328 Bradykinin modulates the fibrogenic actions of hepatic stellate cells

328 Bradykinin modulates the fibrogenic actions of hepatic stellate cells

Posters 122 stellate cells (HSC). Here we demonstrate a direct profibrogenic effect of CVI and the involvement of TGF-b. Aim: To investigate the dir...

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stellate cells (HSC). Here we demonstrate a direct profibrogenic effect of CVI and the involvement of TGF-b. Aim: To investigate the direct profibrogenic effect of CVI on HSC. Methods: 5× 104 cells were grown on plastic or immobilized collagen type VI, synchronized by serum starvation. Cells were treated for 24 h with 50 gg/well collagen VI fragment. Controls were: no treatrnent or 2ng/ml TGF-b, phorbolester or acetic acid. After chloroform extraction mRNA was transcribed to cDNA. Quantification was done by lightCycler@ PCR using TaqMan probes (collagen type I, TIMP-1, Ct-SMA, TGF-b, collagenase 3 and GAPDH) and appropriate primers. Results: In rat HSC collagen I synthesis was 2-fold upregulated by CVI compared to untreated and up to 80% compared to TGF-b treated cells. Kinetic experiments demonstrated a a strong and sustained collagen type I upregulation from 24 h to 50h. Phorbolester treatment depressed collagen I mRNA synthesis to half of the untreated control. After CVI-treatment the expression profiles for TGF-b and 0~-SMA were comparable to that for collagen type I. In contrast to TGF-b collagen type VI treatment leads to a strong TIMP-1 expression. PMA had no effect. MMP-3 and MMP-13 mRNA were strongly downregulated by TGF-b and collagen VI. Discussion: CVI is a profibrogenic factor acting on HSC. It activates these cells (0t-SMA upregulation) and induces TGF-b synthesis which could be the responsible factor for collagen type I synthesis which is upregulated to almost the same extent as induced by addition of TGF-b protein the strongest profibrogenic cytoldne known. In vivo the release of collagen VI fragments could lead to stimulation of TGF-b synthesis and collagen I accumulation as a result of it.

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H A E M A T O P O I E T I C STEM CELLS E N R I C H E D B O N E M A R R O W HOST A POPULATION OF M Y O F I B R O B L A S T S P R E C U R S O R S IN A M U R I N E M O D E L OF LIVER FIBROSIS

F.P. Russo I , E. Amofah I , B. Bigger2 , P. Vig 1, M.R. Alison 3, J.P. Iredale 4, S.J. Forbes I . I Hepatology Unit, St Mary's Hospital, Imperial College

London, London, UK," 2Stem Cell Research, National Blood Service, John t~adclifj'e Hospital, Oxford, UK; 3Department of Diabetes and Metabolic" Medicine, Queen Mary's School of Medicine and Dentistry, London, UK; 4Liver Research Group, University of Southampton, Southampton, UK Background and Aims: A significant proportion of hepatic myofibroblasts are o f bone marrow (BM) origin in human liver fibrosis o f diverse aetiology. In a murine model of chronic liver damage we previously found that approximately 70% of hepatic myofibroblasts were of BM origin. However, it is not known which BM stem cells carl give rise to hepatic cells with a fibrogenic potential. This is important if BM stem cells are to be used clinically for liver directed therapy. Methods: Female C57B1/6 mice were lethally irradiated and then received BM transplants from male donors. Mice received either whole BM or BM enriched for Haen:atopoietic Stem Cells through lineage depletion (Lin-). Immunomagnetic sorting of the whole BM was performed with an antibody cocktail (CD5-, TERll9-, CD45R-, Ly-6G-, CDllb-, Nentrophils-). 4 weeks later mice received intra-peritoneal carbon tetrachloride (CC14) injections every 5 days to induce liver fibrosis. Tissue was analysed for tile proportion of myofibroblasts ((x-Smooth Muscle Actin positive, c~-SMA) that were of BM origin (confirmed by in situ hybridisation for the Y chromosome). By analysing livers from mice that had recovered for 8 weeks after the CC14 induced damage we were able to see if BM cells gave rise to persistently activated myofibroblasts. Results: Although the proportion of myofibroblasts of BM origin was lower in mice that received Lin-BM, all mice with active liver damage had myofibroblasts of BM origin. When mice were allowed to recover for 8 weeks there was a large reduction in tile total nmnber of~-SMA positive myofibroblasts and hepatic scarfing. There was a large reduction in the number of BM derived myofibroblasts and those remaining were mostly of a quiescent phenotype (a-SMA negative and Desmin/Glial Fibrillary Acid Protein positive).

