- Email: [email protected]
490 EVIDENCE OF SNAIL1 TRANSCRIPTION FACTOR CONTRIBUTION TO THE HEPATIC STELLATE CELLS ACTIVATION PROCESS
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genesis and CRP2+/a-SMA+ myofibroblasts were strictly limited to proliferating bile ducts. GFAP+/Desmin+/Vimentin+/a-SMA− HSC appeared in periportal, sinusoidal, and septal zones of fibrotic liver. However, the number of cells did not correlate with the abundant amount of collagen. In parallel, astrocyte like-GFAP+/Desmin−/Vimentin−, but also contractileGFAP+/Desmin+/Vimentin+ HSC with thickened and elongated processes appear in fibrotic areas of the liver sections. Conclusions: 1. The increased CRP2 and unchanged GFAP gene expression in the liver of Abcb4−/ − mice indicate activation but no proliferation of quiescent HSC during fibrogenesis. 2. Co-immunostainings demonstrate GFAP+/Desmin+/Vimentin+/aSMA− HSC in Abcb4−/ − fibrogenesis but also suggest that these cells cannot solely be responsible for the elevated matrix production observed. 3. It has to be elaborated if the contractile- and astrocyte like-HSC phenotypes with elongated and thickened extensions are caused by altered matrix in fibrotic scars or if these cells are actively involved in fibrogenesis. References [1] Popov Y, Patsenker E, Fickert P, Trauner M, Schuppan D. 2005. J Hepatol. 43:1045–1054. [2] Henkel C, Roderfeld M, Weiskirchen R, Berres ML, Hillebrandt S, Lammert F, Meyer HE, Stuhler K, Graf J, Roeb E. 2006. Proteomics 6:6538–6548.
490 EVIDENCE OF SNAIL1 TRANSCRIPTION FACTOR CONTRIBUTION TO THE HEPATIC STELLATE CELLS ACTIVATION PROCESS M. Scarpa1,2 , A. Grillo1,2 , P. Brun1,2 , G. Palu1 , I. Castagliuolo1 , D. Martines2 . 1 Department of Histology, Microbiology and Medical Biotechnologies, 2 Department of Surgical and Gastroenterological Sciences, University of Padua, Padua, Italy E-mail: [email protected] Background and Aims: Following liver injury, hepatic stellate cells (HSC) undergo a process of activation from the quiescent fat-storing phenotype to a highly proliferative myofibroblast-like phenotype. Snail1 is a transcription factor best known for its ability to trigger ephitelial-mesenchymal transition (EMT), to influence mesoderm formation during embryonic development and to favour cell survival. Recent studies have reported Snail1 overexpression in several pathological conditions associated with the deposition of fibrotic tissue and that is expressed in fibroblasts in vivo during the healing process. Our goal was to evaluate the role of Snail1 in HSC transdifferentiation process. Methods: Primary HSC were isolated from livers of healthy mice and cultured on plastic up to 8 days (in vitro activation model) or from livers of CCl4-treated mice and cultured 1 day (in vivo activation model). Activation status of HSC was monitored by assessing mRNA level of HSC activation-related genes a-smooth muscle actin (aSMA) and a(1)I collagen (COL1A1). Snail1 expression was screened by immunostaining and quantitative RT Real Time PCR. RNA interference studies were performed on 2 days cultured HSC by adenoviral delivery of shRNA for Snail1 or LacZ as control; transduced cells were harvested the 8th day of culture for analysis of Snail1 related genes by qRT Real Time PCR and Western Blotting. Results: In vitro studies showed that Snail1 is expressed by HSC and that its transcription is augmented in vitro (8 days culture) and in vivo activated HSC as compared to quiescent HSC (1 day culture). At protein level we could observe the nuclear translocation of Snail1 after the fourth day of culture. Snail1 knock-down in HSC resulted in the significative down-regulation of aSMA and ColIaI mRNA and protein levels. Conclusions: During HSC activation Snail1 is up-regulated and shows a nuclear localization. Snail1 knock-down attenuates the activated phenotype of HSC. Our data support a role for Snail1 transcription factor in liver fibrogenesis and its involvement in HSC transdifferentiation process.
