rennes 1, Rennes, France
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Tzeng TF, Tzeng YC, Cheng YJ, Liou SS, Liu IM. The Ethanol Extract from Lonicera japonica Thunb. Regresses Nonalcoholic Steatohepatitis in a Methionine- and Choline-Deficient Diet-Fed Animal Model. Nutrients 2015; 7:8670-84. [PMID: 26506376 PMCID: PMC4632443 DOI: 10.3390/nu7105423] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2015] [Revised: 09/19/2015] [Accepted: 10/06/2015] [Indexed: 12/22/2022] Open
Abstract
Nonalcoholic steatohepatitis (NASH) is characterized as fat accumulation in the hepatic tissue associated with various degrees of inflammation and progressive fibrosis. The potent anti-inflammatory and ethnopharmacological properties of Lonicera japonica Thunb. (Caprifoliaceae) make it an excellent source of novel medicinal targets for the treatment of NASH. The aim of the study was to investigate the effects of L. japonica ethanol extract (LJEE) on NASH in mice. C57BL/6J mice were fed with methionine-choline-deficient diet (MCDD) for eight weeks to promote the development of NASH. After development of the model, the mice were administered LJEE once daily via oral gavage at doses of 100, 200, or 300 mg/kg for another four weeks. Simultaneous treatments with LJEE (300 mg/kg/day) resulted in pronounced improvements in liver steatosis, ballooning degeneration, and inflammation. LJEE prevented MCDD-induced plasma level increases in aspartate aminotransferase and alanine aminotransferase. LJEE significantly reduced hepatic malondialdehyde level and ameliorated hepatic inflammation and fibrosis in MCDD-fed mice, which were associated with down-regulation of cytochrome P450 2E1 suppression of multiple proinflammatory and profibrotic genes. LJEE can prevent hepatic steatosis by reducing hepatic peroxisome acyl-CoA:diacylglycerol acyltransferase 2 expression, as well as by inducing proliferator-activated receptor α expression. In addition, the LJEE treatments caused significant reduction in the phosphorylated form of Jun N-terminal kinase along with an increase in the phosphorylated level of extra cellular signal-regulated kinase 1/2. Our study demonstrated the protective role of LJEE in ameliorating nutritional steatohepatitis.
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Affiliation(s)
- Thing-Fong Tzeng
- Department of Pharmacy and Master Program, Tajen University, Pingtung, 90741, Taiwan.
| | - Yu-Cheng Tzeng
- St. Dominic's Catholic High School, Kaohsiung, 80288, Taiwan.
| | - Yu-Jou Cheng
- Department of Pharmacy and Master Program, Tajen University, Pingtung, 90741, Taiwan.
| | - Shorong-Shii Liou
- Department of Pharmacy and Master Program, Tajen University, Pingtung, 90741, Taiwan.
| | - I-Min Liu
- Department of Pharmacy and Master Program, Tajen University, Pingtung, 90741, Taiwan.
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Lee HS, Son WC, Ryu JE, Koo BA, Kim YS. Standardized Salvia miltiorrhiza extract suppresses hepatic stellate cell activation and attenuates steatohepatitis induced by a methionine-choline deficient diet in mice. Molecules 2014; 19:8189-211. [PMID: 24941342 PMCID: PMC6271030 DOI: 10.3390/molecules19068189] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2014] [Revised: 06/05/2014] [Accepted: 06/11/2014] [Indexed: 01/07/2023] Open
Abstract
The aim of this study was to examine the effect of standardized extract of Salvia miltiorrhiza (SME) on gene and protein expression of non-alcoholic steatohepatitis (NASH)-related factors in activated human hepatic stellate cells (HSC), and in mice with steatohepatitis induced by a methionine-choline deficient (MCD) diet. Male C57BL/6J mice were placed on an MCD or control diet for 8 weeks and SME (0, 0.1, 0.5 and 1 mg/kg body weight) was administered orally every other day for 4 or 6 weeks. HSCs from the LX-2 cell line were treated with transforming growth factor β-1 (TGF-β1) or TGF-β1 plus SME (0.1–10 μg/mL). To investigate the effect of SME on reactive oxygen species (ROS)-induced condition, LX-2 cells were treated with hydrogen peroxide (H2O2) or H2O2plus SME (0.1–100 μg/mL). MCD administration for 12 weeks increased mRNA expression of tumor necrosis factor (TNF-α), TGF-β1, interleukin-1β (IL-1β), C-reactive protein (CRP), α-smooth muscle actin (α-SMA), type I collagen, matrix metalloproteinase-2 (MMP-2) and MMP-9. TGF-β1-induced LX-2 cells exhibited similar gene expression patterns. SME treatment significantly reduced the mRNA and protein expression of NASH-related factors in the mouse model and HSCs. Histopathological liver analysis showed improved non-alcoholic fatty liver disease (NAFLD) activity and fibrosis score in SME-treated mice. The in vivo studies showed that SME had a significant effect at low doses. These results suggest that SME might be a potential therapeutic candidate for NAFLD treatment.
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Affiliation(s)
- Hak Sung Lee
- Natural Products Research Institute, College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-742, Korea.
| | - Woo-Chan Son
- Department of Pathology, University of Ulsan College of Medicine, Asan Medical Center, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 138-736, Korea.
| | - Jae-Eun Ryu
- Department of Pathology, University of Ulsan College of Medicine, Asan Medical Center, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 138-736, Korea.
| | - Bon Am Koo
- Research Center, Samil Pharmaceutical Co. Ltd., 216 Sandan-ro, Danwon-gu, Ansan 425-852, Korea.
| | - Yeong Shik Kim
- Natural Products Research Institute, College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-742, Korea.
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15
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Dutta-Moscato J, Solovyev A, Mi Q, Nishikawa T, Soto-Gutierrez A, Fox IJ, Vodovotz Y. A Multiscale Agent-Based in silico Model of Liver Fibrosis Progression. Front Bioeng Biotechnol 2014; 2:18. [PMID: 25152891 PMCID: PMC4126446 DOI: 10.3389/fbioe.2014.00018] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2014] [Accepted: 05/14/2014] [Indexed: 01/06/2023] Open
Abstract
Chronic hepatic inflammation involves a complex interplay of inflammatory and mechanical influences, ultimately manifesting in a characteristic histopathology of liver fibrosis. We created an agent-based model (ABM) of liver tissue in order to computationally examine the consequence of liver inflammation. Our liver fibrosis ABM (LFABM) is comprised of literature-derived rules describing molecular and histopathological aspects of inflammation and fibrosis in a section of chemically injured liver. Hepatocytes are modeled as agents within hexagonal lobules. Injury triggers an inflammatory reaction, which leads to activation of local Kupffer cells and recruitment of monocytes from circulation. Portal fibroblasts and hepatic stellate cells are activated locally by the products of inflammation. The various agents in the simulation are regulated by above-threshold concentrations of pro- and anti-inflammatory cytokines and damage-associated molecular pattern molecules. The simulation progresses from chronic inflammation to collagen deposition, exhibiting periportal fibrosis followed by bridging fibrosis, and culminating in disruption of the regular lobular structure. The ABM exhibited key histopathological features observed in liver sections from rats treated with carbon tetrachloride (CCl4). An in silico “tension test” for the hepatic lobules predicted an overall increase in tissue stiffness, in line with clinical elastography literature and published studies in CCl4-treated rats. Therapy simulations suggested differential anti-fibrotic effects of neutralizing tumor necrosis factor alpha vs. enhancing M2 Kupffer cells. We conclude that a computational model of liver inflammation on a structural skeleton of physical forces can recapitulate key histopathological and macroscopic properties of CCl4-injured liver. This multiscale approach linking molecular and chemomechanical stimuli enables a model that could be used to gain translationally relevant insights into liver fibrosis.
