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A simple culture method for liver and intestinal tissue-resident macrophages from neonatal mice. In Vitro Cell Dev Biol Anim 2019; 55:436-444. [PMID: 31119642 DOI: 10.1007/s11626-019-00359-y] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2019] [Accepted: 04/13/2019] [Indexed: 02/08/2023]
Abstract
The liver and intestine contain a remarkably large portion of tissue-resident macrophage cells representing a phenotype that downregulates inflammation and initiates tissue repair. Here, liver and intestinal tissues obtained from neonatal mice were minced, enzymatically digested, and incubated in RPMI1640-based media. In a 2-wk culture, spherical floating cells emerged on a fibroblastic sheet. These cells showed phagocytic activity and F4/80+-CD11b+-CD206+-Arg1+-iNOS--CD209a- phenotype, suggesting that these cells are tissue-resident macrophages. These macrophages proliferated in the co-culture system in the presence of fibroblastic feeder cell layer and absence of supplemental cytokines; the co-culture system did not cause a significant change in the phenotype of cells grown in a 4-wk culture. On the feeder cells, macrophage density was approximately 1.5 × 104/cm2 and the doubling time was approximately 70 h. Based on these observations, we present a simple method for the isolation and propagation of tissue-resident macrophages resembling M2 macrophage from neonatal mice, and this method provides a useful platform for in vitro studies of tissue-resident macrophages.
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The stellate cell system (vitamin A-storing cell system). Anat Sci Int 2017; 92:387-455. [PMID: 28299597 DOI: 10.1007/s12565-017-0395-9] [Citation(s) in RCA: 73] [Impact Index Per Article: 9.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2016] [Accepted: 02/15/2017] [Indexed: 01/18/2023]
Abstract
Past, present, and future research into hepatic stellate cells (HSCs, also called vitamin A-storing cells, lipocytes, interstitial cells, fat-storing cells, or Ito cells) are summarized and discussed in this review. Kupffer discovered black-stained cells in the liver using the gold chloride method and named them stellate cells (Sternzellen in German) in 1876. Wake rediscovered the cells in 1971 using the same gold chloride method and various modern histological techniques including electron microscopy. Between their discovery and rediscovery, HSCs disappeared from the research history. Their identification, the establishment of cell isolation and culture methods, and the development of cellular and molecular biological techniques promoted HSC research after their rediscovery. In mammals, HSCs exist in the space between liver parenchymal cells (PCs) or hepatocytes and liver sinusoidal endothelial cells (LSECs) of the hepatic lobule, and store 50-80% of all vitamin A in the body as retinyl ester in lipid droplets in the cytoplasm. SCs also exist in extrahepatic organs such as pancreas, lung, and kidney. Hepatic (HSCs) and extrahepatic stellate cells (EHSCs) form the stellate cell (SC) system or SC family; the main storage site of vitamin A in the body is HSCs in the liver. In pathological conditions such as liver fibrosis, HSCs lose vitamin A, and synthesize a large amount of extracellular matrix (ECM) components including collagen, proteoglycan, glycosaminoglycan, and adhesive glycoproteins. The morphology of these cells also changes from the star-shaped HSCs to that of fibroblasts or myofibroblasts.
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Kegel V, Deharde D, Pfeiffer E, Zeilinger K, Seehofer D, Damm G. Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells. J Vis Exp 2016:e53069. [PMID: 27077489 DOI: 10.3791/53069] [Citation(s) in RCA: 36] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the "gold standard" for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine(1). Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique. Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation. Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models.
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Affiliation(s)
- Victoria Kegel
- Department of General-, Visceral- and Transplantation Surgery, Charité University Medicine Berlin
| | - Daniela Deharde
- Department of General-, Visceral- and Transplantation Surgery, Charité University Medicine Berlin
| | - Elisa Pfeiffer
- Department of General-, Visceral- and Transplantation Surgery, Charité University Medicine Berlin
| | - Katrin Zeilinger
- Brandenburg Center for Regenerative Therapies, Charité University Medicine Berlin
| | - Daniel Seehofer
- Department of General-, Visceral- and Transplantation Surgery, Charité University Medicine Berlin
| | - Georg Damm
- Department of General-, Visceral- and Transplantation Surgery, Charité University Medicine Berlin;
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Jiang X, Li Z, Jiang S, Tong X, Zou X, Wang W, Zhang Z, Wu L, Tian D. Lipoxin A4 exerts protective effects against experimental acute liver failure by inhibiting the NF-κB pathway. Int J Mol Med 2016; 37:773-80. [PMID: 26865215 DOI: 10.3892/ijmm.2016.2483] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2015] [Accepted: 01/15/2016] [Indexed: 11/05/2022] Open
Abstract
Although rare, acute liver failure (ALF) is associated with high levels of mortality, warranting the development of novel therapies. Nuclear factor-κB (NF-κB), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) play roles in ALF. Lipoxin A4 (LXA4) has been shown to alleviate inflammation in non-hepatic tissues. In the present study, we explored whether LXA4 exerted hepatoprotective effects in a rat model of ALF. A rat model of ALF was generated by intraperitoneal injections of D-galactosamine (300 mg/kg) and lipopolysaccharide (50 µg/kg). Animals were randomly assigned to: control group (no ALF); model group (ALF); and the groups treated with a low dose (0.5 µg/kg), medium dose (1 µg/kg), and high dose (2 µg/kg) of LXA4 (all with ALF); and pyrrolidine dithiocarbamate (PDTC)-treated group (ALF and 100 mg/kg PDTC, an inhibitor of NF-κB). Liver histology was measured using H&E staining, serum levels by ELISA, and liver mRNA expression was measured by RT-PCR for the detection of the pro‑inflammatory cytokines TNF-α and IL-6. Liver cell apoptosis (as measured using the TUNEL method and examining caspase-3 activity), and Kupffer cell NF-κB activity [using an electrophoretic mobility shift assay (EMSA)] were examined. Serum levels of transaminases, TNF-α and interleukin-6 (IL-6) were substantially higher in the model group compared to controls. In the model group, significant increases in TNF-α and IL-6 mRNA expression, TUNEL‑positive cells, and caspase-3 activity in the liver tissue were noted. LXA4 improved liver pathology and significantly decreased the indicators of inflammatory response and apoptosis in a dose-dependent manner. High-dose LXA4 provided better protection than PDTC. LXA4 administration significantly decreased NF-κB expression in hepatocytes and Kupffer cells. These results indicated that LXA4 inhibited NF-κB activation, reduced the secretion of pro-inflammatory cytokines, and inhibited apoptosis of liver cells, thereby exerting protective effects against ALF.
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Affiliation(s)
- Xueqiang Jiang
- Department of Infection, Dongfeng Hospital Affiliated to Hubei Medical University, Shiyan, Hubei 442008, P.R. China
| | - Zhihao Li
- Department of Pharmacy, Dongfeng General Hospital Affiliated to Hubei Medical University, Shiyan, Hubei 442008, P.R. China
| | - Shengfang Jiang
- Center of Reproductive Medicine, People's Hospital Affiliated to Hubei Medical University, Shiyan, Hubei 442000, P.R. China
| | - Xuefei Tong
- Shennong Wudang Institute of Traditional Chinese Medicine, Shiyan Hospital of TCM Affiliated to Hubei University of Chinese Medicine, Shiyan, Hubei 442012, P.R. China
| | - Xiaojing Zou
- Department of Emergency, Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, Hubei 430030, P.R. China
| | - Wan Wang
- Department of Infection, Dongfeng Hospital Affiliated to Hubei Medical University, Shiyan, Hubei 442008, P.R. China
| | - Zhengang Zhang
- Department of Infection, Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, Hubei 430030, P.R. China
| | - Liang Wu
- Department of Infection, Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, Hubei 430030, P.R. China
| | - Deying Tian
- Department of Infection, Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, Hubei 430030, P.R. China
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Zhang C, Lu Y, Zhou H, Lu H, Qian X, Liu X, Wang X, Ding Z, Zhang F, Lu L. Acquiring Kupffer cells in mice using a MACS-based method. Transplant Proc 2015; 47:553-7. [PMID: 25769606 DOI: 10.1016/j.transproceed.2015.01.018] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2014] [Revised: 01/09/2015] [Accepted: 01/28/2015] [Indexed: 12/27/2022]
Abstract
OBJECTIVE This study sought to establish a new method to isolate Kupffer cells (KCs) by magnetic activated cell sorting (MACS). METHODS Nonparenchymal cells were acquired from C57BL/6 mice livers by a perfusion system in vivo and then stained with F4/80(+) fluorescein isothiocyanate and CD11c(-) phycoerythrin antibodies. After incubating with immunomagnetic beads, F4/80(+)CD11c(-) KCs were obtained by MACS selection. The purity was evaluated by flow cytometry, and the morphological features and vitality were analyzed in in vitro cultures. RESULTS Compared with traditional methods, acquiring KCs by MACS was characterized by economy, efficiency, and high purity. The F4/80(+)CD11c(-) KCs cultured in vitro also showed the typical adherent shape and excellent phagocytic ability. CONCLUSIONS With the 2-step method using immunomagnetic beads, we provide a new method by which KCs can be obtained from mouse liver with high purity and distinct phenotype of F4/80(+) CD.
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Affiliation(s)
- C Zhang
- Translational Medicine Research Center of Jiangning Hospital and Liver Transplantation Center of First Affiliated Hospital, Nanjing Medical University, Nanjing, China
| | - Y Lu
- Translational Medicine Research Center of Jiangning Hospital and Liver Transplantation Center of First Affiliated Hospital, Nanjing Medical University, Nanjing, China
| | - H Zhou
- Translational Medicine Research Center of Jiangning Hospital and Liver Transplantation Center of First Affiliated Hospital, Nanjing Medical University, Nanjing, China
| | - H Lu
- Translational Medicine Research Center of Jiangning Hospital and Liver Transplantation Center of First Affiliated Hospital, Nanjing Medical University, Nanjing, China
| | - X Qian
- Translational Medicine Research Center of Jiangning Hospital and Liver Transplantation Center of First Affiliated Hospital, Nanjing Medical University, Nanjing, China
| | - X Liu
- Translational Medicine Research Center of Jiangning Hospital and Liver Transplantation Center of First Affiliated Hospital, Nanjing Medical University, Nanjing, China
| | - X Wang
- Translational Medicine Research Center of Jiangning Hospital and Liver Transplantation Center of First Affiliated Hospital, Nanjing Medical University, Nanjing, China
| | - Z Ding
- Translational Medicine Research Center of Jiangning Hospital and Liver Transplantation Center of First Affiliated Hospital, Nanjing Medical University, Nanjing, China
| | - F Zhang
- Translational Medicine Research Center of Jiangning Hospital and Liver Transplantation Center of First Affiliated Hospital, Nanjing Medical University, Nanjing, China
| | - L Lu
- Translational Medicine Research Center of Jiangning Hospital and Liver Transplantation Center of First Affiliated Hospital, Nanjing Medical University, Nanjing, China.