491 THE BMP-7 PATHWAY IN HEPATIC STELLATE CELLS AND HEPATOCYTES AND ITS ANTAGONISTIC EFFECT ON THE TGF-BETA1 SIGNALING PATHWAY O. Scherner, W.N. Vreden, S.K. Meurer, A.M. Gressner, R. Weiskirchen. Institute of Clinical Chemistry and Pathobiochemistry, University Hospital RWTH Aachen, Aachen, Germany E-mail: [email protected] Background and Aims: Over the last decade it has become evident that TGF-beta1 is the main profibrogenic cytokine in liver fibrosis, which results in activation of hepatic stellate cells (HSC) and massive deposition of collagen in the liver. BMP-7, a submember of the same superfamily is able to counteract the profibrogenic signaling of TGF-beta1 in many different cell types. The expression of BMP-7 and its receptors in HSC and hepatocytes (PC) is controversially discussed in literature. Our aim was to clarify the role of BMP-7 function in liver fibrogenesis. Methods: HSC and PC were isolated following standard procedures. For TGF-beta1 and BMP-7 stimulation cells were cultured and starved before stimulation. Afterwards cells were harvested and analysed by Western blot. For luciferase reporter assay, cells were transiently transfected with (BRE)2-reporter gene, (CAGA)12-reporter gene and rEndoglin receptor. Afterwards cells were lysated and luciferase activity was measured. For immunohistochemistry cells were plated on glass slides and fixated with PFA and incubated with a BMP-7 antibody following a FITC-labelled secondary antibody. Results: We could show that PC express BMP-7 and posses all necessary BMP-7 receptors. HSC posses all necessary BMP-7 receptors too but do not express the cytokine itself. Both cell types are responsive towards BMP-7. We could demonstrate that BMP-7 is counteracting the activation of HSC by TGF-beta1 resulting in a decrease of collagen I and reduced CAGA-luciferase activity. Endoglin is able to inhibit TGF-beta1 signaling in PC and to promote the BMP-7 signaling pathway. BMP-7 itself is upregulated during liver fibrogenesis as indicated through an enhanced (BRE)2-luciferase activity during liver fibrogenesis in vivo. Conclusions: We conclude that BMP-7 is an underestimated hepatic cytokine, which is upregulated during liver fibrogenesis counteracting the profibrogenic TGF-beta1 activities in the liver. BMP-7 directly antagonizes TGF-beta1 induced Smad3 phosphorylation.
492 CCL5 AND CCR5 EXPRESSION AS A SURROGATE MARKER FOR THE STAGE OF FIBROSIS IN PATIENTS WITH CHRONIC HEPATITIS C B. Wang, A. Katsounas, M. Trippler, G. Gerken, J.F. Schlaak. University Hospital of Essen, Essen, Germany E-mail: [email protected];[email protected] Background: Recent data suggested that CCL5 may play an important role in liver fibrosis as its expression is upregulated in the liver of patients with cirrhosis. Therefore, we have studied the expression of CCL5 and its receptor CCR5 in the peripheral blood of patients with chronic hepatitis C and the modulation by antiviral therapy with IFN-a in vivo. Materials and Methods: A total of 36 Caucasian patients with histologically proven chronic hepatitis C were treated with standard combination therapy consisting of pegylated IFN-a2a or pegylated IFN-a2b for 12 months or 6 months in combination with ribavirin (800–1200 mg daily). RNA was isolated (PAXgene, PreAnalytiX) from peripheral blood which was collected 12h before and 12h after the first injection of IFN-a. In addition, 11 patients with HCV-unrelated liver cirrhosis and 10 healthy individuals were used as controls. The transcriptional profile was analysed using human genomic microarrays (Affymetrix HG U133A) and quantitative real-time RT-PCR. Basal, induced and fold change values were subjected to significance analysis (SAM software) and class prediction analysis (PAM software) to identify genes which are differentially regu-
