Aleffi, Sara

Bertolani, Cristiana

Bonacchi, A.

Failli, Paola

Gentilini, Paolo

Laffi, Giacomo

Marra, Fabio

Mazzinghi, B.

Novo, E.

Parola, M.

Petrai, Ilaria

Pinzani, Massimo

Romagnani, Paola

Rotondi, M.

Zamara, E.

English

Florence Research

Category 4c: Molecular and Cellular Biology: HSCS and Fibrosis 99of both CFSC-2G and isolated HSC was inhibited dose-dependently byEMD409915, with complete abrogation of migration at 10-6M. There wasno effect of beta 3 integrin inhibition on cell proliferation in both cell lines.Pre-incubation of the cells with the beta 3 integrin inhibitor completelyblocked PDGF-stimulated phosphorylation of p38 MAPK and FAK.Conclusion: PDGF-BB-induced HSC migration can be completelyblocked in vitro by the beta 3-integrin specific non-peptidic inhibitorEMD409915. Specific integrin inhibition provides a novel promising strat-egy for antifibrotic therapy.324 DIFFERENTIAL EXPRESSION OF TWO ISOFORMS OF THECHEMOKINE RECEPTOR CXCR3 DETERMINES THEULTIMATE EFFECT OF IP-10 ON SURVIVAL OF HUMANHEPATIC STELLATE CELLS (HSC)I. Petrai1, A. Bonacchi1, P. Romagnani2, M. Parola3, B. Mazzinghi2,M. Rotondi2, P. Failli4, S. Aleffi1, C. Bertolani1, E. Novo3, E. Zamara3,M. Pinzani1, G. Laffi1, P. Gentilini1, F. Marra1. 1Dipartimento DiMedicina Interna, University of Florence, Florence, Italy; 2DipartimentoDi Fisiopatologia Clinica, University of Florence, Florence, Italy;3Dipartimento Di Medicina E Oncologia Sperimentale, University ofTurin, Turin, Italy; 4Dipartimento Di Farmacologia Preclinica E Clinica,University of Florence, Florence, ItalyActivated human HSC express the chemokine receptor CXCR3, whichbinds IP-10 and induces cell migration. Recently, an alternatively splicedisoform, CXCR3b, has been described in microvascular endothelial cells.Unlike the “classic” isoform, CXCR3a, CXCR3b leads to growth inhibitionand apoptosis, and mediates angiostatic effects of IP-10. In HSC, the pos-sible role of CXCR3b expression is unknown. In this study we investigatedthe biologic significance of the IP-10/CXCR3 system in HSC, consider-ing the role of CXCR3b. Apoptosis of activated HSC was induced by Fasactivation and/or protein synthesis inhibition by cycloheximide, and wasevaluated with PARP cleavage (western blotting) and caspase 3 activation(fluorometric assay). IP-10 secretion by HSC was measured by ELISA.IP-10 was detectable in HSC supernatants, and increased in response toIFN-gamma. Activating anti-Fas antibody and/or cycloheximide inducedHSC apoptosis, which was inhibited by low concentrations (20ng/ml) ofIP-10. However, the inhibition of apoptosis by IP-10 was variable in dif-ferent cell lines and tended to disappear after prolonged cell culture, whena pro-apoptotic effect was actually observed. To test for this apparent para-dox, expression of CXCR3a and CXCR3b was evaluated in cells that didnot show increased survival with IP-10. Surprisingly, these cells expressedmuch higher levels of CXCR3b than CXCR3a, thus providing a mecha-nism for the reversal of the anti-apoptotic effects by IP-10. We concludethat HSC may express both CXCR3 isoforms, that mediate opposing ef-fects of IP-10 on apoptosis. The ultimate biologic effect of autocrine IP-10secretion depends on the relative expression of the two isoforms.325 HUMAN STEM CELLS CAN REGENERATE ANDTRANSDIFFERANTIATE IN A IMMUNOCOMPETENT RATMODEL OF ACUTE HEPATITISA.C. Piscaglia1, C. Di Campli1, M. Nestola1, S. Rutella2, G. Bonanno2,A. Mariotti2, G. Zannoni3, F.M. Vecchio3, P. Pola4, G. Leone2,G. Gasbarrini1, A. Gasbarrini4. 