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Sørensen KK, Simon‐Santamaria J, McCuskey RS, Smedsrød B. Liver Sinusoidal Endothelial Cells. Compr Physiol 2015; 5:1751-74. [DOI: 10.1002/cphy.c140078] [Citation(s) in RCA: 156] [Impact Index Per Article: 15.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
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Pfeiffer E, Kegel V, Zeilinger K, Hengstler JG, Nüssler AK, Seehofer D, Damm G. Featured Article: Isolation, characterization, and cultivation of human hepatocytes and non-parenchymal liver cells. Exp Biol Med (Maywood) 2015; 240:645-656. [PMID: 25394621 PMCID: PMC4935273 DOI: 10.1177/1535370214558025] [Citation(s) in RCA: 82] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2014] [Accepted: 09/18/2014] [Indexed: 02/06/2023] Open
Abstract
Primary human hepatocytes (PHH) are considered to be the gold standard for in vitro testing of xenobiotic metabolism and hepatotoxicity. However, PHH cultivation in 2D mono-cultures leads to dedifferentiation and a loss of function. It is well known that hepatic non-parenchymal cells (NPC), such as Kupffer cells (KC), liver endothelial cells (LEC), and hepatic stellate cells (HSC), play a central role in the maintenance of PHH functions. The aims of the present study were to establish a protocol for the simultaneous isolation of human PHH and NPC from the same tissue specimen and to test their suitability for in vitro co-culture. Human PHH and NPC were isolated from tissue obtained by partial liver resection by a two-step EDTA/collagenase perfusion technique. The obtained cell fractions were purified by Percoll density gradient centrifugation. KC, LEC, and HSC contained in the NPC fraction were separated using specific adherence properties and magnetic activated cell sorting (MACS®). Identified NPC revealed a yield of 1.9 × 10(6) KC, 2.7 × 10(5) LEC and 4.7 × 10(5) HSC per gram liver tissue, showing viabilities >90%. Characterization of these NPC showed that all populations went through an activation process, which influenced the cell fate. The activation of KC strongly depended on the tissue quality and donor anamnesis. KC became activated in culture in association with a loss of viability within 4-5 days. LEC lost specific features during culture, while HSC went through a transformation process into myofibroblasts. The testing of different culture conditions for HSC demonstrated that they can attenuate, but not prevent dedifferentiation in vitro. In conclusion, the method described allows the isolation and separation of PHH and NPC in high quality and quantity from the same donor.
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Affiliation(s)
- Elisa Pfeiffer
- Department for General, Visceral and Transplantation Surgery, Charité - Universitätsmedizin Berlin, 13353 Berlin, Germany
| | - Victoria Kegel
- Department for General, Visceral and Transplantation Surgery, Charité - Universitätsmedizin Berlin, 13353 Berlin, Germany
| | - Katrin Zeilinger
- Bioreactor Group, Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Charité - Universitätsmedizin Berlin, 13353 Berlin, Germany
| | - Jan G Hengstler
- IfADo - Leibniz Research Centre for Working Environment and Human Factors at Dortmund Technical University, 44139 Dortmund, Germany
| | - Andreas K Nüssler
- Eberhard-Karls University Tübingen, BG Trauma Center, 72076 Tübingen, Germany
| | - Daniel Seehofer
- Department for General, Visceral and Transplantation Surgery, Charité - Universitätsmedizin Berlin, 13353 Berlin, Germany
| | - Georg Damm
- Department for General, Visceral and Transplantation Surgery, Charité - Universitätsmedizin Berlin, 13353 Berlin, Germany
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Targeted delivery of lipid antigen to macrophages via the CD169/sialoadhesin endocytic pathway induces robust invariant natural killer T cell activation. Proc Natl Acad Sci U S A 2013; 110:7826-31. [PMID: 23610394 DOI: 10.1073/pnas.1219888110] [Citation(s) in RCA: 97] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Invariant natural killer T (iNKT) cells induce a protective immune response triggered by foreign glycolipid antigens bound to CD1d on antigen-presenting cells (APCs). A limitation of using glycolipid antigens to stimulate immune responses in human patients has been the inability to target them to the most effective APCs. Recent studies have implicated phagocytic CD169(+) macrophages as major APCs in lymph nodes for priming iNKT cells in mice immunized with glycolipid antigen in particulate form. CD169 is known as sialoadhesin (Sn), a macrophage-specific adhesion and endocytic receptor of the siglec family that recognizes sialic acid containing glycans as ligands. We have recently developed liposomes decorated with glycan ligands for CD169/Sn suitable for targeted delivery to macrophages via CD169/Sn-mediated endocytosis. Here we show that targeted delivery of a lipid antigen to CD169(+) macrophages in vivo results in robust iNKT cell activation in liver and spleen using nanogram amounts of antigen. Activation of iNKT cells is abrogated in Cd169(-/-) mice and is macrophage-dependent, demonstrating that targeting CD169(+) macrophages is sufficient for systemic activation of iNKT cells. When pulsed with targeted liposomes, human monocyte-derived dendritic cells expressing CD169/Sn activated human iNKT cells, demonstrating the conservation of the CD169/Sn endocytic pathway capable of presenting lipid antigens to iNKT cells.
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Cellular and molecular mechanisms regulating the hepatic erythropoietin expression during acute-phase response: a role for IL-6. J Transl Med 2010; 90:1306-24. [PMID: 20458283 DOI: 10.1038/labinvest.2010.85] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023] Open
Abstract
The source of circulating erythropoietin (EPO), the mediators and the mechanisms involved in the upregulation of EPO gene expression during acute-phase reaction are still poorly understood. Acute-phase reaction was induced by either intramuscular turpentine oil (TO) or intraperitoneal lipopolysaccharide (LPS) administration into wild-type and interleukin (IL)-6 knockout (KO) mice. Animals were killed at different time points and blood, liver and muscle tissue were collected. Serum levels of EPO were measured by enzyme-linked immunoadsorbent assay; liver and injured muscle samples were processed for RNA isolation and for protein analysis. EPO, hypoxia-inducible factors 1alpha and 2alpha (HIF-1alpha and HIF-2alpha) mRNA were analyzed by RT-PCR and the protein levels were analyzed by western blot and electrophoretic mobility shift assay. HIF-1alpha and HIF-2alpha localization was performed through immunofluorescence staining. EPO, HIF-1 and HIF-2 gene and protein expression levels were also analyzed in isolated mouse hepatocytes after stimulation with IL-6. In the wild-type animals, EPO serum levels increased dramatically at 12 h after the insults together with the hepatic gene expression. In TO-treated animals, the EPO gene expression reached an 8.2-fold increase at 12 h, and in LPS-treated mice a similar induction was recorded at 6 h (about 4.5-fold increase). In the IL-6KO strain, the upregulation after the inflammatory stimuli was much lower (only 2.0-fold increase). A progressive upregulation of HIF-1alpha and HIF-2alpha was detectable until 6 h after the insults, but only HIF-1alpha upregulation was reduced in IL-6KO mice. In isolated hepatocytes, stimulation with a single dose of IL-6 induced a nuclear accumulation of HIF-1alpha, in parallel with an increase of EPO mRNA. No effect on HIF-2alpha expression was found. IL-6 appears to be the main regulator of EPO gene expression and a major contributor for HIF-1alpha induction in hepatocytes and Kupffer cells during acute-phase response. The increase of HIF-2alpha, predominantly expressed in endothelial cells and fibroblast-like cells, seems not to be affected by the lack of IL-6.
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Young EWK, Simmons CA. Macro- and microscale fluid flow systems for endothelial cell biology. LAB ON A CHIP 2010; 10:143-60. [PMID: 20066241 DOI: 10.1039/b913390a] [Citation(s) in RCA: 152] [Impact Index Per Article: 10.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/18/2023]
Abstract
Recent advances in microfluidics have brought forth new tools for studying flow-induced effects on mammalian cells, with important applications in cardiovascular, bone and cancer biology. The plethora of microscale systems developed to date demonstrate the flexibility of microfluidic designs, and showcase advantages of the microscale that are simply not available at the macroscale. However, the majority of these systems will likely not achieve widespread use in the biological laboratory due to their complexity and lack of user-friendliness. To gain widespread acceptance in the biological research community, microfluidics engineers must understand the needs of cell biologists, while biologists must be made aware of available technology. This review provides a critical evaluation of cell culture flow (CCF) systems used to study the effects of mechanical forces on endothelial cells (ECs) in vitro. To help understand the need for various designs of CCF systems, we first briefly summarize main properties of ECs and their native environments. Basic principles of various macro- and microscale systems are described and evaluated. New opportunities are uncovered for developing technologies that have potential to both improve efficiency of experimentation as well as answer important biological questions that otherwise cannot be tackled with existing systems. Finally, we discuss some of the unresolved issues related to microfluidic cell culture, suggest possible avenues of investigation that could resolve these issues, and provide an outlook for the future of microfluidics in biological research.
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Affiliation(s)
- Edmond W K Young
- Department of Biomedical Engineering, Wisconsin Institutes for Medical Research, University of Wisconsin-Madison, 1111 Highland Avenue, Madison, WI 53705, USA.
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Piscaglia F, Dudás J, Knittel T, Di Rocco P, Kobold D, Saile B, Zocco MA, Timpl R, Ramadori G. Expression of ECM proteins fibulin-1 and -2 in acute and chronic liver disease and in cultured rat liver cells. Cell Tissue Res 2009; 337:449-462. [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] [Key Words] [MESH Headings] [Track Full Text] [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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Affiliation(s)
- Fabio Piscaglia
- Department of Internal Medicine and Gastroenterology, University of Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany
- Present Address: Divisione di Medicina Interna – Bolondi, University of Bologna, Via Albertoni 15, 40138 Bologna, Italy
| | - József Dudás
- Department of Internal Medicine and Gastroenterology, University of Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany
- Present Address: Department of Otorhinolaryngology, Medical University of Innsbruck, Anichstrasse 35, 6020 Innsbruck, Austria
| | - Thomas Knittel
- Department of Internal Medicine and Gastroenterology, University of Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany
| | - Paola Di Rocco
- Department of Internal Medicine and Gastroenterology, University of Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany
| | - Dominik Kobold
- Department of Internal Medicine and Gastroenterology, University of Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany
| | - Bernhard Saile
- Department of Internal Medicine and Gastroenterology, University of Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany
| | - Maria Assunta Zocco
- Department of Internal Medicine and Gastroenterology, University of Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany
| | - Rupert Timpl
- Max-Planck-Institut für Biochemie, 82152 Martinsried, Germany
| | - Giuliano Ramadori
- Department of Internal Medicine and Gastroenterology, University of Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany
- Department of Gastroenterology and Endocrinology, Robert-Koch-Strasse 40, 37075 Göttingen, Germany
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Abstract
Chronic consumption of ethanol induces hepatic steatosis and inflammation, which can eventually lead to more severe liver injury, characterized by fibrosis and cirrhosis. Recruitment of neutrophils to the liver, as well as activation of Kupffer cells, mediates the inflammatory responses observed after chronic ethanol exposure. Kupffer cells, the resident macrophages of the liver, are critical to the onset of ethanol-induced liver injury. Activation of Kupffer cells leads to an increased production of proinflammatory cytokines, such as tumor necrosis factor-alpha and also reactive oxygen species, a process mediated in part by changes in lipopolysaccharide-induced TLR4-dependent signal transduction. The isolation and culture of Kupffer cells is an important technique with which one can elucidate the mechanisms that contribute to alcoholic liver injury.
