Busletta, Chiara

Cannito, Stefania

Colombatto, Sebastiano

David, E

Fagioli, F

Mareschi, Katia

Marra, F

Novo, Erica

Parola, Maurizio

Paternostro, Claudia

Pinzani, M

Povero, Davide

English

Institutional Research Information System University of Turin

POSTERS1033PHARMACOLOGICAL ANTAGONISM OF THE CHEMOKINE CCL2(MCP-1) INHIBITS MACROPHAGE INFILTRATION INTO THE LIVERAND EXPERIMENTAL STEATOSIS IN VIVOC. Baeck1, K. Karlmark1, N. Gassler2, S. Huss3, C. Trautwein1,F. Tacke1. 1Dept of Medicine III, 2Institute of Pathology, UniversityHospital Aachen, Aachen, 3Institute of Pathology, University HospitalBonn, Bonn, GermanyE-mail: cbaeck@ukaachen.deBackground and Aims: Recent experimental models of liver fibrosishighlighted the importance of liver-infiltrating macrophages forperpetuating hepatic inflammation by releasing proinflammatorycytokines and activating hepatic stellate cells (HSC). Monocyte/macrophage infiltration into the liver was found to be criticallyregulated via the chemokine receptor CCR2. In this study,we investigated the pharmacological inhibition of monocytechemoattractant protein-1 (MCP-1, CCL2), the major CCR2 ligand,using a specific MCP-1 antagonist in two models of murine liverfibrosis in vivo.Methods: Liver fibrosis was induced in C57BL/6 wild-type miceby intraperitoneal injections of carbon tetrachloride (CCl4) twiceweekly over 6 weeks or by feeding a methionine-choline-deficientdiet (MCD) over 8 weeks. Mice received anti-MCP-1 (3×/week, s.c.)or a non-specific control drug during liver injury models. CCR2-deficient mice served as controls. The infiltration of immune cellsinto the liver was assessed by FACS and immunohistochemistry. Theintrahepatic cytokine profile was analyzed by ELISA. Fibrosis andfatty degeneration were analyzed by histology (H&E, Sirius Red, oilred O staining), hydroxyproline content, intrahepatic trigylceridesand mRNA expression of fibrosis-related genes.Results: Antagonizing MCP-1 inhibited murine monocytechemotaxis in vitro (Boyden chambers). Upon CCl4- or MCD-induced liver injury in vivo, the infiltration of macrophages intothe liver was significantly decreased in anti-MCP-1 treated mice(by FACS and immunohistological staining; p < 0.01). In line withthe lower level of intrahepatic macrophages, pro-inflammatory,anti-inflammatory and profibrogenic cytokines (TNFa, MCP-1, IFNg,IL-10, IL-6; all p < 0.05) and fibrosis related genes (MCP-1, TNFa,TGFb, alpha-sma; all p < 0.01) were significantly regulated. InMCD challenged mice a lower level of fatty liver degenerationwas detected (oil red O staining, intrahepatic triglyceridemeasurement, p < 0.05) in anti-MCP-1-treated animals. However,overall fibrosis development was not significantly altered by anti-MCP-1 treatment.Conclusion: These results demonstrate the successful pharma-cological inhibition of hepatic monocyte/macrophage infiltrationby blocking MCP-1 during liver damage in vivo. The associateddecreased fatty degeneration suggests that inhibition of MCP-1 maybe an interesting novel approach for pharmacological treatment inliver inflammation and steatohepatitis.1034ONCOSTATIN M STIMULATES DIRECTIONAL MIGRATION OFHUMAN HEPATIC PROFIBROGENIC CELLSC. Busletta1, E. Novo1, C. Paternostro1, K. Mareschi2,3, S. Cannito1,D. Povero1, E. David4, S. Colombatto1, F. Marra5, F. Fagioli3,M. Pinzani5, M. Parola1. 1Dept. Experimental Medicine and Oncology,2Dept. Pediatrics, University of Torino, 3Stem Cell Transplantation &Cellular Therapy Unit, Regina Margherita Hospital, 4PathologyUnit, S. Giovanni Battista Hospital, Torino, 5Dept. Internal Medicineand Centre for Research, Transfer and High Education ’DENOThe’,University of Florence, Florence, ItalyE-mail: chiara.busletta@unito.itBackground and Aims: Myofibroblast like cells (MFs) can originatefrom activated hepatic stellate cells (HSC/MFs), portal fibroblastsand bone marrow-derived mesenchymal stem cells (MSCs). Acritical pro-fibrogenic feature of MFs is represented by theirability to migrate towards the site