POSTERS
genesis and CRP2+/a-SMA+ myofibroblasts were strictly limited to proliferating bile ducts. GFAP+/Desmin+/Vimentin+/a-SMA− HSC appeared in periportal, sinusoidal, and septal zones of fibrotic liver. However, the number of cells did not correlate with the abundant amount of collagen. In parallel, astrocyte like-GFAP+/Desmin−/Vimentin−, but also contractileGFAP+/Desmin+/Vimentin+ HSC with thickened and elongated processes appear in fibrotic areas of the liver sections. Conclusions: 1. The increased CRP2 and unchanged GFAP gene expression in the liver of Abcb4−/ − mice indicate activation but no proliferation of quiescent HSC during fibrogenesis. 2. Co-immunostainings demonstrate GFAP+/Desmin+/Vimentin+/aSMA− HSC in Abcb4−/ − fibrogenesis but also suggest that these cells cannot solely be responsible for the elevated matrix production observed. 3. It has to be elaborated if the contractile- and astrocyte like-HSC phenotypes with elongated and thickened extensions are caused by altered matrix in fibrotic scars or if these cells are actively involved in fibrogenesis. References [1] Popov Y, Patsenker E, Fickert P, Trauner M, Schuppan D. 2005. J Hepatol. 43:1045–1054. [2] Henkel C, Roderfeld M, Weiskirchen R, Berres ML, Hillebrandt S, Lammert F, Meyer HE, Stuhler K, Graf J, Roeb E. 2006. Proteomics 6:6538–6548.
490 EVIDENCE OF SNAIL1 TRANSCRIPTION FACTOR CONTRIBUTION TO THE HEPATIC STELLATE CELLS ACTIVATION PROCESS M. Scarpa1,2 , A. Grillo1,2 , P. Brun1,2 , G. Palu1 , I. Castagliuolo1 , D. Martines2 . 1 Department of Histology, Microbiology and Medical Biotechnologies, 2 Department of Surgical and Gastroenterological Sciences, University of Padua, Padua, Italy E-mail: [email protected] Background and Aims: Following liver injury, hepatic stellate cells (HSC) undergo a process of activation from the quiescent fat-storing phenotype to a highly proliferative myofibroblast-like phenotype. Snail1 is a transcription factor best known for its ability to trigger ephitelial-mesenchymal transition (EMT), to influence mesoderm formation during embryonic development and to favour cell survival. Recent studies have reported Snail1 overexpression in several pathological conditions associated with the deposition of fibrotic tissue and that is expressed in fibroblasts in vivo during the healing process. Our goal was to evaluate the role of Snail1 in HSC transdifferentiation process. Methods: Primary HSC were isolated from livers of healthy mice and cultured on plastic up to 8 days (in vitro activation model) or from livers of CCl4-treated mice and cultured 1 day (in vivo activation model). Activation status of HSC was monitored by assessing mRNA level of HSC activation-related genes a-smooth muscle actin (aSMA) and a(1)I collagen (COL1A1). Snail1 expression was screened by immunostaining and quantitative RT Real Time PCR. RNA interference studies were performed on 2 days cultured HSC by adenoviral delivery of shRNA for Snail1 or LacZ as control; transduced cells were harvested the 8th day of culture for analysis of Snail1 related genes by qRT Real Time PCR and Western Blotting. Results: In vitro studies showed that Snail1 is expressed by HSC and that its transcription is augmented in vitro (8 days culture) and in vivo activated HSC as compared to quiescent HSC (1 day culture). At protein level we could observe the nuclear translocation of Snail1 after the fourth day of culture. Snail1 knock-down in HSC resulted in the significative down-regulation of aSMA and ColIaI mRNA and protein levels. Conclusions: During HSC activation Snail1 is up-regulated and shows a nuclear localization. Snail1 knock-down attenuates the activated phenotype of HSC. Our data support a role for Snail1 transcription factor in liver fibrogenesis and its involvement in HSC transdifferentiation process.