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Affiliation(s)
- Joyeeta Dutta-Moscato
- Department of Biomedical Informatics, University of Pittsburgh , Pittsburgh, PA , USA ; Department of Surgery, University of Pittsburgh , Pittsburgh, PA , USA ; Center for Inflammation and Regenerative Modeling, McGowan Institute for Regenerative Medicine, University of Pittsburgh , Pittsburgh, PA , USA
| | - Alexey Solovyev
- Center for Inflammation and Regenerative Modeling, McGowan Institute for Regenerative Medicine, University of Pittsburgh , Pittsburgh, PA , USA ; Department of Mathematics, University of Pittsburgh , Pittsburgh, PA , USA
| | - Qi Mi
- Center for Inflammation and Regenerative Modeling, McGowan Institute for Regenerative Medicine, University of Pittsburgh , Pittsburgh, PA , USA ; Department of Sports Medicine and Nutrition, University of Pittsburgh , Pittsburgh, PA , USA
| | - Taichiro Nishikawa
- McGowan Institute for Regenerative Medicine, University of Pittsburgh , Pittsburgh, PA , USA ; Department of Surgery, Children's Hospital of Pittsburgh , Pittsburgh, PA , USA
| | - Alejandro Soto-Gutierrez
- McGowan Institute for Regenerative Medicine, University of Pittsburgh , Pittsburgh, PA , USA ; Department of Pathology, University of Pittsburgh , Pittsburgh, PA , USA ; Thomas E. Starzl Transplantation Institute, University of Pittsburgh , Pittsburgh, PA , USA
| | - Ira J Fox
- McGowan Institute for Regenerative Medicine, University of Pittsburgh , Pittsburgh, PA , USA ; Department of Surgery, Children's Hospital of Pittsburgh , Pittsburgh, PA , USA ; Thomas E. Starzl Transplantation Institute, University of Pittsburgh , Pittsburgh, PA , USA
| | - Yoram Vodovotz
- Department of Surgery, University of Pittsburgh , Pittsburgh, PA , USA ; Center for Inflammation and Regenerative Modeling, McGowan Institute for Regenerative Medicine, University of Pittsburgh , Pittsburgh, PA , USA
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16
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Ahsan MK, Mehal WZ. Activation of adenosine receptor A2A increases HSC proliferation and inhibits death and senescence by down-regulation of p53 and Rb. Front Pharmacol 2014; 5:69. [PMID: 24782773 PMCID: PMC3989592 DOI: 10.3389/fphar.2014.00069] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2014] [Accepted: 03/25/2014] [Indexed: 01/28/2023] Open
Abstract
Background and Aims: During fibrosis hepatic stellate cells (HSC) undergo activation, proliferation, and senescence but the regulation of these important processes is poorly understood. The adenosine A2A receptor (A2A) is known to be present on HSC, and its activation results in liver fibrosis. In this study, we tested if A2A has a role in the regulation of HSC proliferation, apoptosis, senescence, and the relevant molecular mechanism. Methods: The ability of adenosine to regulate p53 and Rb protein levels, proliferation, apoptosis and senescence was tested in the human HSC cell line LX-2 and rat primary HSC. Results: Adenosine receptor activation down-regulates p53 and Rb protein levels, increases BrdU incorporation and increases cell survival in LX-2 cells and in primary rat HSC. These effects of NECA were reproduced by an adenosine A2A receptor specific agonist (CGS21680) and blocked by a specific antagonist (ZM241385). By day twenty-one of culture primary rat HSC entered senescence and expressed β-gal which was significantly inhibited by NECA. Furthermore, NECA induced down regulation of p53 and Rb and Rac1, and decreased phosphorylation of p44-42 MAP Kinase in LX-2 cells and primary rat HSC. These effects were reproduced by the cAMP analog 8-Bromo-cAMP, and the adenylyl cyclase activator forskolin, and were blocked by PKA inhibitors. Conclusions: These results demonstrate that A2A receptor regulates a number of HSC fate decisions and induces greater HSC proliferation, reduces apoptosis and senescence by decreasing p53 and Rb through cAMP-PKA/Rac1/p38 MAPK pathway. This provides a mechanism for adenosine induced HSC regulation and liver fibrosis.
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Affiliation(s)
- Md Kaimul Ahsan
- Department of Internal Medicine, Section of Digestive Diseases, Yale University New Haven, CT, USA
| | - Wajahat Z Mehal
- Department of Internal Medicine, Section of Digestive Diseases, Yale University New Haven, CT, USA
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17
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Willemin G, Roger C, Bauduret A, Minehira K. Major Histocompatibility Class II Pathway Is Not Required for the Development of Nonalcoholic Fatty Liver Disease in Mice. Int J Endocrinol 2013; 2013:972962. [PMID: 23710178 PMCID: PMC3655579 DOI: 10.1155/2013/972962] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/12/2013] [Accepted: 03/22/2013] [Indexed: 01/11/2023] Open
Abstract
Single-nucleotide polymorphisms within major histocompatibility class II (MHC II) genes have been associated with an increased risk of drug-induced liver injury. However, it has never been addressed whether the MHC II pathway plays an important role in the development of nonalcoholic fatty liver disease, the most common form of liver disease. We used a mouse model that has a complete knockdown of genes in the MHC II pathway (MHCII(Δ/Δ)). Firstly we studied the effect of high-fat diet-induced hepatic inflammation in these mice. Secondly we studied the development of carbon-tetra-chloride- (CCl4-) induced hepatic cirrhosis. After the high-fat diet, both groups developed obesity and hepatic steatosis with a similar degree of hepatic inflammation, suggesting no impact of the knockdown of MHC II on high-fat diet-induced inflammation in mice. In the second study, we confirmed that the CCl4 injection significantly upregulated the MHC II genes in wild-type mice. The CCl4 treatment significantly induced genes related to the fibrosis formation in wild-type mice, whereas this was lower in MHCII(Δ/Δ) mice. The liver histology, however, showed no detectable difference between groups, suggesting that the MHC II pathway is not required for the development of hepatic fibrosis induced by CCl4.
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Affiliation(s)
- Gilles Willemin
- Department of Physiology, University of Lausanne, Rue du Bugnon 7, 1005 Lausanne, Switzerland
| | - Catherine Roger
- Center for Integrative Genomics, University of Lausanne, 1010 Lausanne, Switzerland
| | - Armelle Bauduret
- Center for Integrative Genomics, University of Lausanne, 1010 Lausanne, Switzerland
| | - Kaori Minehira
- Department of Physiology, University of Lausanne, Rue du Bugnon 7, 1005 Lausanne, Switzerland
- Nestlé Research Center, Vers-chez-les-Blanc, 1000 Lausanne 26, Switzerland
- *Kaori Minehira:
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18
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Song SH, Fukui K, Nakajima K, Kozakai T, Sasaki S, Roh SG, Katoh K. Cloning, expression analysis, and regulatory mechanisms of bovine chemerin and chemerin receptor. Domest Anim Endocrinol 2010; 39:97-105. [PMID: 20399065 DOI: 10.1016/j.domaniend.2010.02.007] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/20/2010] [Revised: 02/16/2010] [Accepted: 02/18/2010] [Indexed: 02/02/2023]
Abstract
Recently, we reported that chemerin, a new adipokine, is highly expressed in the adipose tissue, up-regulated during adipocyte differentiation, and regulates adipogenesis via its own receptor in mice. The objectives of this study were to clone chemerin and its receptor from the adipose tissues of Japanese Black cattle and to investigate the expression of these genes in 16 different tissues. We compared the gene expression of chemerin and its receptor between adipocytes and stromal-vascular (S-V) cells (non-adipocytes) prepared from subcutaneous adipose tissue. In addition, we investigated the mRNA expression levels of chemerin and its receptor in bovine differentiated adipocytes. The DNA sequences of bovine chemerin and its receptor were determined, and they were found to be highly homologous to those of humans, mice, and pigs. The amino acid sequences predicted for the full-length cDNA of bovine chemerin and its receptor were also similar to those of humans, mice, and pigs, suggesting that these genes have similar functions. Bovine chemerin mRNA was highly expressed in the adipose and liver tissues, and the transcripts of chemerin receptor were widely expressed in several tissues including adipose, muscle, liver, and brain tissues. The expression of bovine chemerin mRNA was higher in adipocytes than in S-V cells prepared from adipose tissue. The transcripts of chemerin and its receptor were up-regulated during adipocyte differentiation. Treatment with tumor necrosis factor (TNF)-alpha (10 ng/mL) in bovine differentiated adipocytes increased the mRNA expression of chemerin and its receptor. These results indicate that chemerin, a new adipokine highly expressed in the adipocytes of bovine adipose tissue, is the TNF-alpha-up-regulated gene with a role in adipogenesis.
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Affiliation(s)
- S-H Song
- Laboratory of Animal Physiology, Graduate School of Agricultural Science, Tohoku University, Aoba-ku, Sendai 981-8555, Japan
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19
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Expression of ECM proteins fibulin-1 and -2 in acute and chronic liver disease and in cultured rat liver cells. Cell Tissue Res 2009; 337:449-62. [PMID: 19609566 PMCID: PMC2728066 DOI: 10.1007/s00441-009-0823-9] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2009] [Accepted: 05/25/2009] [Indexed: 01/10/2023]
Abstract
Fibulin-2 has previously been considered as a marker to distinguish rat liver myofibroblasts from hepatic stellate cells. The function of other fibulins in acute or chronic liver damage has not yet been investigated. The aim of this study has been to evaluate the expression of fibulin-1 and -2 in models of rat liver injury and in human liver cirrhosis. Their cellular sources have also been investigated. In normal rat liver, fibulin-1 and -2 were both mainly present in the portal field. Fibulin-1-coding transcripts were detected in total RNA of normal rat liver, whereas fibulin-2 mRNA was only detected by sensitive, real-time quantitative polymerase chain reaction. In acute liver injury, the expression of fibulin-1 was significantly increased (17.23-fold after 48 h), whereas that of fibulin-2 was not modified. The expression of both fibulin-1 and -2 was increased in experimental rat liver cirrhosis (19.16- and 26.47-fold, respectively). At the cellular level, fibulin-1 was detectable in hepatocytes, "activated" hepatic stellate cells, and liver myofibroblasts (2.71-, 122.65-, and 469.48-fold over the expression in normal rat liver), whereas fibulin-2 was restricted to liver myofibroblasts and was regulated by transforming growth factor beta-1 (TGF-beta1) in 2-day-old hepatocyte cultures and in liver myofibroblasts. Thus, fibulin-1 and -2 respond differentially to single and repeated damaging noxae, and their expression is differently present in liver cells. Expression of the fibulin-2 gene is regulated by TGF-beta1 in liver myofibroblasts.
