- Email: [email protected]
1064 UCH-L1: A NOVEL AND FUNCTIONAL MARKER FOR ACTIVATED HEPATIC STELLATE CELLS
POSTERS Methods: Hepatic oval cells were isolated from rats fed a cholinedeficient diet supplemented with ethionine and characterized by flow cytometry. For EMT inducement, the cells were cultured in 10% FBS-DMEM/F12 medium containing 1 ng/ml TGF-b1 with the medium replaced every 2 days. Results: Hepatic oval cells were positive for the progenitor-specific markers a-fetoprotein (AFP), as well as hepatocyte marker albumin and cholangiocyte marker cytokeratin 19. In the presence of TGF-b1 exposure, the cell lost cell contact and flatten down with the cell size increased about 3 to 4 times larger at the 16th day than that at 0 day. Real-time PCR results showed TGF-b1 upregulated snail expression and kept downregulation of e-cadherin, with 3 times more snail and 20% e-cadherin at the 16th day if compared with 0 day, indicating hepatic oval cells experienced a sustained EMT process. During this process, besides the time-dependent increase of extracellular matrix, the expression of AFP and cytokeratin 19 also increased time-dependently to 10 folds and nearly 30 folds, respectively, at the 16th day, while the expression of albumin changed little. Conclusion: The sustained EMT process increased the expression of cytokeratin 19 and AFP in hepatic oval cells, which indicates hepatic progenitors not only participate in liver fibrosis but also may take part in carcinogenesis. 1064 UCH-L1: A NOVEL AND FUNCTIONAL MARKER FOR ACTIVATED HEPATIC STELLATE CELLS C.L. Wilson, F. Oakley, D. Mann. Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK E-mail: [email protected] Background and Aims: Hepatic stellate cells (HSC) are the major cellular source of scar proteins in injured liver and the balance between their survival and death is a determinant of fibrosis progression. Ubiquitination is an important signaling event regulating proliferation, survival and differentiation status of many cell types. The deubiquitinases (DUBs) regulate ubiquitin (Ub) signaling by removal of Ub from target proteins. We reasoned that DUBs may play a critical role in HSC. The aim of this study was to carry out expression profiling for 50 DUBs comparing quiescent (qHSC) and activated (aHSC) cells and then determine the function of DUBs that are induced with HSC activation. Methods: DUB expression was analysed by q-RT-PCR. UCH-L1 expression was further assessed in liver sections from rats treated with CCL4 or olive oil (vehicle), normal human liver and liver from primary billiary cirrhosis (PBC) and hepatocellularcarcinoma (HCC) patients. aHSC were treated with incremental doses of the selective UCH-L1 inhibitor (LDN 57444) at different days of HSC activation and RNA and protein was isolated and analysed for the expression of apoptotic and fibrogenic genes and activation of various signalling pathways. Results: Expression for the majority of DUBs was either repressed or unchanged with HSC activation. However, UCHL1 was induced 500-fold and was abundantly expressed at the protein level in rat and human aHSC. Immunohistochemistry indicated that UCHL1 is selectively expressed in diseased liver (rat and human) and localised to aHSC and the endothelium. Treatment of qHSC with LDN 57444 was without effect, however treatment of aHSC caused increased apoptosis with 90% of cells dying after 24 hrs exposure. Akt is a stimulator of NF-úB activity which is critical for survival of HSC. Western blotting revealed constitutive active Akt (pAkt) in aHSC, which was suppressed following LDN 5744 treatment. Conclusions: HSC activation is associated with a generalised repression of DUB expression. However, UCH-L1 expression is induced in aHSC (in vitro and in vivo) and appears to be required for survival via an Akt/NF-úB pathway. This study therefore reports UCH-L1 as a novel new marker for aHSC and as a regulator of apoptosis and fibrogenesis. S422