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Neubauer K, Lindhorst A, Tron K, Ramadori G, Saile B. Decrease of PECAM-1-gene-expression induced by proinflammatory cytokines IFN-gamma and IFN-alpha is reversed by TGF-beta in sinusoidal endothelial cells and hepatic mononuclear phagocytes. BMC PHYSIOLOGY 2008; 8:9. [PMID: 18466611 PMCID: PMC2396664 DOI: 10.1186/1472-6793-8-9] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/13/2007] [Accepted: 05/08/2008] [Indexed: 12/16/2022]
Abstract
Background and aim The mechanisms of transmigration of inflammatory cells through the sinusoids are still poorly understood. This study aims to identify in vitro conditions (cytokine treatment) which may allow a better understanding of the changes in PECAM (platelet endothelial cell adhesion molecule)-1-gene-expression observed in vivo. Methods and results In this study we show by immunohistochemistry, that there is an accumulation of ICAM-1 (intercellular cell adhesion molecule-1) and ED1 positive cells in necrotic areas of livers of CCl4-treated rats, whereas there are few PECAM-1 positive cells observable. After the administration of CCl4, we could detect an early rise of levels of IFN-γ followed by an enhanced TGF-β protein level. As shown by Northern blot analysis and surface protein expression analysed by flow cytometry, IFN-γ-treatment decreased PECAM-1-gene-expression in isolated SECs (sinusoidal endothelial cells) and mononuclear phagocytes (MNPs) in parallel with an increase in ICAM-1-gene-expression in a dose and time dependent manner. In contrast, TGF-β-treatment increased PECAM-1-expression. Additional administration of IFN-γ to CCl4-treated rats and observations in IFN-γ-/- mice confirmed the effect of IFN-γ on PECAM-1 and ICAM-1-expression observed in vitro and increased the number of ED1-expressing cells 12 h after administration of the toxin. Conclusion The early decrease of PECAM-1-expression and the parallel increase of ICAM-1-expression following CCl4-treatment is induced by elevated levels of IFN-γ in livers and may facilitate adhesion and transmigration of inflammatory cells. The up-regulation of PECAM-1-expression in SECs and MNPs after TGF-β-treatment suggests the involvement of PECAM-1 during the recovery after liver damage.
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Affiliation(s)
- Katrin Neubauer
- University of Göttingen, Department of Internal Medicine, Section of Gastroenterology and Endocrinology, Göttingen, Germany.
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Aprigliano I, Dudas J, Ramadori G, Saile B. Atorvastatin induces apoptosis by a caspase-9-dependent pathway: an in vitro study on activated rat hepatic stellate cells. Liver Int 2008; 28:546-57. [PMID: 18339080 PMCID: PMC2324535 DOI: 10.1111/j.1478-3231.2008.01682.x] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
BACKGROUND Statins are shown to have cholesterol-independent properties such as anti-inflammation and immunomodulation. Activated hepatic stellate cells (HSCs) acquire the capacity to synthesize matrix proteins in damaged liver. We tested the hypothesis that atorvastatin may be capable of inducing apoptosis in HSCs. METHODS Primary cultures of rat HSCs were exposed to atorvastatin, mevalonic acid and U0126. Quantification of living, apoptotic and necrotic HSCs was performed by flow cytometry and laser-scan microscopy. Cell-cycle analysis was performed by flow cytometry. Pro- and anti-apoptotic factors were investigated by Western blot and electrophoresis mobility shift assay. Protease activity of caspases was calculated using a colorimetric kit. RESULTS Atorvastatin leads to a G2-arrest and induces apoptosis in activated HSCs. Atorvastatin-mediated apoptosis could be blocked by co-administration of mevalonic acid and U0126. No effects of atorvastatin on gene expression of CD95, CD95L, NF-kappaB, p53 and p21WAF1 could be observed. Atorvastatin-induced apoptosis in activated HSCs is related to an increased protease activity of caspase-9 and -3. Gene expression of the major proteins of the bcl-system shows that truncated Bid is involved in apoptosis mediated by atorvastatin. By blocking the extracellular signal-regulated protein kinase (ERK1/2) activation by adding U0126, we could prevent the apoptosis induced by atorvastatin. By Western blot we could not detect any change in the activation of c-jun N-terminal kinase (JNK). CONCLUSIONS Atorvastatin induces apoptosis in activated HSCs acting through an ERK-dependent cleavage of Bid and a highly increased protease activity of caspase-9 and -3. JNK is not involved in atorvastatin-mediated apoptosis in HSCs.
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Affiliation(s)
- Isabella Aprigliano
- Department of Internal Medicine, Section of Gastroenterology and Endocrinology, University of Göttingen, Göttingen, Germany
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15
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Dudas J, Mansuroglu T, Batusic D, Saile B, Ramadori G. Thy-1 is an in vivo and in vitro marker of liver myofibroblasts. Cell Tissue Res 2007; 329:503-14. [PMID: 17576600 DOI: 10.1007/s00441-007-0437-z] [Citation(s) in RCA: 63] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2007] [Accepted: 05/09/2007] [Indexed: 12/26/2022]
Abstract
Thy-1, a glycophosphatidylinositol-linked glycoprotein of the outer membrane leaflet, has been described in myofibroblasts of several organs. Previous studies have shown that, in fetal liver, Thy-1 is expressed in a subpopulation of ductular/progenitor cells. The aim of this study has been to investigate whether the liver myofibroblasts belong to the Thy-1-positive subpopulation of the adult liver. The expression of Thy-1 has been studied in normal rat liver, in the rat liver regeneration model following 2-acetylaminofluorene treatment and partial hepatectomy (AAF/PH), and in isolated rat liver cells, at the mRNA and protein levels. In normal rat liver, Thy-1 is detected in sparse cells of the periportal area, whereas 7 days after PH in the AAF/PH model, a marked increase of the number of Thy-1-positive cells is detectable by immunohistochemistry. Comparative immunohistochemical analysis has revealed the co-localization of Thy-1 and smooth muscle actin, but not of Thy-1 and cytokeratin-19, both in normal rat liver and in the AAF/PH model. Investigation of isolated rat liver cell populations has confirmed that liver myofibroblasts are Thy-1-positive cells, whereas hepatocytes, hepatic stellate cells, and liver macrophages are not. Thy-1 is the first cell surface marker for identifying liver myofibroblasts in vivo and in vitro.
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Affiliation(s)
- Jozsef Dudas
- Department of Internal Medicine, Section of Gastroenterology and Endocrinology, Georg August University Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany
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16
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Taubert A, Hermosilla C. Bovine recombinant IFNγ induces endothelial cell gene transcription of immunoregulatory molecules and upregulates PMN and PBMC adhesion on bovine endothelial cells. Vet Res Commun 2007; 32:35-47. [PMID: 17516142 DOI: 10.1007/s11259-007-9001-2] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2007] [Accepted: 04/05/2007] [Indexed: 11/30/2022]
Abstract
Interferon gamma (IFNgamma) is an important modulator of immune responses acting on multiple cell types, such as lymphocytes, macrophages or endothelial cells. We investigated the effects of recombinant bovine IFNgamma on bovine umbilical vein endothelial cells (BUVEC) for the level of polymorphonuclear neutrophil cell (PMN)- and peripheral blood mononuclear cell (PBMC)-adhesion as well as the gene transcription of endothelial cell-derived adhesion molecules (E-selectin, P-selectin, VCAM-1, ICAM-1), chemokines (CXCL1, CXCL8, CXCL10, CCL2, CCL5), GM-CSF, iNOS and COX-2 in comparison to TNFalpha-stimulation. IFNgamma strongly induced PMN and PBMC adhesion on BUVEC involving CD4(+), CD8(+) and gammadelta-TCR(+) (WC1(+)) lymphocytes. Furthermore, IFNgamma-stimulation led to a strong upregulation in the transcription of VCAM-1, ICAM-1, CXCL10 and CCL2 genes and to a low to moderate increase in the E- and P-selectin, CXCL1, CXCL8, CCL5, COX-2 and iNOS gene transcripts, but failed to enhance GM-CSF gene transcription. These results indicate that IFNgamma can be considered an important activator of endothelial cells in the bovine system, most probably by influencing the outcome of inflammatory responses through selective upregulation of immunoregulatory molecules.
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Affiliation(s)
- A Taubert
- Institute of Parasitology, Justus Liebig University Giessen, Rudolf-Buchheim-Str. 2, D-35392 Giessen, Germany.
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17
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DeLeve LD, Wang X, McCuskey MK, McCuskey RS. Rat liver endothelial cells isolated by anti-CD31 immunomagnetic separation lack fenestrae and sieve plates. Am J Physiol Gastrointest Liver Physiol 2006; 291:G1187-9. [PMID: 16782698 DOI: 10.1152/ajpgi.00229.2006] [Citation(s) in RCA: 56] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
The gold standard for the identification of sinusoidal endothelial cells (SEC) is the presence of fenestrae organized in sieve plates, which is characteristic of SEC in vivo. One of the methods currently in use to isolate SEC is immunomagnetic sorting for CD31. However, there is evidence to suggest that CD31 is not present on the surface of differentiated SEC. The present study used scanning electron microscopy to image rat hepatic endothelial cells isolated by anti-CD31 and immunomagnetic sorting and cells isolated by gradient centrifugation and centrifugal elutriation. Cells isolated by elutriation had well-developed fenestrae and sieve plates, whereas cells isolated by anti-CD31 and immunomagnetic sorting had significantly fewer fenestrae organized in sieve plates. In conclusion, cells isolated by anti-CD31 and immunomagnetic sorting lacked the hallmark features of SEC.
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Affiliation(s)
- Laurie D DeLeve
- Research Center for Liver Diseases and the Division of Gastrointestinal and Liver Diseases, Keck School of Medicine at the University of Southern California, Los Angeles, California 90033, USA.