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20
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Everson GT, Balart L, Lee SS, Reindollar RW, Shiffman ML, Minuk GY, Pockros PJ, Govindarajan S, Lentz E, Heathcote EJ. Histological benefits of virological response to peginterferon alfa-2a monotherapy in patients with hepatitis C and advanced fibrosis or compensated cirrhosis. Aliment Pharmacol Ther 2008; 27:542-51. [PMID: 18208570 DOI: 10.1111/j.1365-2036.2008.03620.x] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
BACKGROUND Patients with chronic hepatitis C virus and advanced fibrosis or cirrhosis are at risk for disease progression and hepatic decompensation. AIM To determine the effects on hepatic histology of treatment with peginterferon alfa-2a (90 or 180 mug/week) or interferon alfa-2a (3 million units three times weekly) for 48 weeks in patients with paired biopsies. METHODS Liver biopsies were obtained at baseline and 6 months after end of treatment. Histological and virological responses were compared. RESULTS Patients attaining sustained virological response (n = 40) demonstrated the greatest improvements in fibrosis (-1.0, P < 0.0001) and inflammation (-0.65, P < 0.0001). Patients who cleared hepatitis C virus during treatment, but later relapsed (n = 59), experienced less improvement in fibrosis (-0.04, P < 0.0001) and inflammation (-0.14, P = 0.0768). Nonresponders (n = 85) showed no significant improvement in inflammation or fibrosis. Multiple regression analysis showed that the only factors contributing to improvement in fibrosis were sustained virological response (vs. nonresponder, P = 0.0005; vs. relapse, P = 0.7525) and body mass index < or =30 kg/m2 (P = 0.0995). CONCLUSIONS These findings indicate that virological response to peginterferon alfa-2a improves inflammation and fibrosis in hepatitis C virus patients with advanced fibrosis or cirrhosis. Improving virological response and maintaining ideal body weight are critical for achieving optimal histological outcomes in hepatitis C virus patients.
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Affiliation(s)
- G T Everson
- Section of Hepatology, Department of Medicine, University of Colorado Health Sciences Center, Denver, CO, USA.
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21
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Chang JJ, Thompson AJV, Visvanathan K, Kent SJ, Cameron PU, Wightman F, Desmond P, Locarnini SA, Lewin SR. The phenotype of hepatitis B virus-specific T cells differ in the liver and blood in chronic hepatitis B virus infection. Hepatology 2007; 46:1332-40. [PMID: 17924445 DOI: 10.1002/hep.21844] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
UNLABELLED Hepatitis B virus (HBV)-specific T cells play a key role in clearance of the virus and in the pathogenesis of liver disease. Peripheral blood (n = 25) and liver biopsies (n = 19) were collected from individuals with chronic untreated HBV infection. Whole blood, cultured peripheral blood mononuclear cells (PBMCs), and cultured liver-infiltrating lymphocytes (LILs) were each stimulated with an overlapping peptide library to the whole HBV genome. The expression of T helper 1 (Th1) cytokines [interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and interleukin 2 (IL-2)] and interleukin 10 (IL-10) was analyzed by intracellular cytokine staining and flow cytometry. In ex vivo whole blood, more lymphocytes produced Th1 cytokines than IL-10. When comparing cultured LILs with cultured PBMCs, we found a significantly higher magnitude of CD8(+) T cells from the liver producing IL-10 (P = 0.044), primarily in hepatitis B e antigen positive (HBeAg(+)) individuals. A positive correlation resulted between the magnitude of HBV-specific TNF-alpha(+) CD4(+) T cells in the liver and the degree of liver inflammation and fibrosis (P = 0.002 and P = 0.006, respectively). CONCLUSION The differences in cytokine production from HBV-specific T cells in blood and liver may explain the capacity for HBV to persist in the absence of significant hepatic destruction and highlights the balance between cytokine-mediated viral control and liver damage.
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Affiliation(s)
- J Judy Chang
- Department of Microbiology and Immunology, University of Melbourne, Australia
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22
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Guimarães ELM, Franceschi MFS, Grivicich I, Dal-Pizzol F, Moreira JCF, Guaragna RM, Borojevic R, Margis R, Guma FCR. Relationship between oxidative stress levels and activation state on a hepatic stellate cell line. Liver Int 2006; 26:477-85. [PMID: 16629652 DOI: 10.1111/j.1478-3231.2006.01245.x] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
BACKGROUND/AIMS Oxidative stress plays an important role in liver fibrosis. Under pathological conditions, hepatic stellate cells (HSC) undergo an activation process, developing a myofibroblast-like phenotype from the lipocyte phenotype. In this study, we determined the levels of oxidative stress and proliferation in different activation states of an experimental model of mouse HSC, the GRX cell line. These cells can be induced in vitro to display a more activated state or a quiescent phenotype. METHODS/RESULTS We observed increased oxidative damage and higher levels of reactive oxygen species, measured by thiobarbituric acid reactive species and 2',7'-dichlorofluorescein diacetate, respectively, and diminished catalase activity in activated cells. Activation decreased proliferation and increased the number of cells in G2/M. Antioxidants N-acetylcysteine and Trolox varied in their capacity to correct the oxidative stress and proliferation status. CONCLUSIONS The differences in physiological functions of stellate cell phenotypes suggest a relationship between oxidative stress levels and activation state.
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Affiliation(s)
- E L M Guimarães
- Departamento de Bioquímica, ICBS, UFRGS. Porto Alegre, RS, Brazil
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23
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Migita K, Maeda Y, Abiru S, Nakamura M, Komori A, Yokoyama T, Takii Y, Mori T, Yatsuhashi H, Eguchi K, Ishibashi H. Immunosuppressant FK506 inhibits matrix metalloproteinase-9 induction in TNF-alpha-stimulated human hepatic stellate cells. Life Sci 2005; 78:2510-5. [PMID: 16303143 DOI: 10.1016/j.lfs.2005.10.003] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2005] [Accepted: 10/06/2005] [Indexed: 01/18/2023]
Abstract
Activated hepatic stellate cells (HSCs) play a key role in the pathogenesis of hepatic inflammation and fibrosis through the production of matrix metalloproteinases (MMPs). Cytokines and growth factors are thought to activate HSCs. TNF-alpha has pleiotropic functions in hepatitis, but its role in liver fibrosis remains elusive. In this study we investigated the regulation of tumor necrosis factor-alpha (TNF-alpha) in the expression of MMPs by HSCs. We also examined whether the immunosuppressant FK506 influences the MMPs expression in human HSCs. Human HSCs, LI90, were treated with TNF-alpha in the presence of FK506. Release of MMPs into culture media, levels of MMP-9 mRNA and activation of NF-kappaB were compared between the cells cultured with or without FK506. Stimulation of human HSCs, LI90 cells, with TNF-alpha caused the induction of pro-MMP-9. Further, TNF-alpha stimulation induced the degradation of IkappaB-alpha and resulted in the transcriptional activation of NF-kappaB. FK506 suppressed this TNF-alpha-induced NK-kappaB activation, alone with pro-MMP-9 mRNA and protein induction, in HSC. TNF-alpha contributes to the perpetuation of liver fibrosis through MMP-9 production from HSCs and that FK506 inhibits the induction of MMP-9 through NF-kappaB pathway suggesting the anti-inflammatory properties of FK506.
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Affiliation(s)
- Kiyoshi Migita
- Clinical Research Center, National Hospital Organization (NHO) Nagasaki Medical Center, Kubara 2-1001-1 Omura, Nagasaki, 856-8562, Japan.
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Benten D, Kumaran V, Joseph B, Schattenberg J, Popov Y, Schuppan D, Gupta S. Hepatocyte transplantation activates hepatic stellate cells with beneficial modulation of cell engraftment in the rat. Hepatology 2005; 42:1072-81. [PMID: 16250034 DOI: 10.1002/hep.20889] [Citation(s) in RCA: 61] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
We investigated whether transplanted hepatocytes interact with hepatic stellate cells, as cell-cell interactions could modulate their engraftment in the liver. We transplanted Fischer 344 rat hepatocytes into syngeneic dipeptidyl peptidase IV-deficient rats. Activation of hepatic stellate cells was analyzed by changes in gene expression, including desmin and alpha-smooth muscle actin, matrix proteases and their inhibitors, growth factors, and other stellate cell-associated genes with histological methods or polymerase chain reaction. Furthermore, the potential role of hepatic ischemia, Kupffer cells, and cytokine release in hepatic stellate cell activation was investigated. Hepatocyte transplantation activated desmin-positive hepatic stellate cells, as well as Kupffer cells, including in proximity with transplanted cells. Inhibition of Kupffer cells by gadolinium chloride, blockade of tumor necrosis factor alpha (TNF-alpha) activity with etanercept or attenuation of liver ischemia with nitroglycerin did not decrease this hepatic stellate cell perturbation. After cell transplantation, soluble signals capable of activating hepatic stellate cells were rapidly induced, along with early upregulated expression of matrix metalloproteinases-2, -3, -9, -13, -14, and their inhibitors. Moreover, prior depletion of activated hepatic stellate cells with gliotoxin decreased transplanted cell engraftment. In conclusion, cell transplantation activated hepatic stellate cells, which, in turn, contributed to transplanted cell engraftment in the liver. Manipulation of hepatic stellate cells might provide new strategies to improve liver repopulation after enhanced transplanted cell engraftment.