491 THE BMP-7 PATHWAY IN HEPATIC STELLATE CELLS AND HEPATOCYTES AND ITS ANTAGONISTIC EFFECT ON THE TGF-BETA1 SIGNALING PATHWAY O. Scherner, W.N. Vreden, S.K. Meurer, A.M. Gressner, R. Weiskirchen. Institute of Clinical Chemistry and Pathobiochemistry, University Hospital RWTH Aachen, Aachen, Germany E-mail: [email protected] Background and Aims: Over the last decade it has become evident that TGF-beta1 is the main profibrogenic cytokine in liver fibrosis, which results in activation of hepatic stellate cells (HSC) and massive deposition of collagen in the liver. BMP-7, a submember of the same superfamily is able to counteract the profibrogenic signaling of TGF-beta1 in many different cell types. The expression of BMP-7 and its receptors in HSC and hepatocytes (PC) is controversially discussed in literature. Our aim was to clarify the role of BMP-7 function in liver fibrogenesis. Methods: HSC and PC were isolated following standard procedures. For TGF-beta1 and BMP-7 stimulation cells were cultured and starved before stimulation. Afterwards cells were harvested and analysed by Western blot. For luciferase reporter assay, cells were transiently transfected with (BRE)2-reporter gene, (CAGA)12-reporter gene and rEndoglin receptor. Afterwards cells were lysated and luciferase activity was measured. For immunohistochemistry cells were plated on glass slides and fixated with PFA and incubated with a BMP-7 antibody following a FITC-labelled secondary antibody. Results: We could show that PC express BMP-7 and posses all necessary BMP-7 receptors. HSC posses all necessary BMP-7 receptors too but do not express the cytokine itself. Both cell types are responsive towards BMP-7. We could demonstrate that BMP-7 is counteracting the activation of HSC by TGF-beta1 resulting in a decrease of collagen I and reduced CAGA-luciferase activity. Endoglin is able to inhibit TGF-beta1 signaling in PC and to promote the BMP-7 signaling pathway. BMP-7 itself is upregulated during liver fibrogenesis as indicated through an enhanced (BRE)2-luciferase activity during liver fibrogenesis in vivo. Conclusions: We conclude that BMP-7 is an underestimated hepatic cytokine, which is upregulated during liver fibrogenesis counteracting the profibrogenic TGF-beta1 activities in the liver. BMP-7 directly antagonizes TGF-beta1 induced Smad3 phosphorylation.
492 CCL5 AND CCR5 EXPRESSION AS A SURROGATE MARKER FOR THE STAGE OF FIBROSIS IN PATIENTS WITH CHRONIC HEPATITIS C B. Wang, A. Katsounas, M. Trippler, G. Gerken, J.F. Schlaak. University Hospital of Essen, Essen, Germany E-mail: [email protected];[email protected] Background: Recent data suggested that CCL5 may play an important role in liver fibrosis as its expression is upregulated in the liver of patients with cirrhosis. Therefore, we have studied the expression of CCL5 and its receptor CCR5 in the peripheral blood of patients with chronic hepatitis C and the modulation by antiviral therapy with IFN-a in vivo. Materials and Methods: A total of 36 Caucasian patients with histologically proven chronic hepatitis C were treated with standard combination therapy consisting of pegylated IFN-a2a or pegylated IFN-a2b for 12 months or 6 months in combination with ribavirin (800–1200 mg daily). RNA was isolated (PAXgene, PreAnalytiX) from peripheral blood which was collected 12h before and 12h after the first injection of IFN-a. In addition, 11 patients with HCV-unrelated liver cirrhosis and 10 healthy individuals were used as controls. The transcriptional profile was analysed using human genomic microarrays (Affymetrix HG U133A) and quantitative real-time RT-PCR. Basal, induced and fold change values were subjected to significance analysis (SAM software) and class prediction analysis (PAM software) to identify genes which are differentially regu-