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18
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Quondamatteo F, Krick W, Hagos Y, Krüger MH, Neubauer-Saile K, Herken R, Ramadori G, Burckhardt G, Burckhardt BC. Localization of the sulfate/anion exchanger in the rat liver. Am J Physiol Gastrointest Liver Physiol 2006; 290:G1075-81. [PMID: 16357056 DOI: 10.1152/ajpgi.00492.2005] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Although the sulfate/anion transporter (sat-1; SLC26A1) was isolated from a rat liver cDNA library by expression cloning, localization of sat-1 within the liver and its contribution to the transport of sulfate and organo sulfates have remained unresolved. In situ hybridization and immunohistochemical studies were undertaken to demonstrate the localization of sat-1 in liver tissue. RT-PCR studies on isolated hepatocytes and liver endothelial and stellate cells in culture were performed to test for the presence of sat-1 in these cells. In sulfate uptake and efflux experiments, the substrate specificity of sat-1 was evaluated. Sat-1 mRNA was found in hepatocytes and endothelial cells. Sat-1 protein was localized in sinusoidal membranes and along the borders of hepatocytes. The canalicular region and bile capillaries were not stained. Sulfate uptake was only slightly affected by sulfamoyl diuretics or organo sulfates. Sulfate efflux from sat-1-expressing oocytes was enhanced in the presence of bicarbonate, indicating sulfate/bicarbonate exchange. Estrone sulfate was not transported by sat-1. Sat-1 may be responsible for the uptake of inorganic sulfate from the blood into hepatocytes to enable sulfation reactions. In hepatocytes and endothelial cells, sat-1 may also supply sulfate for proteoglycan synthesis.
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Affiliation(s)
- Fabio Quondamatteo
- Abteilung Histologie, Georg August Universität Göttingen, Humboldtallee 23, Göttingen 37073, Germany
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19
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Gressner AM, Lahme B, Meurer SK, Gressner O, Weiskirchen R. Variable expression of cystatin C in cultured trans-differentiating rat hepatic stellate cells. World J Gastroenterol 2006; 12:731-8. [PMID: 16521186 PMCID: PMC4066123 DOI: 10.3748/wjg.v12.i5.731] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To study the expression of cystatin C (CysC), its regulation by transforming growth factor-β1 (TGF-β1) and platelet-derived growth factor (PDGF) and the potential interference of CysC with TGF-β1 signaling in this special cell type.
METHODS: We evaluated the CysC expression in cultured, profibrogenic hepatic stellate cells and trans-differentiated myofibroblasts by Northern and Western blotting and confocal laser scanning microscopy.
RESULTS: CysC was increased significantly in the course of trans-differentiation. Both TGF-β1 and PDGF-BB suppressed CysC expression. Furthermore, CysC secretion was induced by the treatment with TGF-β1. Although CysC induced an increased binding affinity of TGF-β receptor type III (beta-glycan) as assessed by chemical cross-linking with [125I]-TGF-β1, it did not modulate TGF-β1 signal transduction as shown by evaluating the Smad2/3 phosphorylation status and [CAGA]-MLP-luciferase reporter gene assay. Interestingly, the shedding of type III TGF-β receptor beta-glycan was reduced in CysC-treated cells. Our data indicated that CysC expression was upregulated during trans-differentiation.
CONCLUSION: Increased CysC levels in the serum of patients suffering from liver diseases are at least partially due to a higher expression in activated hepatic stellate cells. Furthermore, TGF-β1 influences the secretion of CysC, highlighting a potentially important role of cysteine proteases in the progression of hepatic fibrogenesis.
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Affiliation(s)
- Axel M Gressner
- Institute of Clinical Chemistry and Pathobiochemistry, RWTH Aachen University, D-52074 Aachen, Germany.
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20
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Charalampopoulos I, Androulidaki A, Minas V, Chatzaki E, Tsatsanis C, Notas G, Xidakis C, Kolios G, Kouroumalis E, Margioris AN, Gravanis A. Neuropeptide urocortin and its receptors are expressed in rat Kupffer cells. Neuroendocrinology 2006; 84:49-57. [PMID: 17090973 DOI: 10.1159/000096827] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/29/2006] [Accepted: 09/01/2006] [Indexed: 01/13/2023]
Abstract
The stress neuropeptides, corticotropin-releasing hormone (CRH) and urocortin (UCN), modulate the inflammatory response via the hypothalamus-pituitary-adrenal axis and locally, in a paracrine manner, act on mast and macrophage cells. Kupffer cells (KCs) are the resident macrophages of the liver. They represent the bulk of tissue macrophages in the body and they are the first to face invading noxious agents reaching the body via the portal circulation. The aim of the present report was to study the expression of the CRH system in rat KC and test its functionality. Our findings are as follows: (1) In highly purified KCs the transcripts of UCN, of its receptors CRHR1, CRHR2 and that of the pseudoreceptor CRH-binding protein (CRHBP) were present while that of CRH was not detectable. (2) Similarly, immunoreactive UCN, CRHR1, CRHR2 and CRHBP were easily detectable by immunohistochemistry and immunofluorescence in sections of whole rat liver (localized in KC) as well as in purified KC while CRH was again not detectable. (3) Exposure of purified KC to CRH or UCN suppressed lipopolysaccharide-induced tumor necrosis factor alpha production, an effect completely prevented by the CRHR1 and CRHR2 receptor antagonist astressin. Our data demonstrate the presence of UCN and its receptors in rat KC, the absence of CRH, and the functionality of these receptors. We propose that a UCN-based system may affect local inflammatory phenomena in the liver acting in a paracrine manner.
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21
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Timmers M, Vekemans K, Vermijlen D, Asosingh K, Kuppen P, Bouwens L, Wisse E, Braet F. Interactions between rat colon carcinoma cells and Kupffer cells during the onset of hepatic metastasis. Int J Cancer 2004; 112:793-802. [PMID: 15386374 DOI: 10.1002/ijc.20481] [Citation(s) in RCA: 50] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Liver sinusoids harbor populations of 2 important types of immunocompetent cells, Kupffer cells (KCs) and natural killer (NK) cells, which are thought to play an important role in controlling hepatic metastasis in the first 24 hr upon arrival of the tumor cells in the liver. We studied the early interaction of KCs, NK and CC531s colon carcinoma cells in a syngeneic rat model by confocal laser scanning microscopy. Results showed a minority of KCs (19% periportal and 7% pericentral) involved in the interaction with 94% of tumor cells and effecting the phagocytosis of 92% of them. NK cell depletion decreased the phagocytosis of tumor cells by KCs by 33% over a period of 24 hr, leaving 35% of the cancer cells free, as compared to 6% in NK-positive rats. Surviving cancer cells were primarily located close to the Glisson capsule, suggesting that metastasis would initiate from this region.
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Affiliation(s)
- Maarten Timmers
- Laboratory for Cell Biology and Histology, Free University of Brussels, Brussels, Belgium
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22
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Saile B, DiRocco P, Dudas J, El-Armouche H, Sebb H, Eisenbach C, Neubauer K, Ramadori G. IGF-I induces DNA synthesis and apoptosis in rat liver hepatic stellate cells (HSC) but DNA synthesis and proliferation in rat liver myofibroblasts (rMF). J Transl Med 2004; 84:1037-49. [PMID: 15156158 DOI: 10.1038/labinvest.3700116] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Several lines of evidence suggest a role of insulin-like growth factor I (IGF-I) in the regulation of apoptosis. Up to now its impact on many specific cells is unknown. We therefore studied the effect of IGF-I on two similar mesenchymal matrix-producing cell types of the liver, the hepatic stellate cells (HSC) and the myofibroblasts (rMF). The present study aimed to reveal the influence of IGF-I on cell cycle and apoptosis of HSC and rMF and to elucidate responsible signaling. While IGF-I significantly increased DNA synthesis in HSC, cell number decreased and apoptosis increased. In rMF IGF-I also increased DNA synthesis, which is, however, followed by proliferation. Blocking extracellular signal regulating kinase (ERK) revealed that in HSC, bcl-2 upregulation and bax downregulation are effected downstream of ERK, whereas downregulation of NFkappaB and consecutive of bcl-xL is mediated upstream. In the rMF upregulation of both, the antiapoptotic bcl-2 and bcl-xL is mediated upstream of ERK. The expression of the proapoptotic bax is not regulated by IGF-I in rMF. The studies demonstrate a completely different effect and signaling of IGF-I in two morphologically and functionally similar matrix-producing cells of the liver.
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Affiliation(s)
- Bernhard Saile
- Department of Internal Medicine, Section of Gastroenterology and Endocrinology, University of Göttingen, Göttingen, Germany
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23
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Fukui H, Koike T, Saheki A, Sonoke S, Seki J. A novel delivery system for amphotericin B with lipid nano-sphere (LNS®). Int J Pharm 2003; 265:37-45. [PMID: 14522116 DOI: 10.1016/s0378-5173(03)00404-6] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
A low-dose therapeutic system with a lipid emulsion for amphotericin B (AmB), a potent antifungal drug, was studied. Lipid nano-sphere (LNS), a small-particle lipid emulsion, was taken up by the liver to a lesser extent than was a conventional lipid emulsion. As a result, LNS yielded higher plasma concentrations of a radiochemical tracer than did the conventional lipid emulsion. LNS was therefore judged to be a suitable carrier for a low-dose therapeutic system for AmB, and LNS incorporating AmB (LNS-AmB) was prepared. LNS-AmB was found to be a homogeneous emulsion with mean particle diameters ranging from 25 to 50 nm. LNS-AmB yielded higher plasma concentrations of AmB than did Fungizone, a conventional intravenous dosage form of AmB, after intravenous administration to mice, rats, dogs, and monkeys. This difference between LNS-AmB and Fungizone was also observed for constant intravenous infusion. In contrast to Fungizone, LNS-AmB showed a linear relationship between dose and AUC. These pharmacokinetic characteristics of LNS-AmB make it a suitable candidate for an effective low-dose therapeutic system for AmB.
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Affiliation(s)
- Hiroshi Fukui
- R&D Administration Department, Nippon Shinyaku Co. Ltd., 14, Nishinosho-Monguchi-cho, Kisshoin, Minami-ku, Kyoto 601-8550, Japan.
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24
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Neubauer K, Baruch Y, Lindhorst A, Saile B, Ramadori G. Gelsolin gene expression is upregulated in damaged rat and human livers within non-parenchymal cells and not in hepatocytes. Histochem Cell Biol 2003; 120:265-75. [PMID: 14574581 DOI: 10.1007/s00418-003-0564-x] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 07/21/2003] [Indexed: 12/17/2022]
Abstract
Gelsolin, a 90-kDa protein, was suggested to be involved in cell motility, to inhibit apoptosis and to have a protective role for tissue. This study intends to analyse the modulation of cytoplasmic gelsolin expression in damaged rat and human livers and to identify its cellular sources. In the normal liver gelsolin-immunoreactive cells could be identified along vessel walls and along the sinusoids. In cultured rat hepatic stellate cells (HSCs), liver myofibroblasts (MFs), mononuclear cells (MCs) and sinusoidal endothelial cells (SECs), but not in hepatocytes, gelsolin expression could be detected by immunostaining and Northern blot analysis. In acute CCl4-induced liver damage there was no gelsolin positivity detectable in necrotic areas. However, in human fulminant hepatic failure positivity in the necrotic areas was detected. In chronically damaged rat and human livers gelsolin-immunoreactive cells could be identified within the fibrotic septa. Northern blot analysis revealed an increase of the gelsolin-specific transcript level under conditions of acute and chronic human or rat liver damage. The amount of gelsolin-specific transcripts in SECs and large MCs isolated from damaged rat livers increased in comparison to cells obtained from normal rats. However, the amount of gelsolin-specific transcripts in small MCs (representing recruited inflammatory cells) decreased. In conclusion, SECs, MCs, MFs and HSCs, but not hepatocytes, express gelsolin. In the damaged liver all tested cell populations but the inflammatory cells and the hepatocytes are responsible for the enhanced gelsolin expression.