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Affiliation(s)
- Daniel Benten
- Department of Medicine, Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY 10461, USA
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25
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Lang A, Sakhnini E, Fidder HH, Maor Y, Bar-Meir S, Chowers Y. Somatostatin inhibits pro-inflammatory cytokine secretion from rat hepatic stellate cells. Liver Int 2005; 25:808-16. [PMID: 15998432 DOI: 10.1111/j.1478-3231.2005.01057.x] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
BACKGROUND/AIMS Activated hepatic stellate cells (HSCs) have been implicated in hepatic fibrosis. Somatostatin (SOM) has an immunomodulatory role. The aim of this study was to assess the secretion of pro-inflammatory cytokines by HSCs and to determine the effect of SOM on the secretion of these mediators. METHODS Activated rat HSCs were evaluated for their secretion of IL-1beta, IL-8, and TNF-alpha using ELISA. RNA protection assay was used to determine cytokine mRNA levels. The expression of chemokine and cytokine mRNA and the secretion of these mediators were assessed following incubation with SOM or octreotide. RESULTS HSCs spontaneously secreted IL-1beta, IL-8, and TNF-alpha. This secretion was augmented following stimulation by IL-1beta and TNF-alpha. SOM inhibited the spontaneous and TNF-alpha-induced secretion of IL-1beta, IL-8, and TNF-alpha and suppressed the expression of IL-1beta and TNF-alpha mRNA. Octreotide suppressed the secretion of IL-1beta and IL-8. CONCLUSIONS These observations indicate that SOM exerts an inhibitory immunomodulatory effect on HSCs.
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Affiliation(s)
- Alon Lang
- Department of Gastroenterology and Liver Diseases, Chaim Sheba Medical Center, Tel-Hashomer, Israel.
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26
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Yu MC, Chen CH, Liang X, Wang L, Gandhi CR, Fung JJ, Lu L, Qian S. Inhibition of T-cell responses by hepatic stellate cells via B7-H1-mediated T-cell apoptosis in mice. Hepatology 2004; 40:1312-21. [PMID: 15565659 DOI: 10.1002/hep.20488] [Citation(s) in RCA: 242] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
In the injured liver, hepatic stellate cells (HSCs) secrete many different cytokines, recruit lymphocytes, and thus participate actively in the pathogenesis of liver disease. Little is known of the role of HSCs in immune responses. In this study, HSCs isolated from C57BL/10 (H2b) mice were found to express scant key surface molecules in the quiescent stage. Activated HSCs express major histocompatibility complex class I, costimulatory molecules, and produce a variety of cytokines. Stimulation by interferon gamma (IFN-gamma) or activated T cells enhanced expression of these molecules. Interestingly, addition of the activated (but not quiescent) HSCs suppressed thymidine uptake by T cells that were stimulated by alloantigens or by anti-CD3-mediated T-cell receptor ligation in a dose-dependent manner. High cytokine production by the T cells suggests that the inhibition was probably not a result of suppression of their activation. T-cell division was also found to be normal in a CFSE dilution assay. The HSC-induced T-cell hyporesponsiveness was associated with enhanced T-cell apoptosis. Activation of HSCs was associated with markedly enhanced expression of B7-H1. Blockade of B7-H1/PD-1 ligation significantly reduced HSC immunomodulatory activity, suggesting an important role of B7-H1. In conclusion, the bidirectional interactions between HSCs and immune cells may contribute to hepatic immune tolerance.
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Affiliation(s)
- Ming-Chin Yu
- Thomas E. Starzl Transplantation Institute and Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA 15261, USA
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27
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Ramadori G, Saile B. Inflammation, damage repair, immune cells, and liver fibrosis: specific or nonspecific, this is the question. Gastroenterology 2004; 127:997-1000. [PMID: 15362057 DOI: 10.1053/j.gastro.2004.07.041] [Citation(s) in RCA: 59] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
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Abstract
AIM: To appraise the effect of sea buckthorn (Hippophae rhamnoides) on cirrhotic patients.
METHODS: Fifty cirrhotic patients of Child-Pugh grade A and B were randomly divided into two groups: Group A as the treated group (n = 30), taking orally the sea buckthorn extract, 15 g 3 times a day for 6 mo. Group B as the control group (n = 18), taking vitamin B complex one tablet, 3 times a day for 6 mo. The following tests were performed before and after the treatment in both groups to determine LN, HA, collagens types III and IV, cytokines IL-6 and TNFα, liver serum albumin, total bile acid, ALT, AST and prothrombin time.
RESULTS: The serum levels of TNFα, IL-6, laminin and type IV collagen in group A were significantly higher than those in the control group. After a course of sea buckthorn treatment, the serum levels of LN, HA, collagen types III and IV, total bile acid (TBA) decreased significantly as compared with those before and after treatment in the control group. The sea buckthorn notably shortened the duration for normalization of aminotransferases.
CONCLUSION: Sea buckthorn may be a hopeful drug for prevention and treatment of liver fibrosis.
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Affiliation(s)
- Ze-Li Gao
- Department of Gastroenterology, Baogang Hospital, Shanghai Second Medical University, Shanghai 201900, China.
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29
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Benlloch S, Beltrán B, Moreno R, Berenguer M. [Fibrogenesis and liver transplantation]. GASTROENTEROLOGIA Y HEPATOLOGIA 2003; 26:381-95. [PMID: 12809575 DOI: 10.1016/s0210-5705(03)70375-6] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/20/2023]
Affiliation(s)
- S Benlloch
- Servicio de Medicina Digestiva. Hospital La Fe. Valencia. España
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30
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Ide M, Yamate J, Machida Y, Nakanishi M, Kuwamura M, Kotani T, Sawamoto O. Emergence of different macrophage populations in hepatic fibrosis following thioacetamide-induced acute hepatocyte injury in rats. J Comp Pathol 2003; 128:41-51. [PMID: 12531686 DOI: 10.1053/jcpa.2002.0603] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023]
Abstract
Macrophages may play a role in fibrogenesis. The kinetics and distribution of different macrophage populations were investigated immunohistochemically in hepatic lesions following acute hepatocyte injury induced in F344 rats by a single injection of thioacetamide (TAA) (300 mg/kg body weight, intraperitoneally). Hepatocyte degeneration or necrosis induced by TAA occurred mainly in the perivenular areas of hepatic lobules as early as post-injection (PI) days 1 and 3; fibrotic lesion development began in the damaged areas on day 1, and peaked on day 5; thereafter (PI days 7 and 10), the fibrotic areas decreased and were replaced by regenerated hepatocytes on PI days 15 and 20, indicating a remodelling process. In this rat model, the number of macrophages reacting with ED1 antibody (specific for exudate macrophages), ED2 (recognizing cell membrane antigens of resident macrophages, including Kupffer cells) and OX6 (recognizing MHC class II antigens expressed in antigen-presenting macrophages and dendritic cells) began to increase on PI day 1, peaking on PI day 3. The numbers gradually decreased on PI days 5 and 7; however, the statistically significant increase was maintained in respect of ED1-positive cells up to PI day 20, whereas no significant increase in ED2- and OX6-positive cells remained from PI day 10 onwards. Interestingly, of the ED1-, ED2- and OX6-positive cells, the OX6-positive cells were the least numerous. ED1- and OX6-positive cells appeared exclusively in the injured perivenular areas, whereas ED2-positive cells were present mainly in the mid-zonal areas and in smaller numbers in the perivenular areas. These findings indicated differences in kinetics and distribution between macrophage populations appearing in hepatic fibrosis. In addition, RT-PCR revealed that mRNA expression of osteopontin, a factor for induction and maintenance of macrophages in inflammation, was markedly increased on PI days 5, 7 and 10, suggesting a role in the pathogenesis of hepatic fibrosis.
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Affiliation(s)
- M Ide
- Department of Veterinary Pathology, Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, Gakuencho 1-1, Sakai, Osaka 599-8531, Japan
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31
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Varela-Rey M, Montiel-Duarte C, Osés-Prieto JA, López-Zabalza MJ, Jaffrèzou JP, Rojkind M, Iraburu MJ. p38 MAPK mediates the regulation of α1(I) procollagen mRNA levels by TNF-α and TGF-β in a cell line of rat hepatic stellate cells1. FEBS Lett 2002; 528:133-8. [PMID: 12297293 DOI: 10.1016/s0014-5793(02)03276-3] [Citation(s) in RCA: 80] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
The role of members of the mitogen-activated protein kinase (MAPK) family on tumor necrosis factor alpha (TNF-alpha)-mediated down-regulation of col1a1 gene was studied. TNF-alpha increased extracellular-regulated kinase and Jun-N-terminal kinase phosphorylation, but these effects were not related to its inhibitory effect on alpha1(I) procollagen (col1a1) mRNA levels. Phosphorylation of p38 MAPK was decreased in response to TNF-alpha, and the specific p38 MAPK inhibitor SB203580 mimicked the effect of TNF-alpha on col1a1 mRNA levels. Transforming growth factor beta (TGF-beta) increased p38 MAPK phosphorylation and SB203580 prevented the induction of col1a1 mRNA levels by TGF-beta. These results suggest that p38 MAPK plays an important role in regulating the expression of col1a1 in hepatic stellate cells in response to cytokines.
