- Email: [email protected]
927 Antiapoptotic Effect of Insulin on Activated Hepatic Stellate Cells is Dependent on AKT and P70s6k
donor gender, donor age, donor height, African American donor, and donor DM. When controlling for the year of LT and transplant center in addition to the issues discussed above, it was found that HCV had a signif'ly increased risk of graft failure in both strata of # LTs received. The hazard ratio (HR) for the effect of HCV on the 1o LT increased signif'ly over time (p<.0001). At 1yr: HR=1.55 (95%CI 1.5-1.6), 3yrs: HR=1.82 (95%CI 1.7-2.0), 5yrs: HR=1.96 (95%CI 1.8-2.1) 7yrs: HR=2.06 (95%CI 1.9-2.2). The HR for the effect of HCV on Re-LT did not, however, vary signif'ly over time and was 1.5 (95%CI 1.2-1.8, p<.0001). After 1,512 days, the effect of HCV on 1o LT was signif'ly greater than that on Re-LT (p<.05). CONCLUSIONS: HCV status was found to be associated with worse graft survival for 1o LT (HR increased over time) and for 1st re-LT after controlling for known covariates associated w/ graft survival and pts who had Re-LT. The HR increased signif'ly over time for 1o LT for the effect of HCV on 1o LT and did not vary signif'ly over time for effect of HCV on Re-LT. After 1,512 days, the effect of HCV on 1o LT was signif'ly greater than that on ReLT. An analysis with a larger proportion of Re-LTs with longer follow-up is recommended.
0,04) , regardless of viremia, donor and recipient features, genotype, distance from OLT, olt-recurrence interval, fibrosis stage and treatment dose and duration. Conclusions: EOT response to antiviral treatment for post-OLT HCV recurrence is significantly higher among Cyclosporin treated recipients, however no differences in SVR and patient survival was observed. 926 Bid and Bim are Essential Regulators Involving the Intrinsic Pathway of Apoptosis in Hepatocytes in the Absence of Anti-Apoptotic BCL-2 Family Proteins Takahiro Kodama, Hayato Hikita, Tsukasa Kawaguchi, Satoshi Tanaka, Hinako Tsunematsu, Kumiko Nishio, Takatoshi Nawa, Minoru Shigekawa, Satoshi Shimizu, Wei Li, Takuya Miyagi, Atsushi Hosui, Tomohide Tatsumi, Tatsuya Kanto, Naoki Hiramatsu, Tetsuo Takehara
845
Hepatocyte apoptosis via the intrinsic pathway, also known as the mitochondrial pathway, is involved in the pathophysiology of various liver diseases and regulated by a fine balance between anti- and pro-apoptotic core Bcl-2 family proteins. We previously reported that hepatocyte-specific disruption of anti-apoptotic Bcl-2 family members, Bcl-xL or Mcl-1, caused spontaneous hepatocyte apoptosis, which was completely prevented in the additional deficiency of Bak and Bax, downstream apoptosis executioners (Hikita H, Kodama T, et al., Hepatology 2009). Meanwhile, BH3-only proteins are known to function as a pro-apoptotic sensor and precede this intrinsic apoptosis, but their actual involvement and requisites for hepatocyte intrinsic apoptosis machinery remained unclear. In the present study, we examined whether hepatocyte apoptosis via the intrinsic pathway can be induced by just a lack of Bcl-xL or/and Mcl-1, or requires the BH3-only proteins In Vivo. Hepatocyte apoptosis in the absence of Bcl-xL was significantly ameliorated by an additional deficiency of Bid or Bim and completely prevented by both as evidenced by TUNEL staining of liver sections and serum alanine transaminase (ALT) levels (Bcl-xL-/-, 537 ± 184 IU/L; Bcl-xL-/-Bid-/-, 64 ± 31 IU/L; Bcl-xL-/-Bim-/-, 114 ± 61 IU/L; Bcl-xL-/-Bid-/-Bim-/-, 27 ± 3 IU/L). In consistent with these findings, Bid/Bim double knockout mice did not show any hepatocyte apoptosis compared to Bim knockout mice upon administration of ABT-737 (100 mg/kg), which could inhibit Bcl-xL (Serum ALT levels: Bim-/-, 83 ± 33 IU/L; Bid-/-Bim-/-, 31 ± 15 IU/L). In Vivo administration of mcl-1 siRNA decreased Mcl-1 expression in the liver but did not cause hepatocyte apoptosis in the Bid/Bim double knockout mice. In addition, Bid/Bim/Bcl-xL triple knockout mice could survive even upon siRNA-mediated In Vivo knockdown of Mcl1 in the liver, in sharp contrast to our previous report that inhibition of both Bcl-xL and Mcl-1 led to lethal hepatocyte apoptosis In Vivo (Hikita H, Kodama T, et al., Hepatology 2010). These findings indicated that the BH3-only proteins, Bid and Bim, are essential for hepatocyte apoptosis via the intrinsic pathway in the absence of anti-apoptotic Bcl-2 family proteins, suggesting clinical implication that inhibition of these BH3-only proteins may be useful for preventing unfavorable hepatocyte apoptosis involved in a variety of liver diseases.