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Affiliation(s)
- Katrin Neubauer
- Department of Internal Medicine, Section of Gastroenterology and Endocrinology, Georg-August-University Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany
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25
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Saile B, Eisenbach C, El-Armouche H, Neubauer K, Ramadori G. Antiapoptotic effect of interferon-alpha on hepatic stellate cells (HSC): a novel pathway of IFN-alpha signal transduction via Janus kinase 2 (JAK2) and caspase-8. Eur J Cell Biol 2003; 82:31-41. [PMID: 12602946 DOI: 10.1078/0171-9335-00285] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
The hepatic stellate cell (HSC), the pericyte of the liver sinusoids belongs to the mesenchymal cells of the liver. Damaging noxae induce a transformation from the quiescent (vitamin A-storing cell) to the activated (connective tissue-producing cell) state. The balance between proapoptotic and surviving factors decides about the fate of the activated HSC. Interferon-alpha (IFN-alpha) has been shown to elicit antiproliferative and/or antifibrogenic effects in various cell types of mesenchymal origin. We therefore investigated the effect of IFN-alpha on primary cultured rat HSC in their quiescent (day 2) and activated state (day 7). IFN-alpha significantly inhibited spontaneous apoptosis in activated HSC in vitro and simultaneously inhibited cell cycle progression by inducing a G1 arrest. The effect of IFN-a is not accompanied by a modulation of CD95, CD95L, p53, p21(WAF1), p27, bcl-2, bcl-xL, bax, NFkappaB, or IkappaB gene expression. Surprisingly, the IFN-alpha effect could be abolished completely by blocking JAK2 activity or JAK2 translation. The downregulating effect of IFN-alpha on the activity of caspase-8 and caspase-3 could also be neutralized using tyrphostin AG490 or JAK-2 antisense. Taken together IFN-alpha inhibits apoptosis of activated HSC by activation of JAK2 which inhibits the caspase-8 apoptosis pathway.
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Affiliation(s)
- Bernhard Saile
- Department of Internal Medicine, Section of Gastroenterology and Endocrinology, University of Göttingen, Göttingen, Germany
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26
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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: 33] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
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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27
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Weiskirchen R, Moser M, Weiskirchen S, Erdel M, Dahmen S, Buettner R, Gressner AM. LIM-domain protein cysteine- and glycine-rich protein 2 (CRP2) is a novel marker of hepatic stellate cells and binding partner of the protein inhibitor of activated STAT1. Biochem J 2001; 359:485-96. [PMID: 11672422 PMCID: PMC1222169 DOI: 10.1042/0264-6021:3590485] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
Activation of hepatic stellate cells is considered to be the main step in the development of liver fibrosis, which is characterized by the transition of quiescent vitamin-A-rich cells to proliferative, fibrogenic and contractile myofibroblasts. The identification of regulatory genes during early cell activation and transdifferentiation is essential to extend our knowledge of hepatic fibrogenesis. In liver, the gene CSRP2 is exclusively expressed by stellate cells, whereas no transcripts are detectable in hepatocytes, sinusoidal endothelial cells or Kupffer cells. The early activation of stellate cells induced by platelet-derived growth factor is accompanied by an enhanced expression of CSRP2. During later stages of transdifferentiation, the expression of CSRP2 in these cells is suppressed in vitro and in vivo. The CSRP2-encoded cysteine- and glycine-rich double-LIM-domain protein (CRP)2 is proposed to function as a molecular adapter, arranging two or more as yet unidentified protein constituents into a macromolecular complex. To identify these proteins and assign a cellular function to CRP2, a human cDNA library was screened with full-length CRP2 as bait in a yeast two-hybrid screen. The protein inhibitor of activated STAT1 ('PIAS1') was shown to associate selectively with the C-terminal LIM domain of CRP2. Physical interaction of both proteins in the cellular environment was confirmed by co-localization experiments with confocal laser scanning microscopy and co-immunoprecipitation analysis. These results establish CRP2 as a potential new factor in the JAK/STAT-signalling pathway and suggest that the suppression of CSRP2 might be a prerequisite for the myofibroblastic transition of hepatic stellate cells.
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Affiliation(s)
- R Weiskirchen
- Institute of Clinical Chemistry and Pathobiochemistry, RWTH-University Hospital, Pauwelsstrasse 30, D-52074 Aachen, Germany.
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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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Shah V, Cao S, Hendrickson H, Yao J, Katusic ZS. Regulation of hepatic eNOS by caveolin and calmodulin after bile duct ligation in rats. Am J Physiol Gastrointest Liver Physiol 2001; 280:G1209-16. [PMID: 11352814 DOI: 10.1152/ajpgi.2001.280.6.g1209] [Citation(s) in RCA: 63] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
In carbon tetrachloride-induced liver cirrhosis, diminution of hepatic endothelial nitric oxide synthase (eNOS) activity may contribute to impaired hepatic vasodilation and portal hypertension. The mechanisms responsible for these events remain unknown; however, a role for the NOS-associated proteins caveolin and calmodulin has been postulated. The purpose of this study is to characterize the expression and cellular localization of the NOS inhibitory protein caveolin-1 in normal rat liver and to then examine the role of caveolin in conjunction with calmodulin in regulation of NOS activity in cholestatic portal hypertension. In normal liver, caveolin protein is expressed preferentially in nonparenchymal cells compared with hepatocytes as assessed by Western blot analysis of isolated cell preparations. Additionally, within the nonparenchymal cell populations, caveolin expression is detected within both liver endothelial cells and hepatic stellate cells. Next, studies were performed 4 wk after bile duct ligation (BDL), a model of portal hypertension characterized by prominent cholestasis, as evidenced by a significant increase in serum cholesterol in BDL animals. After BDL, caveolin protein levels from detergent-soluble liver lysates are significantly increased as assessed by Western blot analysis. Immunoperoxidase staining demonstrates that this increase is most prominent within sinusoids and venules. Additionally, caveolin-1 upregulation is associated with a significant reduction in NOS catalytic activity in BDL liver lysates, an event that is corrected with provision of excess calmodulin, a protein that competitively binds eNOS from caveolin. We conclude that, in cholestatic portal hypertension, caveolin may negatively regulate NOS activity in a manner that is reversible by excess calmodulin.
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Affiliation(s)
- V Shah
- Gastrointestinal Research Unit, Mayo Clinic, Rochester, MN 55905, USA.
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Kaneo Y, Tanaka T, Nakano T, Yamaguchi Y. Evidence for receptor-mediated hepatic uptake of pullulan in rats. J Control Release 2001; 70:365-73. [PMID: 11182206 DOI: 10.1016/s0168-3659(00)00368-0] [Citation(s) in RCA: 130] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
Fluorescein-labeled pullulan (FP-60; MW 58,200) was prepared by reaction with FITC according to the method of de Belder and Granath. The hepatic distribution of FP-60 was examined using a specific high-performance size-exclusion chromatography. Intravenously administered FP-60 was rapidly eliminated from the blood circulation followed by an appreciable distribution to the liver. A marked dose-dependency was seen in the hepatic uptake of FP-60 which was markedly reduced by the coadministration of both asialofetuin and arabinogalactan. Measurement of the hepatocellular localization demonstrated the overwhelming distribution of FP-60 in the parenchymal liver cell fraction. Furthermore, microscopic examination revealed that FP-60 was effectively endocytosed by the parenchymal liver cells. Radiolabeled pullulan ([(125)I]P-60) was prepared by (125)I-labeling the tyramine derivative of pullulan which was synthesized by the cyano-transfer method. [(125)I]P-60 was predominantly accumulated in sliced rat liver tissue at 37 degrees C, which was drastically inhibited by the addition of both asialofetuin and arabinogalactan. The kinetic parameters of the specific binding of [(125)I]P-60 to monolayered hepatocytes at 0 degrees C were almost identical to those for asialofetuin. The binding of [(125)I]P-60 to isolated parenchymal cells was significantly inhibited by arabinogalactan and asialofetuin, however dextran, the same glucan as pullulan, did not affect the binding of [(125)I]P-60. It was found that pullulan, which is bound to the asialoglycoprotein receptor with high affinity, is subsequently internalized to the hepatocyte via receptor-mediated endocytosis.
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Affiliation(s)
- Y Kaneo
- Laboratory of Biopharmaceutics, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Fukuyama, Hiroshima 729-0292, Japan.
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31
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Masson S, Daveau M, François A, Bodenant C, Hiron M, Ténière P, Salier JP, Scotté M. Up-regulated expression of HGF in rat liver cells after experimental endotoxemia: a potential pathway for enhancement of liver regeneration. Growth Factors 2001; 18:237-50. [PMID: 11519823 DOI: 10.3109/08977190109029113] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
A lipopolysaccharide (LPS)-induced inflammation prior to an hepatic resection has been shown to enhance liver regeneration in rat. The aim of the present study was to investigate the expression of hepatocyte growth factor (HGF) and its c-Met receptor under such experimental conditions. Animals were submitted to a two-third hepatectomy or a LPS challenge carried out 12 h prior to resection. Non parenchymal and parenchymal cells were isolated from livers obtained at various times post-hepatectomy. Quantitative RT-PCR for HGF and c-Met mRNAs were performed from total liver or purified cell fractions and HGF mRNA was also analyzed by in situ RT-PCR on liver sections. A LPS challenge alone induced a marked up-regulation of HGF mRNA level in whole liver and isolated hepatocytes. Furthermore, when partial hepatectomy (PH) was preceded by a LPS challenge, an increase of HGF mRNA level was seen in whole liver and contrasted with a decreased level in non parenchymal cells. These results were confirmed by in situ RT-PCR. In isolated hepatocytes from endotoxemic rats, the mRNA level for the LPS-specific membranous receptor mCD14 was markedly up-regulated and even more so when LPS was followed by PH. Moreover, a TNFalpha challenge alone induced an up-regulation of HGF mRNA in hepatocytes and a down-regulation in non parenchymal cells (NPCs). Overall, when a LPS challenge is given prior to PH the major source of hepatic HGF appears to be the hepatocyte itself rather than NPCs. An autocrine HGF/c-Met loop which promotes the proliferative potential of the hepatic parenchymal cell and participates in liver regeneration is postulated.