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Affiliation(s)
- M Varela-Rey
- Department of Biochemistry, University of Navarra, C/Irunlarrea 1, 31008 Pamplona, Spain
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Abstract
The wall of the liver sinusoid is made of highly specialized cells, the hepatic stellate cells (HSC) which together with the sinusoidal endothelial cells represent a loose barrier to the corpusculate part of the blood flowing through the liver. Quiescent stellate cells (quiescent HSC) store Vitamin A; "activated" stellate cells become involved in the reaction to acute or chronic noxae damaging the liver parenchyma. Activated HSC show increased protein synthesis capacity, increased DNA-synthesis and acquire a myofibroblast-like phenotype. Under similar conditions liver myofibroblasts (MF) of the portal field and of the pericentral area may also become "activated" by increasing protein synthesis, DNA synthesis and cell division. They express the fibulin-2 gene and produce large amounts of IL-6. In contrast to "activated" HSC they do not undergo spontaneous apoptosis in vitro and do not express the CD95-ligand gene. So far no definite prove has been found for a "transdifferentiation" of HSC to myofibroblasts. On the contrary an increasing amount of data support the conviction that HSC and MF represent two similar but not identical cell populations the latter being comparable to those of other organs.
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Affiliation(s)
- G Ramadori
- Department of Internal Medicine, Section of Gastroenterology and Endocrinology, Georg-August University Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany.
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Saile B, Matthes N, Neubauer K, Eisenbach C, El-Armouche H, Dudas J, Ramadori G. Rat liver myofibroblasts and hepatic stellate cells differ in CD95-mediated apoptosis and response to TNF-alpha. Am J Physiol Gastrointest Liver Physiol 2002; 283:G435-44. [PMID: 12121892 DOI: 10.1152/ajpgi.00441.2001] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Hepatic stellate cells (HSC), particularly activated HSC, are thought to be the principle matrix-producing cell of the diseased liver. However, other cell types of the fibroblast lineage, especially the rat liver myofibroblasts (rMF), also have fibrogenic potential. A major difference between the two cell types is the different life span under culture conditions. Although nearly no spontaneous apoptosis could be shown in rMF cultures, 18 +/- 2% of the activated HSC (day 7) were apoptotic. Compared with activated HSC, CD95R was expressed in 70% higher amounts in rMF. CD95L could only be detected in activated HSC. Stimulation of the CD95 system by agonistic antibodies (1 ng/ml) led to apoptosis of all rMF within 2 h, whereas activated HSC were more resistant (5.3 h/ 40% of total cells). Although transforming growth factor-beta downregulated apoptosis in both activated HSC and rMF, tumor necrosis factor-alpha (TNF-alpha) upregulated apoptosis in rMF. Lack of spontaneous apoptosis and CD95L expression in rMF and the different reaction on TNF-alpha stimulation reveal that activated HSC and rMF belong to different cell populations.
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Affiliation(s)
- Bernhard Saile
- Department of Internal Medicine, Section of Gastroenterology and Endocrinology, University of Göttingen, Germany
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34
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Kitamura K, Nakamoto Y, Akiyama M, Fujii C, Kondo T, Kobayashi K, Kaneko S, Mukaida N. Pathogenic roles of tumor necrosis factor receptor p55-mediated signals in dimethylnitrosamine-induced murine liver fibrosis. J Transl Med 2002; 82:571-83. [PMID: 12003998 DOI: 10.1038/labinvest.3780452] [Citation(s) in RCA: 52] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
TNF-alpha has pleiotropic functions, but its role in liver fibrosis has not yet been clarified. To understand the pathophysiologic role of the TNF-alpha/TNF receptor (TNFR) p55 signals in liver fibrosis, 10 mg/kg of dimethylnitrosamine, a specific hepatotoxicant, was administered twice a week into the peritoneal cavity of both TNFRp55 knock-out (KO) and wild-type mice, and the severity of fibrosis was monitored histologically and biochemically. In wild-type mice, histologic analysis demonstrated evident fibrotic changes 1 week after the initiation of dimethylnitrosamine administration, consistent with increased liver collagen contents. Concomitantly, the numbers of Kupffer cells and activated hepatic stellate cells (HSCs) were increased in liver tissue. On the contrary, fibrotic changes were attenuated and the numbers of Kupffer cells and HSCs were decreased in TNFRp55-KO mice. Moreover, gene expression of TNF-alpha and monocyte chemoattractant protein-1, which are involved in Kupffer cell activation or migration, was decreased in the liver of TNFRp55-KO mice. Collectively, TNFRp55-mediated signals may regulate activation of Kupffer cells and HSCs and eventually enhance fibrotic process.
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MESH Headings
- Animals
- Antigens, CD/genetics
- Antigens, CD/metabolism
- Chemokine CCL2/genetics
- Chemokine CCL2/metabolism
- Collagen/drug effects
- Collagen/metabolism
- DNA Primers/chemistry
- Dimethylnitrosamine/toxicity
- Female
- Gene Expression/drug effects
- Kupffer Cells/metabolism
- Kupffer Cells/pathology
- Liver/drug effects
- Liver/metabolism
- Liver/pathology
- Liver Cirrhosis, Experimental/genetics
- Liver Cirrhosis, Experimental/metabolism
- Liver Cirrhosis, Experimental/pathology
- Matrix Metalloproteinases/metabolism
- Mice
- Mice, Inbred BALB C
- Mice, Knockout
- RNA, Messenger/analysis
- Receptors, Tumor Necrosis Factor/deficiency
- Receptors, Tumor Necrosis Factor/genetics
- Receptors, Tumor Necrosis Factor/metabolism
- Receptors, Tumor Necrosis Factor, Type I
- Reverse Transcriptase Polymerase Chain Reaction
- Signal Transduction
- Specific Pathogen-Free Organisms
- Tissue Inhibitor of Metalloproteinases/metabolism
- Tumor Necrosis Factor-alpha/genetics
- Tumor Necrosis Factor-alpha/metabolism
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Affiliation(s)
- Kazuya Kitamura
- Department of Gastroenterology, Graduate School of Medicinal Sciences, Division of Molecular Bioregulation, Cancer Research Institute, Kanazawa University, Kanazawa, Japan
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35
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Levy MT, McCaughan GW, Marinos G, Gorrell MD. Intrahepatic expression of the hepatic stellate cell marker fibroblast activation protein correlates with the degree of fibrosis in hepatitis C virus infection. LIVER 2002; 22:93-101. [PMID: 12028401 DOI: 10.1034/j.1600-0676.2002.01503.x] [Citation(s) in RCA: 101] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/01/2023]
Abstract
BACKGROUND Activated hepatic stellate cells (HSCs), recognised by their alpha smooth muscle actin immunoreactivity, are primarily responsible for liver fibrosis. However, the presence of alpha smooth muscle actin positive HSCs is not always associated with the development of liver fibrosis. Recently, other markers of human HSCs including the gelatinase fibroblast activation protein (FAP) and glial fibrillary acidic protein have been identified. AIMS We examined the relationship between the expression of these HSC markers and the severity of liver injury in patients with chronic hepatitis C virus infection. METHODS Liver tissue from 27 patients was examined using immunohistochemistry. Linear correlation analysis was used to compare staining scores with the stage and grade of liver injury. RESULTS-CONCLUSIONS FAP expression, seen at the tissue-remodelling interface, was strongly and significantly correlated with the severity of liver fibrosis. A weaker correlation was seen between glial fibrillary acidic protein expression and fibrosis stage. This contrasted with the absence of a relationship between alpha smooth muscle actin and the fibrotic score. A correlation was also observed between FAP expression and necroinflammatory score. In summary, FAP expression identifies a HSC subpopulation at the tissue-remodelling interface that is related to the severity of liver fibrosis.
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Affiliation(s)
- M T Levy
- A.W. Morrow Gastroenterology and Liver Centre, Royal Prince Alfred Hospital, Centenary Institute of Cancer Medicine and Cell Biology and the University of Sydney, Australia.
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36
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Rigamonti C, Andorno S, Maduli E, Morelli S, Pittau S, Nicosia G, Boldorini R, Sartori M. Iron, hepatic stellate cells and fibrosis in chronic hepatitis C. Eur J Clin Invest 2002; 32 Suppl 1:28-35. [PMID: 11886429 DOI: 10.1046/j.1365-2362.2002.0320s1028.x] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
BACKGROUND/AIMS In patients with chronic hepatitis C, hepatic iron concentration correlates with liver fibrosis. However, it is not clear whether this correlation merely reflects the presence of more active disease, or iron exacerbates chronic hepatitis C virus (HCV)-induced damage through activation of hepatic stellate cells and regeneration of hepatocytes. MATERIALS AND METHODS We studied 72 HCV-positive patients, staged according to the Ishak's score system. We measured hepatic iron concentration with spectrophotometry and evaluated the number of hepatic stellate cells (using monoclonal antibody against alpha smooth muscle actin) and proliferating hepatocytes (using monoclonal antibody against Ki67). Iron and ferritin serum levels were also determined. RESULTS Hepatic iron concentration correlated statistically with ferritin serum level (r = 0.59, P < 0.001), with grading (r = 0.47, P < 0.001) and staging (r = 0.51, P < 0.001) scores for chronic hepatitis in the whole group of patients. Hepatic iron concentration correlated positively with stellate cell number (r = 0.55, P = 0.004) and Ki67-positive hepatocyte number (r = 0.36, P = 0.08) in patients with chronic hepatitis C and low grading score (< 3). CONCLUSIONS In patients with chronic hepatitis C and low grading score, hepatic iron could play a role in the activation of hepatic stellate cells and in the progression of fibrosis.