Conclusions: The BM contributes sig;nificantly to the hepatic myofibroblast population. Even BM that is enriched for Haernatopoietic Stem Cells contains myofibroblast precursors. Following injury recovery the BM derived myofibroblasts decreased in number and were of a less activated phenotype. Clinical programmes using BM stern cells therapy for liver disease should consider the fibrogenic potential of BM stem cells.

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BRADYKININ M O D U L A T E S THE FIBROGENIC A C T I O N S OF HEPATIC STELLATE CELLS

P. Sancho-Bru, R. Bataller, J. Colrnenero, V Khurdayan, M. Moreno, V Arroyo, E Gin,s. Liver Unit, Hospital Clinic, University of Barcelona

School of Medicine, IDIBAPS, Barcelona, Spain Background: Brady!dnin (BK) is a vasoactive substance with antifibrotic properties that partially mediates the beneficial effect of angiotensinconverting ettzyme (ACE) inhibitors in renal fibrosis. BK binds to the ubiquitous receptor B2 and to B1 receptors, which are expressed in inflammatory conditions. It is unknown whether normal and fibrotic livers express BK receptors to regulate hepatic fibrogenesis. We hypothesize that BK participates in liver fibrogenesis and exerts antifibrotic effects in hepatic stellate cells (HSC), a major fibrogenic cell type in the injured liver. Ahns: to investigate tile expression of BK receptors in normal and fibrotic livers and to explore the biological effects of BK in primary cultures of HSC. Methods and Results: B 1 and B2 receptors were assessed in liver from both sham-operated and bile duct ligated (2 weeks) rats by immunohistochemistry. B 1 receptors were barely detected in normal livers, while they were markedly expressed in fibrotic livers. B1 expression co-localized with activated HSC. B2 receptors were expressed in both normal and fibrotic livers. We next investigated whether HSC are targets for BK. Human primary HSC activated in culture highly expressed both B 1 and B2 receptors, as assessed by western blot analysis. Stimulation offluo-41 oaded HSC with BK (10 -6 to 10-8 M) resulted in a marked dose-dependent increase in intracellular calcium concentration, as analyzed with confocal microscopy. This effect was completely blocked by pre-incubating cells with HOE-140, a specific B2 receptor antagonist. BK induced a transient stimulation of ERK2 and c-jun, as assessed by western blot analysis. BK also stimulated NFI~B, as assessed by NF~zB dependent luciferase reporter gone assay. We finally fiwestigated whether BK modulates the fibrogenic properties of HSC. BK reduced procollagen al(I) and TGFb 1 gene expression, while TIMP-1 expression was unaffected, as assessed by real time PCR. Finally, BK increased by 70% the gelatinolytic action of HSC, as assessed by zymogram analysis. Conclusions: BK receptors are expressed in chronically damaged livers and BK modulates the fibrogenic properties of HSC by reducing collagen gone expression and increasing fibrinolysis. Therefore, BK may modulate the pathogenic actions of HSCs during liver fibrogenesis.

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C H A R A C T E R I Z A T I O N O F THE H U M A N ZINC FINGER PROTEIN 267 P R O M O T E R : ESSENTIAL R O L E OF N U C L E A R FACTOR Y

K. Hu, M. Fink, M. Froh, E. Gaebele, C. Hellerbrand, M. Muehlbauer, R. Wiest, J Schoelmerich, 13. Schnabl. Department of Internal Medicine L

University of Regensburg, Regensburg, Germany Background: Liver fibrosis results from excessive deposition of extracellular matrix proteins secreted by activated hepatic stellate cells (HSCs). The activation process is accompanied by modulation of various transcription factors, including zinc finger protein 267 (ZNF267). Recently, we have shown that ZNF267 is up-regulated during the activation process of human HSCs and functions as a negative transcriptional regulator of MMP-10, which might promote liver fibrogenesis through diminished