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Affiliation(s)
- S Masson
- INSERM Unit 519, and Institut Fédératif de Recherches Multidisciplinaires sur les Peptides, Rouen, France
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Tsutusmi A, Shiota G, Yamazaki H, Kunisada T, Terada T, Kawasaki H. Accelerated growth of hepatocytes in association with Up-regulation of cyclin E in transgenic mice expressing the dominant negative form of retinoic acid receptor. Biochem Biophys Res Commun 2000; 278:229-35. [PMID: 11071877 DOI: 10.1006/bbrc.2000.3786] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Retinoids play an important role in pathogenesis of liver diseases. To clarify the functional role of retinoic acid (RA) in liver, we developed transgenic mice (Tg) which express the dominant negative form of retinoic acid receptor (RARE) in liver. Here, we report that proliferation of hepatocytes in RARE Tg is greatly enhanced and that cyclin E is up-regulated in RARE Tg. Liver weight, liver/body weight, and proliferating cell nuclear antigen (PCNA) labeling index in RARE Tg were significantly increased, compared to those in wild-type mice (P < 0.01, each). Cell cycle analysis showed that 2N DNA content cells and aneuploid area between 2N and 4N DNA, reflecting S phase cells, were significantly increased in RARE Tg, compared to wild-type mice (P < 0.01, each). Of G1 phase-related proteins including cyclins, cyclin-dependent protein kinases (CDKs) and cyclin-dependent protein kinase inhibitors (CKIs), cyclin E mRNA and protein was up-regulated in liver from RARE Tg by reverse transcription polymerase chain reaction and Western blot analysis. Furthermore, the immunoprecipitation with anti-cdk2 antibody, followed by Western blot analysis with anti-cyclin E antibody indicated that cyclin E/cdk2 complex is increased in liver of RARE Tg. The results of the present study suggest that cyclin E in association with cdk2 governs cell cycle progression through G1 in hepatocytes where function of RA is inhibited.
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Affiliation(s)
- A Tsutusmi
- Second Department of Internal Medicine, Faculty of Medicine, Tottori University, Yonago 683-8504, Japan
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Shah V, Chen AF, Cao S, Hendrickson H, Weiler D, Smith L, Yao J, Katusic ZS. Gene transfer of recombinant endothelial nitric oxide synthase to liver in vivo and in vitro. Am J Physiol Gastrointest Liver Physiol 2000; 279:G1023-30. [PMID: 11053000 DOI: 10.1152/ajpgi.2000.279.5.g1023] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) contributes to hepatic vascular homeostasis. The aim of this study was to examine whether delivery of an adenoviral vector encoding eNOS gene to liver affects vasomotor function in vivo and the mechanism of NO production in vitro. Rats were administered adenoviruses encoding beta-galactosidase (AdCMVLacZ) or eNOS (AdCMVeNOS) via tail vein injection and studied 1 wk later. In animals transduced with AdCMVLacZ, beta-galactosidase activity was increased in the liver, most prominently in hepatocytes. In AdCMVeNOS-transduced animals, eNOS protein levels and catalytic activity were significantly increased. Overexpression of eNOS diminished baseline perfusion pressure and constriction in response to the alpha(1)-agonist methoxamine in the perfused liver. Transduction of cultured hepatocytes with AdCMVeNOS resulted in the targeting of recombinant eNOS to a perinuclear distribution and binding with the NOS-activating protein heat shock protein 90. These events were associated with increased ionomycin-stimulated NO release. In summary, this is the first study to demonstrate successful delivery of the recombinant eNOS gene to liver in vivo and in vitro with ensuing NO production.
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Affiliation(s)
- V Shah
- Gastrointestinal Research Unit and Anesthesia Research Unit, Mayo Clinic, Rochester, Minnesota 55905, USA.
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Neubauer K, Ritzel A, Saile B, Ramadori G. Decrease of platelet-endothelial cell adhesion molecule 1-gene-expression in inflammatory cells and in endothelial cells in the rat liver following CCl(4)-administration and in vitro after treatment with TNFalpha. Immunol Lett 2000; 74:153-64. [PMID: 10996391 DOI: 10.1016/s0165-2478(00)00203-0] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
UNLABELLED Platelet-endothelial cell adhesion molecule (PECAM-1), a member of the Ig superfamily is strongly expressed at endothelial cell-cell junctions, on most leukocytes and on monocytes. PECAM-1 has been implicated as a key mediator of the transendothelial migration of leukocytes and monocytes. To further define the physiological role of PECAM-1, we studied the modulation of PECAM-1-expression in a model of liver inflammation in both mononuclear cells (MCs) and sinusoidal endothelial cells (SECs). In normal rat liver sections, PECAM-1 immunohistology indicated a sinusoidal pattern similar to the ICAM-1 staining. Both, SECs, small and large MCs isolated from control rats express PECAM-1 as demonstrated by immunocytochemistry, flow cytometry, and Northern blot analysis. Immunohistochemical studies on liver sections from CCl(4)-treated animals indicated, that in the areas of necrosis 24-48 h after a single administration of the toxin, there was an accumulation of LFA-1-, ED1- (marker for rat monocytes) and ICAM-1-positive, but ED2-(marker for tissue macrophages)-negative inflammatory cells. Most of these cells were PECAM-1-negative. In situ hybridization indicated that there is no accumulation of PECAM-1 specific transcripts after CCl(4) treatment within the pericentral region. Immunocytology, flow cytometry, and Northern blot analysis of MCs and SECs isolated at different times after the administration of CCl(4) revealed a decrease of PECAM-1 gene expression in MCs and in SECs, whereas ICAM-1 expression increased. As TNFalpha has been shown to be upregulated early after CCl(4) administration, the influence of TNFalpha on PECAM-gene-expression was analyzed. TNFalpha treatment of cultured rat SECs and of small and large MCs from normal liver decreased PECAM-1 specific transcript level in parallel to the increase of ICAM-1 transcript level. CONCLUSIONS Early production of TNFalpha after liver injury could induce an increased ICAM-1-expression and a decreased PECAM-1 expression, which might be essential for the transmigration of inflammatory cells into the parenchyma.
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Affiliation(s)
- K Neubauer
- University of Göttingen, Department of Internal Medicine, Section of Gastroenterology and Endocrinology, Georg-August-University Göttingen, Robert-Koch-Strasse 40, D-37075, Göttingen, Germany
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Neubauer K, Wilfling T, Ritzel A, Ramadori G. Platelet-endothelial cell adhesion molecule-1 gene expression in liver sinusoidal endothelial cells during liver injury and repair. J Hepatol 2000; 32:921-32. [PMID: 10898312 DOI: 10.1016/s0168-8278(00)80096-3] [Citation(s) in RCA: 37] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/30/2023]
Abstract
BACKGROUND/AIMS Platelet-endothelial cell adhesion molecule (PECAM)-1 is suggested to be critical for transmigration processes. It is a matter of debate whether PECAM-1 is expressed in liver sinusoids and whether it is involved in liver inflammation. METHODS Indirect immunostaining and in situ hybridization was used to analyze PECAM-1 gene expression in normal and diseased rat and human livers as well as in isolated rat sinusoidal endothelial cells (SECs), hepatic stellate cells and hepatocytes. At various time points after the administration of CCl4 (6 h, 24 h, 48 h, and 72 h), PECAM-1 gene expression was analyzed in livers and in SECs by immunostaining, and Northern blot analysis. RESULTS In normal rat or human livers PECAM-1 immunoreactivity was detected along the sinusoids in a pattern similar to ICAM-1 staining. PECAM-1 specific transcripts were detected in freshly isolated and cultured SECs. After a single CCl4-administration, PECAM-1 immunoreactivity did not increase along the sinusoids in contrast to the early increase of ICAM-1. Northern blot analysis indicated that PECAM-1 expression in liver tissue and in isolated SECs does not increase after a single administration of CCl4, whereas ICAM-1 steady-state level increased after 6 h. In diseased human livers PECAM-1 was detectable along the sinusoids, within inflammatory infiltrates and within fibrotic septa. Neither in acutely nor chronically diseased human livers was an obvious increase of PECAM-1 immunoreactivity detectable. CONCLUSIONS PECAM-1 is expressed by SECs. In contrast to ICAM-1, PECAM-1 transcript level is not enhanced during liver damage.
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Affiliation(s)
- K Neubauer
- University of Göttingen, Department of Internal Medicine, Section of Gastroenterology and Endocrinology, Germany
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36
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Panin LE, Usynin IF, Khar'kovskii AV. Effect of tetrahydrocortisol on protein biosynthesis in hepatocytes. Bull Exp Biol Med 2000. [DOI: 10.1007/bf02434794] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/24/2022]
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37
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Matsumoto T, Sakurai MH, Kiyohara H, Yamada H. Orally administered decoction of Kampo (Japanese herbal) medicine, "Juzen-Taiho-To" modulates cytokine secretion and induces NKT cells in mouse liver. IMMUNOPHARMACOLOGY 2000; 46:149-61. [PMID: 10647873 DOI: 10.1016/s0162-3109(99)00166-6] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
The effects of orally administered decoction of Juzen-Taiho-To (JTT; Si-Quan-Da-Bu-Tang in Chinese) on cytokine production in hepatic lymphocytes were studied in mice. JTT was found to increase interferon gamma (IFN-gamma), as well as interleukin-4 (IL-4), IL-5 and IL-6 secretion from stimulated hepatic lymphocytes, whereas IL-2 secretion was reduced. The number of IFN-gamma- and IL-4-spot forming cells (SFC) were not changed by administration of JTT. These results suggest that modulation of cytokine secretion by JTT might not be due to changes in the number of cytokine secreting cells within liver lymphocytes. CD4/CD8 ratio and alphabeta/gammadelta T cell receptor (TCR) ratio in hepatic lymphocytes were not changed. However, flow cytometric analysis revealed that the population of CD3 positive intermediate cells in NK positive cells (NKT cells) was increased after oral administration of JTT. The population of CD3int IL-2Rbeta+ cells was also increased. The induction of NKT cells by JTT was reduced by injection of 2-chloroadenosine. JTT enhanced transcription of IL-12 mRNA in liver. From these results, it may be concluded that a rise in NKT cell population contributes, at least partially, to the modulating effect of JTT on cytokine production in liver lymphocytes, and macrophages. The production of IL-12 in liver may also contribute to this NKT induction.