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Affiliation(s)
- Cristina Rigamonti
- Clinica Medica Generale, Università del Piemonte Orientale A. Avogadro, A.O. Maggiore della Carità, Corso Massini 18, 28100 Novara, Italy.
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37
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Fernández-Martínez E, Morales-Ríos MS, Pérez-Álvarez V, Muriel P. Effects of thalidomide and 3-phthalimido-3-(3,4-dimethoxyphenyl)-propanamide on bile duct obstruction-induced cirrhosis in the rat. Drug Dev Res 2002. [DOI: 10.1002/ddr.10022] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
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Sohara N, Trojanowska M, Reuben A. Oncostatin M stimulates tissue inhibitor of metalloproteinase-1 via a MEK-sensitive mechanism in human myofibroblasts. J Hepatol 2002; 36:191-9. [PMID: 11830330 DOI: 10.1016/s0168-8278(01)00265-3] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
BACKGROUND/AIMS We previously showed that in cultured human myofibroblasts (hMFBs), Oncostatin M (OSM)-stimulated collagen accumulation is associated with increased tissue inhibitor of metalloproteinase (TIMP)1 message. However, the mechanism is unknown. METHODS hMFBs were isolated by outgrowth from cirrhotic liver explants and cultured. Using OSM (10 ng/ml) stimulation, with and without PD98059 (PD, a specific mitogen-activated protein kinase/extracellular signal-related kinase (MEK) inhibitor), we measured: TIMP-1 protein in culture medium by Western blot, TIMP-1 mRNA levels and stability by Northern analysis, TIMP-1 promoter activity (including transcription site mutation analysis), DNA binding activity to nuclear proteins by electrophoretic mobility shift assay (EMSA), and total and phosphorylated MAP kinase in hMFB extracts by Western blot. RESULTS OSM stimulation of hMFBs increased TIMP-1 protein production 1.69-fold, TIMP-1 mRNA levels 2.36-fold, promoter activity 2.22-fold, TIMP-1 message stability, and phosphorylation of mitogen-activated protein kinase (MAPK). PD inhibited OSM-mediated stimulation of TIMP-1 protein, mRNA, promoter activity, phosphorylation of MAPK, and TIMP-1 message stability. An SP-1 transcription site of the TIMP-1 promoter is essential for OSM induction of TIMP-1 promoter activity. EMSA demonstrates that this site binds to transcriptional factors SP-1 and SP-3. CONCLUSIONS OSM stimulates the TIMP-1 axis in hMFBs in vitro via a MEK-MAP kinase cascade.
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Affiliation(s)
- Naondo Sohara
- Division of Gastroenterology and Hepatology, Department of Medicine, Medical University of South Carolina, 96 Jonathan Lucas Street, Suite 210, Charleston, SC 29425, USA
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39
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Ide M, Yamate J, Machida Y, Sawamoto O, Nakanishi M, Kuwamura M, Kotani T, Sakuma S. Macrophage Populations, Myofibroblastic Cells, and Extracellular Matrix Accumulation in Chronically-Developing Rat Liver Cirrhosis Induced by Repeated Injection of Thioacetamide. J Toxicol Pathol 2002. [DOI: 10.1293/tox.15.19] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Affiliation(s)
- Mika Ide
- Department of Veterinary Pathology, Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University
| | - Jyoji Yamate
- Department of Veterinary Pathology, Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University
| | - Yuko Machida
- Department of Veterinary Pathology, Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University
| | - Osamu Sawamoto
- Department of Veterinary Pathology, Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University
| | - Masako Nakanishi
- Department of Veterinary Pathology, Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University
| | - Mitsuru Kuwamura
- Department of Veterinary Pathology, Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University
| | - Takao Kotani
- Department of Veterinary Pathology, Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University
| | - Sadashige Sakuma
- Department of Veterinary Pathology, Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University
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40
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Saile B, Matthes N, El Armouche H, Neubauer K, Ramadori G. The bcl, NFkappaB and p53/p21WAF1 systems are involved in spontaneous apoptosis and in the anti-apoptotic effect of TGF-beta or TNF-alpha on activated hepatic stellate cells. Eur J Cell Biol 2001; 80:554-61. [PMID: 11561906 DOI: 10.1078/0171-9335-00182] [Citation(s) in RCA: 114] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Activated hepatic stellate cells (HSC) are thought to play a pivotal role in development of liver fibrosis which takes place in chronic liver diseases. Previous studies have shown that "activated" rat HSC undergo spontaneous apoptosis probably through the CD95/CD95L pathway. TGF-beta as well as TNF-alpha reduced spontaneous apoptosis and CD95L expression. The aim of this study was to investigate the possible mechanisms responsible for the spontaneous apoptosis and for the anti-apoptotic effect of TGF-beta and TNF-alpha on activated HSC. While bcl-2, bax, NFkappaB and p53 gene expression were spontaneously upregulated, bcl-xL and p21WAF1 gene expression decreased and IkappaB remained unchanged during the activation process in vitro. TGF-beta as well as TNF-alpha induced activation of NFKB and upregulated bcl-xL. The latter was inhibited by overexpression of IkappaB. By suppressing spontaneous apoptosis TGF-beta as well as TNF-alpha inhibited p53 gene expression while that of the p21WAF1 gene was increased. We conclude that TGF-beta as well as TNF-alpha may act as surviving factors for activated rat HSC not only through reduction of CD95L gene expression but also by upregulating the anti-apoptotic factors NFKB, bcl-xL and p21WAF1 and by downregulating the proapoptotic factor p53. The interaction with these factors may lead to the generation of new antifibrotic drugs.
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Affiliation(s)
- B Saile
- University of Göttingen, Department of Internal Medicine, Section of Gastroenterology and Endocrinology, Germany
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41
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Abstract
Cytokines comprise a group of small proteins released from cells in order to influence the function of other cells. By binding to highly specific cell-surface receptors, they trigger a vast array of intracellular signalling cascades. Cytokines have been described as interleukins, growth factors, interferons and chemokines. Unlike hormones, which act in a similar way, cytokines are produced by many different types of cell and act on many other types. Most of them are produced only after certain stimuli. The most intense field of cytokine activity is without doubt host defence. The liver resembles a central organ of cytokine activity due to the fact that it hosts hepatocytes, which are highly susceptible to the activity of cytokines in a variety of physiological and pathophysiological processes. Moreover, the non-parenchymal cells of the liver, in particular Kupffer cells (KCs), the resident tissue macrophages of the liver, are able to synthesize a variety of cytokines that may act systemically on any other organ of the body, or in a paracrine manner on hepatocytes and other non-parenchymal liver cells. A classic example of how cytokines act can be observed during the acute phase reaction discussed in this article. The role of cytokines in liver development, acute liver injury, liver regeneration, liver fibrosis and liver metastasis is also discussed.
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Affiliation(s)
- G Ramadori
- Department of Gastroenterology and Endocrinology, Georg-August-University Göttingen, Göttingen, Germany.
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42
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Apte MV, Phillips PA, Fahmy RG, Darby SJ, Rodgers SC, McCaughan GW, Korsten MA, Pirola RC, Naidoo D, Wilson JS. Does alcohol directly stimulate pancreatic fibrogenesis? Studies with rat pancreatic stellate cells. Gastroenterology 2000; 118:780-94. [PMID: 10734030 DOI: 10.1016/s0016-5085(00)70148-x] [Citation(s) in RCA: 179] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
BACKGROUND & AIMS Activated pancreatic stellate cells have recently been implicated in pancreatic fibrogenesis. This study examined the role of pancreatic stellate cells in alcoholic pancreatic fibrosis by determining whether these cells are activated by ethanol itself and, if so, whether such activation is caused by the metabolism of ethanol to acetaldehyde and/or the generation of oxidant stress within the cells. METHODS Cultured rat pancreatic stellate cells were incubated with ethanol or acetaldehyde. Activation was assessed by cell proliferation, alpha-smooth muscle actin expression, and collagen synthesis. Alcohol dehydrogenase (ADH) activity in stellate cells and the influence of the ADH inhibitor 4-methylpyrazole (4MP) on the response of these cells to ethanol was assessed. Malondialdehyde levels were determined as an indicator of lipid peroxidation. The effect of the antioxidant vitamin E on the response of stellate cells to ethanol or acetaldehyde was also examined. RESULTS Exposure to ethanol or acetaldehyde led to cell activation and intracellular lipid peroxidation. These changes were prevented by the antioxidant vitamin E. Stellate cells exhibited ethanol-inducible ADH activity. Inhibition of ADH by 4MP prevented ethanol-induced cell activation. CONCLUSIONS Pancreatic stellate cells are activated on exposure to ethanol. This effect of ethanol is most likely mediated by its metabolism (via ADH) to acetaldehyde and the generation of oxidant stress within the cells.