1065 TARGETED THERAPY FOR LIVER CIRRHOSIS USING HYALURONIC ACID (HA) CONJUGATED TGFb1 SIRNA S.K. Yoon1 , S.W. Hong1 , P.S. Sung1 , C.-H. Park1 , M.J. Song1 , J.M. Yang1 , S.W. Choi1 , C.D. Lee1 , Y.S. Lee1 , S.K. Hahn2 . 1 Department of Internal Medicine & WHO Collaborating Center of Viral Hepatitis, The Catholic University of Korea, Seoul, 2 Departments of Materials Science and Engineering, Pohang University of Science and Technology (POSTECH), Pohang, Republic of Korea E-mail: [email protected] Background and Aims: Transforming growth factor-b1 (TGF- b1) plays a major role in the development of liver cirrhosis. We previously demonstrated that hyaluronic acid (HA) derivatives might be a novel drug delivery carrier for the treatment of liver cirrhosis. In this study, we investigated the therapeutic effect of HA-conjugated TGFb1 siRNA in vitro and in vivo. Methods: To verify effect of siTGFb1-HA-g-PEISS (siTGFb1-HA) complex in vitro, we transfected siTGFb1-HA complex and siTGFb1g-PEISS (siTGFb1-PEISS) complex into HSC-T6 cells. Cirrhotic mice model was established using CCl4 treatment for 8 weeks. Furthermore, to know the liver targeting of HA derivatives in mice, Q Dot-HA was injected normal mice and cirrhotic mice, and was evaluated by real-time bio-imaging. Next, siTGFb1-HA complex was injected to cirrhotic mice and therapeutic effect was assessed using Western blot and masson’s trichrome stain. Results: TGF-b1 expression was more significantly decreased in siTGFb1-HA complex transfected cells compared to siTGFb1-PEISS complex transfected cells in ELISA. In the experiment of drug delivery of HA, Q Dot-HA was mainly accumulated in the liver and its clearance was slower in cirrhotic mice than normal mice. The expression of TGFb1 was more significantly decreased in siTGFb1HA complex injected mice than siTGFb1-PEISS complex injected mice. Moreover, the anti-fibrotic effect was significantly higher in siTGFb1-HA complex injected mice compared to siTGFb1-PEISS complex injected mice. Conclusion: These results demonstrate the feasibility of HA derivatives as novel target specific and long acting drug delivery carriers in the liver, and suggest that HA conjugating siTGFb1 may be promising target therapy for liver cirrhosis through enhancement of efficient delivery. 1066 INHIBITION OF TISSUE INHIBITOR OF METALLOPROTEINASE-1 BY RECOMBINANT ADENO-ASSOCIATED VIRUSES CARRYING SIRNA PREVENTS LIVER FIBROSIS IN RATS M. Cong, P. Wang, T. Liu, Y. Bai, J. Jia, J. Chen, H. You. Beijing Friendship Hospital, Capital Medical University, Beijing, China E-mail: [email protected] Background and Aims: Elevated tissue inhibitor of metalloproteinase (TIMP)-1 expression contributes to excess production of extracellular matrix in liver fibrosis. The present study constructed a recombinant adeno-associated virus (AAV) carrying siRNA of TIMP-1 and investigated the effects of administration of this recombinant AAV in carbon tetrachloride (CCl4 )-induced rat liver fibrosis model. Methods: An siRNA which had the strongest suppression effect was cloned into the AAV vector and constructed the AAV/siRNA-TIMP-1. Rats were randomly divided into olive oil control group, CCl4 treated group, AAV/EGFP administration group and recombinant AAV/siRNA-TIMP-1 administration group (10 rats/group), which were administered the recombinant AAV at the end of 1st week and 2nd week after CCl4 -treatment. At the 4th weekend, the rats were sacrificed for detection of histological changes of liver tissue, the concentration of type I and type III collagen in serum, liver tissue hydroxyproline content, expression of TIMP-1, MMP2, MMP13, a-SMA and TGF-b.