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Affiliation(s)
- T Matsumoto
- Oriental Medicine Research Center, The Kitasato Institute, Tokyo, Japan
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38
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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: 258] [Impact Index Per Article: 9.9] [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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39
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Neubauer K, Krüger M, Quondamatteo F, Knittel T, Saile B, Ramadori G. Transforming growth factor-beta1 stimulates the synthesis of basement membrane proteins laminin, collagen type IV and entactin in rat liver sinusoidal endothelial cells. J Hepatol 1999; 31:692-702. [PMID: 10551394 DOI: 10.1016/s0168-8278(99)80350-x] [Citation(s) in RCA: 63] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
BACKGROUND/AIMS It is suggested that during fibrogenesis as well as during carcinogenesis of the liver, the hepatic microvascular phenotype is transformed from sinusoids - which lack a basement membrane--into continuous capillaries which rest on a basement membrane. As transforming growth factor (TGF)-beta1 seems to be the most effective mediator in the stimulation of matrix protein synthesis, we were interested in the modulation of basement membrane proteins collagen type IV, laminin, and entactin expression by TGF-beta1 in liver sinusoidal endothelial cells (SECs), especially since a stimulation of the synthesis of collagen type IV but not of entactin and laminin by TGF-beta1 has been demonstrated in a fibrosarcoma cell line. METHODS The synthesis of the basement membrane (BM) proteins entactin, laminin, and collagen type IV and of the extracellular matrix (ECM) proteins tenascin and fibronectin with or without TGF-beta1--stimulation was analyzed by immunostaining, immunoprecipitation of endogenously labeled proteins and Northern blot analysis of total RNA extracted from freshly isolated or cultured SECs from rat or guinea pig livers. Furthermore, SECs were isolated from acutely and chronically CCl4-damaged rat livers and were analyzed for matrix protein expression. RESULTS SECs were adherent 24 h after isolation and formed confluent monolayers on day 4 of primary culture. Specific immunoprecipitates and specific transcripts for the BM proteins entactin, laminin, and collagen type IV and for ECM proteins tenascin and fibronectin were detectable in freshly isolated or cultured SECs. The synthesis of all tested BM proteins and ECM proteins was stimulated at least 3-fold by TGF-beta1. In SECs isolated after CCl4-induced acute and chronic liver damage, increased levels of matrix protein transcripts were detectable. CONCLUSIONS The stimulation of the synthesis of all BM-proteins by TGF-beta1 in vitro and the accumulation of ECM transcripts in SECs isolated from CCl4-treated livers, suggests that SECs are involved in the formation of a basement membrane during the "capillarization" of the sinusoids during liver disease.
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Affiliation(s)
- K Neubauer
- Department of Internal Medicine, University of Göttingen, Germany
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Masson S, Scotté M, François A, Coeffier M, Provot F, Hiron M, Ténière P, Fallu J, Salier JP, Daveau M. Changes in growth factor and cytokine mRNA levels after hepatectomy in rat with CCl(4)-induced cirrhosis. THE AMERICAN JOURNAL OF PHYSIOLOGY 1999; 277:G838-46. [PMID: 10516150 DOI: 10.1152/ajpgi.1999.277.4.g838] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/14/2023]
Abstract
Cirrhotic liver is considered to regenerate less actively than normal liver after hepatic resection. However, the mechanisms responsible for this impaired regeneration and the cross talk of implicated factors still remain unclear. In the present study, mRNA levels for cyclins, growth factors, and cytokines were quantitatively assessed by a RT-PCR method at different times after hepatectomy in order to determine the relationships between these factors and the impaired regenerative process observed in cirrhotic liver. In our model of CCl(4)-induced cirrhosis, mRNA levels for cyclins and thymidine kinase provide evidence for the impaired and delayed hepatic regeneration. Moreover, we observed a significant decrease in interleukin (IL)-6 and tumor necrosis factor-alpha mRNA and a significant increase for IL-1beta mRNA. No significant change of hepatocyte growth factor (HGF) mRNA level was detected, contrasting with the decrease both at mRNA and protein levels in the expression of the c-Met/HGF receptor. Therefore, the impaired regeneration of the cirrhotic liver is associated not only with a lowered level of signals that normally promote liver growth but also with a strong decrease in c-Met receptor despite a normal expression of its specific ligand.
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Affiliation(s)
- S Masson
- Institut National de la Santé et de la Recherche Médicale Unité 519 and Institut Fédératif de Recherches Multidisciplinaires sur les Peptides, 76 183 Rouen, Centre Hospitalier et Universitaire, Rouen, France
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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: 49] [Impact Index Per Article: 1.9] [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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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: 125] [Impact Index Per Article: 4.8] [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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Lohmann R, Souba WW, Bode BP. Rat liver endothelial cell glutamine transporter and glutaminase expression contrast with parenchymal cells. THE AMERICAN JOURNAL OF PHYSIOLOGY 1999; 276:G743-50. [PMID: 10070052 DOI: 10.1152/ajpgi.1999.276.3.g743] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/11/2023]
Abstract
Despite the central role of the liver in glutamine homeostasis in health and disease, little is known about the mechanism by which this amino acid is transported into sinusoidal endothelial cells, the second most abundant hepatic cell type. To address this issue, the transport of L-glutamine was functionally characterized in hepatic endothelial cells isolated from male rats. On the basis of functional analyses, including kinetics, cation substitution, and amino acid inhibition, it was determined that a Na+-dependent carrier distinct from system N in parenchymal cells, with properties of system ASC or B0, mediated the majority of glutamine transport in hepatic endothelial cells. These results were supported by Northern blot analyses that showed expression of the ATB0 transporter gene in endothelial but not parenchymal cells. Concurrently, it was determined that, whereas both cell types express glutamine synthetase, hepatic endothelial cells express the kidney-type glutaminase isozyme in contrast to the liver-type isozyme in parenchymal cells. This represents the first report of ATB0 and kidney-type glutaminase isozyme expression in the liver, observations that have implications for roles of specific cell types in hepatic glutamine homeostasis in health and disease.
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Affiliation(s)
- R Lohmann
- Division of Surgical Oncology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114-2696, USA
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Blanc MC, Housset C, Lasnier E, Rey C, Capeau J, Giboudeau J, Poupon R, Vaubourdolle M. Direct cytotoxicity of hypoxia-reoxygenation towards sinusoidal endothelial cells in the rat. LIVER 1999; 19:42-9. [PMID: 9928765 DOI: 10.1111/j.1478-3231.1999.tb00008.x] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/10/2023]
Abstract
AIMS/BACKGROUND Sinusoidal endothelial cells are the primary target of ischemia-reperfusion injury following liver preservation. The present study was undertaken to examine the susceptibility of sinusoidal endothelial cells to hypoxia-reoxygenation and the potential role of oxygen free radicals in the induction of cell injury. METHODS Sinusoidal endothelial cells were isolated from rat liver. After 2 3 days of primary culture, the cells were exposed to hypoxia (N2/CO2 95/5) for 120 min and reoxygenation (O2/CO2 95/5) for 90 min. Control cells were exposed to hypoxia alone, to 95% O2 alone or were maintained under normoxic conditions. Human umbilical vein endothelial cells were used as a model of vascular endothelial cells and submitted to the same protocol. Cell viability and lipid peroxidation were assessed by LDH leakage and malondialdehyde production, respectively. In order to test the potential role of xanthine oxidase and mitochondrial dysfunction in cell injury, the cells were treated with allopurinol and potassium cyanide (KCN) respectively. RESULTS The different gaseous treatments did not affect LDH leakage in human umbilical vein endothelial cells. In sinusoidal endothelial cells, the sequential hypoxia-reoxygenation caused a significant increase in LDH release, malondialdehyde production and xanthine oxidase activity while hypoxia alone had no effect except on xanthine oxidase activity. Allopurinol inhibited xanthine oxidase without preventing cell injury or lipid peroxidation in this latter cell type. CONCLUSIONS The results suggest that sinusoidal endothelial cells, as opposed to vascular endothelial cells, are susceptible to a direct cytotoxic effect of hypoxia-reoxygenation. This effect occurs in combination with an increase in xanthine oxidase activity and lipid peroxidation, although cell injury is mediated at least in part by mechanisms independent of xanthine oxidase such as mitochondrial dysfunction.
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Affiliation(s)
- M C Blanc
- Service de Biochimie A, INSERM U402 Hôpital Saint-Antoine, Paris, France
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Lemberg A, Calabrese G, Majowicz M, Peredo H, Scorticati C, Filinger E, Perazzo JC. Prostanoid production in endothelial and Kupffer liver cells from monocrotaline intoxicated rats. Hum Exp Toxicol 1998; 17:564-9. [PMID: 9821020 DOI: 10.1177/096032719801701007] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
UNLABELLED A single dose of monocrotaline, a pyrrolizidine alkaloid, was injected into rats in order to produce 25 (Group I) and 45 (Group II) days later a progressive and so called delayed liver injury. The present study investigated the prostanoid production of Kupffer cells and endothelial cells separated from Monocrotaline and saline (Group III) injected rat livers. Kupffer cells: formation of 6 keto Prostaglandin F1 alpha, the major prostacycline metabolite, gradually decreased in Groups I vs II (P < 0.01) and in both Groups I and II vs Controls (P < 0.01). In addition Prostaglandin F2 alpha showed a significant increase in Groups I and II when compared to Group III, (P < 0.001), and Thromboxane B2 was present in both Groups of Monocrotaline treated animals, while it was not detectable in the control Group III. Endothelial cells: 6 keto Prostaglandin F1 alpha decreased in Groups 1 vs II. This differences was significant when compared, and compared to controls (Group III, P < 0.001). Prostaglandin E2 was detected only in Groups I and II. Prostaglandin F2 alpha and Thromboxane B2 could not be detected in any Group. Ultramicroscopy showed morphological cell damage in nonparenchymal cells in Monocrotaline intoxication in Group II, rats sacrificed 45 days after the injection, while it shows normal features in those treated animals sacrificed 25 days after the injection, as well as in control group. CONCLUSION A single Monocrotaline injection produces, 25 and 45 days later, severe and progressive alterations in the prostanoid production in Kupffer and Endothelial cells, while ultramicroscopic alterations was only observed 45 days after the injection of Monocrotaline. A decreased production of vasodilators and the presence of vasoconstrictor prostanoids that can participate in the production of the circulatory derangements enhancing liver injury and portal hypertension were also observed.