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Affiliation(s)
- M V Apte
- Pancreatic Research Group, Prince of Wales Hospital and University of New South Wales, Sydney, Australia
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43
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Levy MT, Trojanowska M, Reuben A. Oncostatin M: a cytokine upregulated in human cirrhosis, increases collagen production by human hepatic stellate cells. J Hepatol 2000; 32:218-26. [PMID: 10707861 DOI: 10.1016/s0168-8278(00)80066-5] [Citation(s) in RCA: 43] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
BACKGROUND/AIMS Hepatic stellate cells are predominantly responsible for the increased extracellular matrix seen in cirrhosis. The cytokine oncostatin M has been implicated in fibrogenesis in vitro in other cell types and in vivo in other tissues, although its effect on hepatic stellate cells or in cirrhosis is unknown. METHODS To examine the effect of oncostatin M on collagen production by human hepatic stellate cells in culture, collagen protein was measured and collagen alpha2(1) mRNA was quantified by Northern analysis. Tissue inhibitor of metalloproteinase-1 (an inhibitor of collagen degradation) mRNA was measured in response to oncostation M stimulation. To explore the potential biological significance of this work to human liver disease, oncostatin M messenger RNA in normal and cirrhotic human liver was measured. RESULTS Oncostatin M induced in a 2-fold increase in collagen secretion. The potency of induction of collagen protein secretion was equal to that observed after transforming growth factor beta stimulation. An increase in endogenous collagen alpha2(1) mRNA could not be detected. This suggested a post-transcriptional mechanism for the increase in collagen protein. In response to oncostatin M stimulation, there was a 2-fold increase in the tissue inhibitor or metalloproteinase-1 mRNA. Oncostatin M mRNA was detected in 6/6 cirrhotic livers and 1/7 normal livers after 28 PCR cycles. CONCLUSION These results suggest that oncostatin M expression is upregulated in cirrhosis where it may have a role as a profibrogenic cytokine in hepatic stellate cells.
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Affiliation(s)
- M T Levy
- Division of GI/Hepatology, Medical University of South Carolina, Charleston, USA.
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Knittel T, Kobold D, Dudas J, Saile B, Ramadori G. Role of the Ets-1 transcription factor during activation of rat hepatic stellate cells in culture. THE AMERICAN JOURNAL OF PATHOLOGY 1999; 155:1841-8. [PMID: 10595913 PMCID: PMC1866949 DOI: 10.1016/s0002-9440(10)65502-2] [Citation(s) in RCA: 24] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
During liver tissue repair, hepatic stellate cells (HSCs), a pericyte-like nonparenchymal liver cell population, transform from a quiescent status (resting HSCs) into myofibroblast like cells (activated HSCs); the latter is the principal matrix-synthesizing cell of the liver. Although several factors have been shown to be involved in this important process, the molecular mechanisms regulating HSC activation are still under investigation. To identify key regulatory proteins involved in the HSC activation process, we used different mRNA display technologies, with cDNAs prepared from HSCs at different stages of in vitro activation. With the latter technique, the transcription factor Ets-1 was detected through its down-regulation during activation. As confirmed by Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, mRNAs coding for Ets-1 were present in the highest amounts in freshly isolated HSCs and in HSCs 2 days after plating (classified as resting HSCs/early activated HSCs) and were diminished in HSCs 7 days after plating (activated cells). Ets-1 protein was present in HSC-lysates, as assessed by Western blot, and bound to an oligonucleotide containing the Ets-1 consensus cis-acting motif, as demonstrated by electrophoretic mobility shift assay. Ets-1 binding activity peaked in nuclear extracts prepared from resting/early activated cells and was diminished in extracts derived from fully activated cells. In contrast, binding activity of the transcription factors TFIID, AP-1, and SP-1 was highest in activated HSCs and only barely detectable in resting/early activated HSCs. By Northern blot and RT-PCR analysis, Ets-1-specific transcripts were present in parenchymal and other nonparenchymal liver cells too, illustrating that hepatic Ets-1 expression is not specific or restricted to HSCs. However, the unique pattern of Ets-1 binding activity present in resting versus activated HSCs and its known implications for cellular differentiation and tissue remodeling suggest that Ets-1 could be of crucial importance for HSC activation and hepatic tissue repair.
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Affiliation(s)
- T Knittel
- Department of Internal Medicine, Section of Gastroenterology and Endocrinology, University of Göttingen, Göttingen, Germany.
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Knittel T, Kobold D, Saile B, Grundmann A, Neubauer K, Piscaglia F, Ramadori G. Rat liver myofibroblasts and hepatic stellate cells: different cell populations of the fibroblast lineage with fibrogenic potential. Gastroenterology 1999; 117:1205-21. [PMID: 10535885 DOI: 10.1016/s0016-5085(99)70407-5] [Citation(s) in RCA: 282] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
BACKGROUND & AIMS Hepatic stellate cells (HSCs) are considered the principal matrix-producing cells of the damaged liver. However, other cell types of the fibroblast lineage that have not yet been characterized are also involved in liver tissue repair and fibrogenesis. METHODS We established cultures of cells of the fibroblast lineage, termed rat liver myofibroblasts, and analyzed their phenotypical and functional properties in comparison with HSCs. RESULTS HSCs and rat liver myofibroblasts were discernible by morphological criteria and growth behavior. Prolonged subcultivation of rat liver myofibroblasts was achieved, but HSCs were maintained in culture at maximum until second passage. HSCs were characterized by expression of glial fibrillary acidic protein, desmin, and vascular cell adhesion molecule 1, which were almost completely absent in rat liver myofibroblasts. For synthetic properties, HSCs and rat liver myofibroblasts displayed mostly overlapping properties with 4 striking differences. The complement-activating protease P100 and the protease inhibitor alpha(2)-macroglobulin were preferentially expressed by HSCs, whereas interleukin 6-coding messenger RNAs and the extracellular matrix protein fibulin 2 were almost exclusively detectable in rat liver myofibroblasts. CONCLUSIONS The data show that morphologically and functionally different fibroblastic populations, HSCs and rat liver myofibroblasts, can be derived from liver tissue. HSCs may not represent the single matrix-producing cell type of the fibroblast lineage in the liver.
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Affiliation(s)
- T Knittel
- Section of Gastroenterology, Department of Internal Medicine, University of Göttingen, Göttingen, Germany
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Saile B, Matthes N, Knittel T, Ramadori G. Transforming growth factor beta and tumor necrosis factor alpha inhibit both apoptosis and proliferation of activated rat hepatic stellate cells. Hepatology 1999; 30:196-202. [PMID: 10385656 DOI: 10.1002/hep.510300144] [Citation(s) in RCA: 121] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Transforming growth factor beta (TGF-beta) as well as tumor necrosis factor alpha (TNF-alpha) gene expression are up-regulated in chronically inflamed liver. These cytokines were investigated for their influence on apoptosis and proliferation of activated hepatic stellate cells (HSCs). Spontaneous apoptosis in activated HSC was significantly down-regulated by 53% +/- 8% (P <.01) under the influence of TGF-beta and by 28% +/- 2% (P <.05) under the influence of TNF-alpha. TGF-beta and TNF-alpha significantly reduced expression of CD95L in activated HSCs, whereas CD95 expression remained unchanged. Furthermore, HSC apoptosis induced by CD95-agonistic antibodies was reduced from 96% +/- 2% to 51 +/- 7% (P <.01) by TGF-beta, and from 96% +/- 2% to 58 +/- 2% (P <.01) by TNF-alpha, suggesting that intracellular antiapoptotic mechanisms may also be activated by both cytokines. During activation, HSC cultures showed a reduced portion of cells in the G0/G1 phase and a strong increment of G2-phase cells. This increment was significantly inhibited (G1 arrest) by administration of TGF-beta and/or TNF-alpha to activated cells. In liver sections of chronically damaged rat liver (CCl4 model), using desmin and CD95L as markers for activated HSC, most of these cells did not show apoptotic signs (TUNEL-negative). Taken together, these findings indicate that TGF-beta and/or TNF-alpha both inhibit proliferation and also apoptosis in activated HSC in vitro. Both processes seem to be linked to each other, and their inhibition could represent the mechanism responsible for prolonged survival of activated HSC in chronic liver damage in vivo.