Journal of Hepatology 2011 vol. 54 | S363–S534
1065 TARGETED THERAPY FOR LIVER CIRRHOSIS USING HYALURONIC ACID (HA) CONJUGATED TGFb1 SIRNA S.K. Yoon1 , S.W. Hong1 , P.S. Sung1 , C.-H. Park1 , M.J. Song1 , J.M. Yang1 , S.W. Choi1 , C.D. Lee1 , Y.S. Lee1 , S.K. Hahn2 . 1 Department of Internal Medicine & WHO Collaborating Center of Viral Hepatitis, The Catholic University of Korea, Seoul, 2 Departments of Materials Science and Engineering, Pohang University of Science and Technology (POSTECH), Pohang, Republic of Korea E-mail: [email protected] Background and Aims: Transforming growth factor-b1 (TGF- b1) plays a major role in the development of liver cirrhosis. We previously demonstrated that hyaluronic acid (HA) derivatives might be a novel drug delivery carrier for the treatment of liver cirrhosis. In this study, we investigated the therapeutic effect of HA-conjugated TGFb1 siRNA in vitro and in vivo. Methods: To verify effect of siTGFb1-HA-g-PEISS (siTGFb1-HA) complex in vitro, we transfected siTGFb1-HA complex and siTGFb1g-PEISS (siTGFb1-PEISS) complex into HSC-T6 cells. Cirrhotic mice model was established using CCl4 treatment for 8 weeks. Furthermore, to know the liver targeting of HA derivatives in mice, Q Dot-HA was injected normal mice and cirrhotic mice, and was evaluated by real-time bio-imaging. Next, siTGFb1-HA complex was injected to cirrhotic mice and therapeutic effect was assessed using Western blot and masson’s trichrome stain. Results: TGF-b1 expression was more significantly decreased in siTGFb1-HA complex transfected cells compared to siTGFb1-PEISS complex transfected cells in ELISA. In the experiment of drug delivery of HA, Q Dot-HA was mainly accumulated in the liver and its clearance was slower in cirrhotic mice than normal mice. The expression of TGFb1 was more significantly decreased in siTGFb1HA complex injected mice than siTGFb1-PEISS complex injected mice. Moreover, the anti-fibrotic effect was significantly higher in siTGFb1-HA complex injected mice compared to siTGFb1-PEISS complex injected mice. Conclusion: These results demonstrate the feasibility of HA derivatives as novel target specific and long acting drug delivery carriers in the liver, and suggest that HA conjugating siTGFb1 may be promising target therapy for liver cirrhosis through enhancement of efficient delivery. 1066 INHIBITION OF TISSUE INHIBITOR OF METALLOPROTEINASE-1 BY RECOMBINANT ADENO-ASSOCIATED VIRUSES CARRYING SIRNA PREVENTS LIVER FIBROSIS IN RATS M. Cong, P. Wang, T. Liu, Y. Bai, J. Jia, J. Chen, H. You. Beijing Friendship Hospital, Capital Medical University, Beijing, China E-mail: [email protected] Background and Aims: Elevated tissue inhibitor of metalloproteinase (TIMP)-1 expression contributes to excess production of extracellular matrix in liver fibrosis. The present study constructed a recombinant adeno-associated virus (AAV) carrying siRNA of TIMP-1 and investigated the effects of administration of this recombinant AAV in carbon tetrachloride (CCl4 )-induced rat liver fibrosis model. Methods: An siRNA which had the strongest suppression effect was cloned into the AAV vector and constructed the AAV/siRNA-TIMP-1. Rats were randomly divided into olive oil control group, CCl4 treated group, AAV/EGFP administration group and recombinant AAV/siRNA-TIMP-1 administration group (10 rats/group), which were administered the recombinant AAV at the end of 1st week and 2nd week after CCl4 -treatment. At the 4th weekend, the rats were sacrificed for detection of histological changes of liver tissue, the concentration of type I and type III collagen in serum, liver tissue hydroxyproline content, expression of TIMP-1, MMP2, MMP13, a-SMA and TGF-b.
Journal of Hepatology 2011 vol. 54 | S363–S534