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Affiliation(s)
- A Lemberg
- Cátedra de Fisiopatolociá, CONICET, Buenos Aires, Argentina
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Geerts A, Niki T, Hellemans K, De Craemer D, Van Den Berg K, Lazou JM, Stange G, Van De Winkel M, De Bleser P. Purification of rat hepatic stellate cells by side scatter-activated cell sorting. Hepatology 1998; 27:590-8. [PMID: 9462662 DOI: 10.1002/hep.510270238] [Citation(s) in RCA: 87] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
In this study, we present a new method to obtain pure, viable, freshly isolated hepatic stellate cells. Stellate cells were purified by cell sorting using their high side scatter (SSC) of incident light. Purity of the cells was established by light and transmission electron microscopy (TEM). Starting from stellate cells that were 50% to 70% enriched by centrifugation in 11% Nycodenz, the cell purity after sorting was found to be 96.6% +/- 2.9%. Viability of the sorted cells was 90.8% +/- 2.2% as measured by the Trypan blue exclusion test and was confirmed by cell culturing. Per hour of sorting, 1.4 +/- 0.4 million stellate cells were obtained. Sorting runs of up to 4 hours were practically feasible, resulting in yields of 5 to 6 million cells per rat liver. Cells attached to plastic substratum within 24 hours. Subsequently, they spread and underwent spontaneous transition into myofibroblast-like cells. The purity of sorted cells was documented by reverse-transcriptase polymerase chain reaction (RT-PCR) experiments using specific primer pairs for messenger RNA (mRNA) species that were only present in parenchymal (preproalbumin), endothelial (endothelial cell nitric oxide synthase [eNOS]), stellate (desmin), or Kupffer cells (77- to 88-kd fucose receptor). Contaminating mRNA species were absent in sorted stellate cells. Next, we examined freshly sorted stellate cells by Western blotting to confirm the presence of relevant cytoskeletal proteins. Cells were positive for vimentin, desmin, and glial fibrillary acidic protein (GFAP), but negative for alpha-smooth muscle actin (alpha-SMA). Sorted and cultured cells were immunophenotyped for the presence of collagen types I, III, and IV, laminin, and the cytoskeletal proteins, alpha-SMA, desmin, vimentin, and GFAP. At 90 hours in culture, cells expressed all the investigated extracellular matrix proteins. Desmin was present in 82% +/- 1%, vimentin in 96% +/- 2.5%, and GFAP in 91% +/- 4.5% of cells. Alpha-SMA was present in 91% +/- 2% of cultured cells. We conclude that cell sorting based on SSC of incident light is a convenient method to obtain virtually pure stellate cells that can be used for direct analysis or for culturing. Although the yields obtained with this method are lower than with standard methods, and additional equipment is required, SSC-activated sorting offers the possibility of very pure cells when essential for analyses based on sensitive detection methods such as RT-PCR.
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Affiliation(s)
- A Geerts
- Laboratory for Cell Biology and Histology, Free University Brussels, Brussels-Jette, Belgium
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Spolarics Z, Wu JX. Role of glutathione and catalase in H2O2 detoxification in LPS-activated hepatic endothelial and Kupffer cells. THE AMERICAN JOURNAL OF PHYSIOLOGY 1997; 273:G1304-11. [PMID: 9435555 DOI: 10.1152/ajpgi.1997.273.6.g1304] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
The present study investigated the effect of lipopolysaccharide (LPS; from Escherichia coli, 2 mg/kg body wt ip) on selected aspects of the antioxidant status in Kupffer and sinusoidal endothelial cells. Cells were isolated 18 h after the injection of saline or LPS. In fresh suspension cultures, cellular reduced glutathione (GSH) and H2O2 were determined by monochlorobimane, and 2',7'-dichlorofluorescein diacetate, respectively, using a fluorescence plate reader. LPS injection increased GSH content two- to threefold in Kupffer cells compared with cells from control rats. Cellular GSH content was higher in endothelial than Kupffer cells. However, LPS did not increase GSH content in endothelial cells. Addition of H2O2 (40-200 microM) to Kupffer or endothelial cells caused a transient decrease in GSH, which was more pronounced in cells from control rats (approximately 45% drop) than in LPS-exposed cells (approximately 25% drop). Depleted GSH levels were accompanied by a proportional increase in cellular H2O2. After inhibition of catalase by 3-amino-1,2,4-triazole, the presence of 0.2 mM H2O2 depleted GSH content by 75% and 40% in Kupffer cells from saline- or LPS-injected rats, respectively. The same treatments caused a similar 50% decrease in both activated and control endothelial cells. LPS decreased catalase activity by 45% in Kupffer cells, whereas it had no effect on catalase in endothelial cells. Glutathione reductase activity was not altered by LPS in either cell type. These data show that in activated Kupffer cells the elevated level of cellular glutathione plays an augmented role in the protection against reactive oxygen species, whereas the contribution of catalase to H2O2 detoxification is attenuated. In LPS-stimulated endothelial and Kupffer cells, the efficient maintenance of GSH is consistent with upregulated production of reducing power through the hexose phosphate shunt observed previously.
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Affiliation(s)
- Z Spolarics
- Department of Anatomy, Cell Biology, and Injury Sciences, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark 07103, USA
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De Bleser PJ, Niki T, Rogiers V, Geerts A. Transforming growth factor-beta gene expression in normal and fibrotic rat liver. J Hepatol 1997; 26:886-93. [PMID: 9126804 DOI: 10.1016/s0168-8278(97)80257-7] [Citation(s) in RCA: 132] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
BACKGROUND/AIMS Transforming growth factor-beta (TGF-beta) is considered to be an important mediator in the development of fibrosis in several chronic liver diseases. To understand the mechanism(s) by which TGF-beta exerts its action(s), we investigated the cellular distribution of TGF-beta(1,2,3) transcripts in normal and carbon tetrachloride (CCl4)-induced fibrotic rat liver. METHODS Parenchymal, sinusoidal endothelial, Kupffer and stellate cells were isolated and purified. The exact cellular composition of each isolate was determined by transmission electron microscopy. Expression of TGF-beta(1,2,3) transcripts was investigated using Northern hybridization analysis. Hybridization signals were quantified by scanning densitometry and corrected for: (i) differences in extractable RNA per cell type, (ii) signal contribution from contaminating cells, and (iii) differences in loading, capillary transfer and hybridization. RESULTS In normal liver, TGF-beta1 mRNA was predominantly expressed in Kupffer cells, exhibiting values approximately 9-fold higher than those in stellate cells. No expression was found in endothelial and parenchymal cells. Signals for TGF-beta2 and TGF-beta3 were much weaker when compared to TGF-beta1. In Kupffer cells, the level of TGF-beta2 was approximately 4-fold higher than in stellate cells. Little expression was found in endothelial cells. TGF-beta3 expression could only be detected in stellate cells. TGF-beta2 and TGF-beta3 was not expressed in parenchymal cells. In fibrotic liver, TGF-beta1 mRNA was strongly expressed in all the sinusoidal cells. TGF-beta2 and TGF-beta3 could no longer be detected. When compared to the level of expression in normal stellate cells, the level of TGF-beta1 increased 12-fold in stellate cells from fibrotic livers, and 6-fold in endothelial cells. In Kupffer cells, the level of expression remained unchanged. CONCLUSIONS (i) In both normal and fibrotic liver, TGF-beta1 is the most abundant isoform, (ii) in normal liver, TGF-beta1 is expressed strongly by Kupffer cells and moderately by stellate cells, TGF-beta2 expression is highest in Kupffer cells, followed by stellate cells and endothelial cells. TGF-beta3 is expressed by stellate cells, (iii) in fibrotic liver, the level of TGF-beta1 expression increases selectively in stellate cells and endothelial cells. This suggests an important role, not only for stellate, but also for endothelial cells in fibrogenesis.
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Affiliation(s)
- P J De Bleser
- Laboratory for Cell Biology and Histology, Free University Brussels (V.U.B.), Belgium.
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Yabe N, Matsui H. Ampelopsis brevipedunculata (Vitaceae) extract stimulates collagen synthesis through superoxide generation in the serum-free cultures of rat dermal fibroblasts and Ito cells. JOURNAL OF ETHNOPHARMACOLOGY 1997; 56:67-76. [PMID: 9147256 DOI: 10.1016/s0378-8741(96)01508-5] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/22/2023]
Abstract
We describe the effects of an ethanol-extracted fraction of berries of Ampelopsis brevipedunculata (Maxim.) Trautv. (Vitaceae), a plant used in folk medicine to treat liver disease, on the synthesis of non-collagenous proteins and collagen by rat collagen-producible cells such as dermal fibroblasts and liver non-parenchymal Ito cells. The generation of superoxide and hydroxyl radical was assessed by measuring the reduction of cytochrome c and the formation of thiobarbituric acid-reactive substances from deoxyribose, respectively. The synthesis of non-collagenous proteins and collagen as evaluated by measuring the extent of [3H]tryptophan incorporation into a total protein fraction of culture products and the [3H]proline-incorporating rate into a collagenase-digestible protein fraction, respectively. Both types of cells promptly synthesized only collagen in response to a dialyzable fraction of the extract. Major activity to generate oxygen free radicals accumulated in the dialyzable fraction whereas activity to decrease ferrous iron-mediated generation of the radicals accumulated in an undialyzable fraction of the extract. Stimulation of collagen synthesis was caused by superoxide because addition of superoxide dismutase but not pyruvate, an antioxidant of hydrogen peroxide, or dimethyl sulfoxide, an antioxidant of the hydroxyl radical, abrogated the stimulatory effect. The extract may arrest the progress of liver injury mediated by oxygen free radicals generated in the presence of ferrous iron.
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Affiliation(s)
- N Yabe
- Department of Hygiene, Dokkyo University School of Medicine, Tochigi, Japan
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Cattan P, Zhang B, Braet F, Atia N, Conti F, Conjeaud H, Weill B, Chereau C, Houssin D, Calmus Y. Comparison between aortic and sinusoidal liver endothelial cells as targets of hyperacute xenogeneic rejection in the pig to human combination. Transplantation 1996; 62:803-10. [PMID: 8824481 DOI: 10.1097/00007890-199609270-00018] [Citation(s) in RCA: 21] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
Abstract
Endothelial cells of aortic origin are usually used in vitro as targets of hyperacute xenogeneic rejection, although endothelial cells from organs may have different properties. The sensitivities of aortic and liver endothelial cells to hyperacute xenogeneic rejection were compared in the pig to human combination. Sinusoidal liver endothelial cells were isolated and purified by collagenase perfusion of pig livers, sedimentation on a percoll gradient and selective adherence. Purity and viability of isolated liver endothelial cells after adherence were 85+/-6% and >95%, respectively. Endothelial cells from pig aortae (purity and viability >95%) were isolated by scraping. Immunoblotting analysis of xenoantigens on liver and aortic endothelial cell membranes preparations showed identical patterns. The strongest bands revealed by human IgM were located between 110 and 135 kD, while human IgG detected two major bands at 115 and 75kD. The membrane expression of xenoantigens recognized by human sera, analyzed by flow cytometry, was significantly lower on liver than on aortic endothelial cells (IgM: P=0.0006; IgG: P=0.0009). However, the complement-dependent cytotoxic activity of human sera was the same whether liver (54.5+/-1.4%) or aortic endothelial cells (50.0+/-4.2%) were used as targets. Taken together, those results allow the use of aortic instead of sinusoidal liver endothelial cells in the characterization of pig antigens recognized by human natural antibodies.
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Affiliation(s)
- P Cattan
- Laboratoire de Recherche Chiurgicale, Faculté de Médecine Cochin-Port-Royal, Université Paris V, France
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