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Affiliation(s)
- B Saile
- University of Göttingen, Department of Internal Medicine, Section of Gastroenterology and Endocrinology, Göttingen, Germany
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Piscaglia F, Knittel T, Kobold D, Barnikol-Watanabe S, Di Rocco P, Ramadori G. Cellular localization of hepatic cytochrome 1B1 expression and its regulation by aromatic hydrocarbons and inflammatory cytokines. Biochem Pharmacol 1999; 58:157-65. [PMID: 10403529 DOI: 10.1016/s0006-2952(99)00066-0] [Citation(s) in RCA: 43] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/17/2022]
Abstract
Cytochrome P450 1B1 (CYP1B1) is an activator of several xenobiotics and is induced in the liver upon experimental exposure to aromatic hydrocarbons. Since its cellular localization and regulation are incompletely clarified, Cyp1B1 expression and inducibility by 9,10-dimethyl-1,2-benzanthracene (DMBA) and inflammatory cytokines were investigated in different rat liver cell populations in vitro and in the liver during hepatocellular injury. Expression of Cyp1B1 was studied by Northern blot analysis in hepatic stellate cells (HSCs), myofibroblasts (MFs), Kupffer cells (KCs), and hepatocytes at various time points of primary cultures and in acutely damaged rat liver (carbon tetrachloride model). Enzyme inducibility was assessed by incubation of cells with DMBA as well as, in the case of HSCs, with tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor beta1 (TGFbeta1). Cyp1B1 messengers were expressed at high levels by HSCs and MFs, whereas constitutive expression was not detectable in KCs or in hepatocytes. Cyp1B1-specific mRNA were expressed at highest levels in HSCs at an early stage of activation (2 days after plating) and were diminished upon further activation. DMBA strongly enhanced Cyp1B1 gene expression in HSCs, MFs, and in hepatocytes at day 3 of primary cultures, but not in hepatocytes at day 1, or in KCs. The inflammatory cytokine TNF-alpha enhanced the Cyp1B1 gene expression in HSCs, either when administered alone or in addition to DMBA, while TGFbeta1 did not affect Cyp1B1 expression, even after DMBA induction. We conclude that HSCs and MFs seem to be the major cellular sources of hepatic Cyp1B1 expression and that the constitutive expression of the Cyp1B1 gene and the responsiveness to DMBA stimulation differ between mesenchymal and parenchymal liver cells, indicating a cell-specific regulation of Cyp1B1 gene expression. Interestingly, TNF-alpha is a potent stimulator of the Cyp1B1 gene in HSCs and acts in concert with DMBA.
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Affiliation(s)
- F Piscaglia
- Department of Internal Medicine, University of Göttingen, Germany
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Guerret S, Desmoulière A, Chossegros P, Costa AM, Badid C, Trépo C, Grimaud JA, Chevallier M. Long-term administration of interferon-alpha in non-responder patients with chronic hepatitis C: follow-up of liver fibrosis over 5 years. J Viral Hepat 1999; 6:125-33. [PMID: 10607223 DOI: 10.1046/j.1365-2893.1999.00148.x] [Citation(s) in RCA: 37] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
In chronic hepatitis C, previous data have shown that short-term treatment with interferon-alpha (IFN-alpha) can reduce collagen deposition in the liver independently of the viral response. The aim of this work was to determine, in non-responder patients, the long-term effect of IFN-alpha on liver fibrosis according to the total administered dose and the fibrotic stage. Fibrosis was investigated on liver biopsies from 24 non-responder patients with chronic hepatitis C retreated with successive courses of IFN-alpha. The degree of liver fibrosis was assessed on three successive biopsies, performed before IFN-alpha treatment and 1 and 5 years later, in 13 and 11 patients, respectively, treated for less (mean: 7.5 months, 313 MU) and more (mean: 21.8 months, 791 MU) than 1 year. For each biopsy, fibrosis was assessed using a histological semiquantitative fibrosis scoring system and by morphometry after picrosirius red staining. Regardless of the dose and duration of IFN-alpha therapy, a slight decrease of fibrosis was observed in patients 5 years after starting treatment. In cirrhotic patients, a short treatment induced an improvement followed by a relapse of fibrosis in 57%, and only 43% of patients showed constant collagen regression over the 5 years of follow-up. On the contrary, after prolonged therapy, a progressive and significant decrease occurred throughout the follow-up period in all patients (P = 0.045). Long-term treatment with IFN-alpha is therefore associated with regression of liver fibrosis, particularly in cirrhotic patients. These promising results need to be confirmed in a larger series of patients.
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Affiliation(s)
- S Guerret
- Laboratoire d'Anatomie et Cytologie Pathologiques, Laboratoire Marcel Mérieux, Lyon, France
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Knittel T, Mehde M, Kobold D, Saile B, Dinter C, Ramadori G. Expression patterns of matrix metalloproteinases and their inhibitors in parenchymal and non-parenchymal cells of rat liver: regulation by TNF-alpha and TGF-beta1. J Hepatol 1999; 30:48-60. [PMID: 9927150 DOI: 10.1016/s0168-8278(99)80007-5] [Citation(s) in RCA: 253] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/18/2023]
Abstract
BACKGROUND/AIMS Although matrix metalloproteinases (MMPs) and their specific inhibitors (TIMPs) play an essential role in liver injury associated with tissue remodeling, the cellular origin of MMPs/TMPs within the liver remains to be clarified. METHODS Different liver cell populations were analysed with respect to their expression by reverse transcription-polymerase chain reaction, Northern blot analysis and zymography. RESULTS MMP and TIMP coding transcripts were detectable in all liver cell types by reverse transcription-polymerase chain reaction; however, the cellular expression levels were markedly different as assessed by Northern blot analysis. Gelatinase-B was predominantly expressed in Kupffer cells, gelatinase-A in hepatic stellate cells and rat liver myofibroblasts and stromelysins-1, -2 as well as collagenase in hepatic stellate cells. Membrane type-1 MMP (MMP-14) was found in significant amounts in all liver cells. TIMP-1 coding m-RNAs were present mainly in hepatic stellate cells and rat liver myofibroblasts, TIMP-2 additionally in Kupffer cells, while TIMP-3 expression was detectable only in hepatocytes. During in vitro activation of hepatic stellate cells, MMP expression was mostly downregulated, while TIMP expression was enhanced, thereby providing an explanation for matrix accumulation co-localised with these cells during chronic liver injury. In general, TNF-alpha stimulated both MMP and TIMP expression of hepatic stellate cells, while TGF-beta1 induced TIMP expression only. CONCLUSIONS Collectively these data demonstrate that all resident liver cells are involved in matrix degradation to some extent and that hepatic stellate cells play an important role in matrix breakdown in addition to matrix synthesis. The cytokine-specific regulation of MMP/TIMP expression in hepatic stellate cells suggests that the initial matrix breakdown following liver injury might be enhanced by TNF-alpha, while diminished matrix degradation during chronic tissue injury might be due to the action of TGF-beta1 through TIMP induction.
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Affiliation(s)
- T Knittel
- Department of Internal Medicine, University of Göttingen, Germany
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Knittel T, Dinter C, Kobold D, Neubauer K, Mehde M, Eichhorst S, Ramadori G. Expression and regulation of cell adhesion molecules by hepatic stellate cells (HSC) of rat liver: involvement of HSC in recruitment of inflammatory cells during hepatic tissue repair. THE AMERICAN JOURNAL OF PATHOLOGY 1999; 154:153-67. [PMID: 9916930 PMCID: PMC1853435 DOI: 10.1016/s0002-9440(10)65262-5] [Citation(s) in RCA: 102] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
Hepatic stellate cells (HSC), a pericyte-like nonparenchymal liver cell population, are regarded as the principal matrix-synthesizing cells of fibrotic liver. They might also play a role during liver inflammation. The present study analyzed (i) expression of cell adhesion molecules (CAMs) mediating cell infiltration, like intercellular adhesion molecule-1 (I-CAM-1) and vascular cell adhesion molecule-1 (V-CAM-1), by HSC, (ii) CAM regulation in HSC by growth factors and inflammatory cytokines, and (iii) CAM expression in situ during liver inflammation, using immunochemistry and Northern blot analysis. I-CAM-1 and V-CAM-1 expression was present in HSC in vitro and in cells located in the sinusoidal/perisinusoidal area of normal liver. Growth factors, eg, transforming growth factor-beta1, down-regulated I-CAM-1- and V-CAM-1-coding mRNAs and stimulated N-CAM expression of HSC. In contrast, inflammatory cytokines like tumor necrosis factor-alpha reduced N-CAM-coding mRNAs, whereas induction of I-CAM-1- and V-CAM-1-specific transcripts increased several fold. In situ, messengers specific for I-CAM-1 and V-CAM-1 were induced 3 hours after CCl4 treatment (thereby preceding mononuclear cell infiltration starting at 12 hours), were expressed at maximal levels 9-12 hours after CCl4 application, and decreased afterwards. I-CAM-1 and V-CAM-1 immunoreactivity increased in a linear fashion starting 3 hours after CCl4-induced liver injury, was detected in highest amounts at 24-48 hours characterized by maximal cell infiltration, and returned to baseline values at 96 hours. Interestingly, the induction/repression of CAM-specific messengers paralleled the time kinetics of tumor necrosis factor-alpha transforming growth factor-beta1 expression in injured liver. HSC might be important during the onset of hepatic tissue injury as proinflammatory elements and might interact with I-CAM-1 and V-CAM-1 ligand-bearing cells, namely lymphocyte function-associated antigen-1- or Mac-1/very late activation antigen-4-positive inflammatory cells, thereby modulating the recruitment and migration of mononuclear cells within the perisinusoidal space of diseased livers.
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Affiliation(s)
- T Knittel
- Department of Internal Medicine, University of Göttingen, Germany
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