of injury and to align withnascent and established fibrotic septa in response to severalpolypeptides, hypoxia or reactive oxygen species (ROS). Along theselines, oncostatin M (OSM), a cytokine expressed in cirrhotic liverand belonging to the interleukin-6 family, has been reported toorchestrate hypoxia-modulated processes in the liver (development,regeneration, angiogenesis) involving HIF-1 and to be potentiallyinvolved in the progression of chronic liver diseases (CLDs). In thisstudy we investigated signaling mechanisms regulating migrationof human HSC/MFs and human, MF-like and bone marrow-derived,MSCs in response to oncostatin M.Methods: Signal transduction was evaluated by integrating cell andmolecular biology techniques, whereas non-oriented migration andchemotaxis were assessed by wound healing assay and the modifiedBoyden’s chambers assay, respectively. Morphological analysis wasperformed by immunohistochemistry (IHC) on liver specimens fromHCV cirrhotic patients (Metavir F4).Results: Exposure of pro-fibrogenic cell types to humanrecombinant OSM resulted in increased non-oriented migrationand chemotaxis. Moreover, IHC revealed positive stain for OSMin hepatocytes of cirrhotic nodules, mainly in the proximity offibrotic septa. The following features, common to both HSC/MFsand MSCs were demonstrated by employing specific experimentalapproaches:a. early intracellular ROS generation and activation of Ras/ErkJNK1/2 as well as of STAT1 and STAT3 signaling pathways andinvolvement of hypoxia-inducible factor-1a (HIF-1a);b. OSM-dependent migration, which was significantly inhibited byapocynin, indicating NADPH-oxidase as a major source of ROS;c. OSM-dependent motogenic action that, similarly to hypoxia,appeared to be exerted in HSC/MFs and MSCs through a biphasicmechanism requiring early generation of ROS and late HIF1-dependent expression and release of VEGF.Conclusions: OSM, which is expressed by hepatocytes in cirrhoticparenchyma, may contribute to fibrogenesis by stimulatingdirectional migration of profibrogenic human hepatic MFs througha biphasic, redox- and HIF-1a/VEGF-dependent mechanism.1035THE ANTIFIBROTIC EFFECT OF SORAFENIB IS DUE TO DECREASEDHSCS ACTIVATION MEDIATED BY ANGIOGENESIS BLOCKAGES. De Minicis, L. Agostinelli, C. Rychlicki, S. Saccomanno, L. Trozzi,I. Pierantonelli, A. Benedetti, G. Svegliati-Baroni. Department ofGastroenterology, Polytechnic University of Marche, Ancona, ItalyE-mail: s.deminicis@yahoo.itBackground: Liver cirrhosis represents the pathological responseof the liver to all forms of chronic injuries and may lead tohepatocellular carcinoma. Recent evidence have shown that kinaseinhibitors are able to interfere with liver cancer developmentand spreading by inhibiting angiogenesis. Angiogenesis is a keymechanism in both cancer and fibrosis development. Althoughsorafenib treatment has been shown to reduce liver fibrosis invitro and in vivo, the role of this molecule in interfering with themechanisms of fibrogenesis are still under investigation.Aim: To investigate the role of sorafenib, a multikinase inhibitor, ina mouse model of liver fibrogenesis.Methods: Wild-type mice were treated by i.p. injection of CCl4(0.2ml/kg three times a week) for five weeks to induce fibrosis.Mice received orally either sorafenib (at the dose of 50mg/kgof body-weight) or vehicle starting 1 week later than fibrosis-induction.Results: Liver fibrosis induced by CCL4 was significantly reducedby sorafenib administration. Morphometry after Sirius red stainingshowed reduced collagen deposition in CCL4-treated mice exposedto sorafenib, in comparison to control mice treated with CCL4 andJournal of Hepatology 2011 vol. 54 | S363–S534 S411

Oncostatin M stimulates directional migration of human hepatic pro-fibrogenic cells.

https://iris.unito.it/bitstream/2318/88299/1/1-s2.0-S0168827811610362-main.pdf

Oncostatin M stimulates directional migration of human hepatic pro-fibrogenic cells.

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