1Internal Medicine, Catholic Univeristy,Gemelli Hospital, Rome, Italy; 2Haematology, Catholic Univeristy,Gemelli Hospital, Rome, Italy; 3Pathologic Anatomy, Catholic Univeristy,Gemelli Hospital, Rome, Italy; 4Medical Pathology, Catholic Univeristy,Gemelli Hospital, Rome, ItalyBackground and aim: Tissue homeostasis and turnover are guaranted bythe stem proliferating reserve. Several studies, performed with immunode-ficient animals, suggested that a particular degree of plasticity is shown bythe haematopoietic stem cell (HSC) compartment, that could be a sourcefor liver regeneration. We aimed to explore the hepatic differentiation po-tential of human HSC also in immunocompetent rats after a toxic liverdamage.Materials and methods: Winstar rats were so sorted: Group A: liver dam-age with Allyl Alcohol (AA) intraperitoneally (ip) injection and killing at2, 4, 7 days after. Group B: human HSC injection (ip) and killing at 1,3, 6 days after. Group C: liver damage (AA, ip); human HSC injection(ip) after 1 day and killing at 1, 3, 6 days after HSC injection. Group D:sacrifice without any treatment. Livers, spleens and bone marrows wereanalysed for human HSC detection using Flow Cytometry (FCM); liverswere also tested with immunohistochemistry (IHC) to demonstrate HSCtransdifferentiation into hepatocytes.Results: FCM confirmed the presence of human HSC (groups B and C)and revealed their selective recruitment into the damaged livers (group C)in comparison with the controls (group B). IHC demonstrated the HSCcapability to transdifferentiate into hepatic cells.Conclusions: Our study demonstrated that HSC are able to contribute inliver regeneration after an acute toxic damage in not-myeloablated recipi-ents, opening doors to speculations about their future applications for hu-man liver diseases, without the need of immunosuppressive pre-treatments.326 STIMULATION OF INTERSTITIAL COLLAGENASE ACTIVITYBY HALOFUGINONE IN HEPATIC STELLATE CELLS ISDEPENDENT ON ACTIVATION OF P38 AND NF KAPPA BY. Popov1, E. Patsenker1, E. Niedobitek1, M. Bauer1, M. Pines2,D. Schuppan1. 1Dept. of Medicine I, University of Erlangen, Erlangen,Germany; 2Institute For Animal Science, Bet Dagan, IsraelBackground: The semisynthetic plant alkaloid Halofuginone (HAL) wasreported to ameliorate rat liver fibrosis. We recently found that the antifi-brotic potential of HAL is likely to be associated with a strong MMP in-duction in activated hepatic stellate cells (HSCs). Our present study aimedto investigate molecular mechanisms involved in this effect.Methods: The HSC line CFSC-2G and isolated rat HSCs were incubatedwith increasing concentrations of HAL. mRNA steady state levels of MMP-3, -13 were quantified by real-time PCR. Interstitial collagenase activitieswere assessed in conditioned media. p38 MAPK and nuclear factor kappaB (NF kappa B) pathways were evaluated by western blotting (WB) ofcell lysates and nuclear extracts with phospho-p38 and p65 NF kappa Bantibodies, respectively. SB 203580 and MG132 were used to block p38and NF kappa B signaling.Results: Interstitial collagenase activity of HSC cultures increased dose-and time-dependently up to 3-fold in the presence of HAL at 20-200 nM.At 200 nM HAL rapidly up-regulated MMP-3 and MMP-13 (interstitialcollagenase) mRNA expression 10-50-fold in CFSC-2G and HSC, whichwas dose-dependently inhibited by pre-incubation of cells with both SB203580 and MG132 prior to treatment with HAL. Activation of p38 andNF kappa B pathways was confirmed by WB in HSC treated with HAL for0,5-6 hours.Conclusions: 1. HAL stimulates interstitial collagenolytic activity of HSCin a hitherto unmatched way via activation of p38 MAPK and NF kappa Bpathways; 2. HAL is a highly promising drug for combination therapy ofliver fibrosis.327 FLUORESCENCE RESONANCE ENERGY TRANSFER (FRET)BETWEEN TIMP-1 AND MMP-9 REVEALED INTRACELLULARCOMPLEX FORMATION IN LIVER CELLSM. Roderfeld1, B. Giese2, G. Muller-Neven2, B. Jansen1, S. Matern1,E. Roeb1. 1Clinic For Medicine III, University Hospital Aachen, Aachen,Germany; 2Dept. of Biochemistry, University Hospital Aachen, Aachen,GermanyBackground: Expression of tissue inhibitor of metalloproteinases-1(TIMP-1) is closely correlated with liver fibrogenesis. In the context ofTIMP-1-antagonisation by matrix metalloproteinases (MMPs) we inves-

Differential expression of two isoforms of the chemokine receptor CXCR3 determines the ultimate effect of IP-10 on survival of human hepatic stellate cells (HSC)

https://flore.unifi.it/bitstream/2158/778415/1/Petrai2004.pdf

Differential expression of two isoforms of the chemokine receptor CXCR3 determines the ultimate effect of IP-10 on survival of human hepatic stellate cells (HSC)

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