AASLD Abstracts
Use of Telaprevir Plus PEG Interferon/Ribavirin for Null Responders Post OLT With Advanced Fibrosis/Cholestatic Hepatitis C Paul Y. Kwo, Marwan Ghabril, Marco A. Lacerda, Rakesh Vinayek, Jonathan A. Fridell, A. Joseph Tector, Rodrigo M. Vianna Hepatitis C is the most common indication for OLT worldwide and aggressive recurrence of hepatitis C remains problematic with low SVR rates when therapy with PEGIFN/RBV (PR) is administered. To date, there is limited data on treatment of G1 HCV infection with DAA therapy in combination with PR and there are substantial drug-drug interactions with telaprevir(TVR)/calcineurin inhibitors (CNIs). Our AIM was to review our experience with telaprevir addition to PR in treatment of aggressive hepatitis C in null responders to PR post OLT. Methods: Adult patients who developed recurrent HCV infection following OLT with null response to PR for at least 12 weeks (< 2 log reduction) post-OLT received 4 week lead-in PEG-IFN alfa-2b (1.0 μg/kg/wk) plus RBV (600-1000 mg/day) followed by addition of telaprevir 750 q8. All patients who were not on cyclosporine modified (CYA) were converted to CYA from tacrolimus prior to initiation of HCV therapy. On the first day of TVR, patients received 25 mg CYA with trough levels titrated to 100 ng/ml . HCV RNA measured every 4 weeks by Cobas TaqMan HCV Test. Results. Seven patients (3 M, 4 F), mean age 56 years, have been treated thus far, 4 patients have received 12 weeks of TVR after lead-in. Three were < 1 year post-OLT, 5 had cirrhosis and two bridging fibrosis.Treatment paramenters are described in table. All patients required ribavirin dose reduction, EPO, ¾ GSF, mean packed-red-blood-cell requirement was 10 U per pt during TVR treatment. One patient who reached futility at week 4 of TVR therapy has continued PR and has viral level of HCV RNA <25 IU/ml, undetectable at week 24.Mild-moderate rash noted in all pts with 1 patient having perianal pain requiring narcotics. No supra/sub-therapeutic CYA levels encountered, though 2 patients were hospitalized for anemia and one for decompensated cirrhosis. Conclusions. Conversion to CYA followed by four week PR lead-in with addition of telaprevir for 12 weeks can lead to significant clearance rates in null responders with advanced fibrosis though high rates of anemia/RBV dose reduction, growth factor, and transfusion requirements were noted. CYA Interactions were easily managed by CYA dose adjustment. Twelve week TVR treatment data will be available for 7 patients in 5/2012. HCV RNA, RBV, CYA, Hgb weeks 0-16
927 Antiapoptotic Effect of Insulin on Activated Hepatic Stellate Cells is Dependent on AKT and P70s6k Cindy X. Cai, Hema Buddha, Amara J. Nidimusili, Vanita T. Talkad, Bruce R. Bacon, Brent A. Tetri Background: Hyperinsulinemia is a hallmark feature of nonalcoholic steatohepatitis (NASH). Insulin might play a role in NASH-related hepatic fibrosis and cirrhosis. Hepatic stellate cells (HSC) are central to the development and maintenance of fibrosis. We previously showed that insulin has differential profibrogenic effects in the initiation and perpetuation phase of HSC activation via the insulin-activated PI3K-Akt-p70S6K pathway. We investigated whether insulin may also be involved in regulating the resolution phase of activated HSC, mainly HSC apoptosis, a potential target for antifibrotic therapy. Aims: To investigate the molecular mechanisms of insulin-mediated antiapoptotic effect on activated HSC. Methods: Primary rat HSCs and the human immortalized HSC cell line, LX-2 were used in this study. Primary HSCs were isolated by sequential pronase and collagenase digestion, separated by density gradient centrifugation. Apoptosis was induced by proteasome inhibitor MG132 and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Cells were treated with MG132 or TRAIL in the absence or presence of insulin (20 μU/ml, 100 μU/ml, 1 mU/ml) followed by analysis of Annexin V FITC labeling, nuclear fragmentation/condensation, and immunoblotting for caspase 3, IκBα, phosphorylated Akt and p70S6K. Nuclear translocation of NFκB was visualized by immunofluorescence. Cell proliferation was measured by BrdU incorporation and collagen I was measured by ELISA. Some cells were pretreated with the insulin receptor tyrosine kinase inhibitor AG1024, PI3K inhibitor LY294002 or the mammalian target of rapamycin 1 (mTOR1) inhibitor rapamycin. Results: MG132 and TRAIL induced apoptosis in both human and rat HSCs, as detected by increased Annexin V labeling, nuclear fragmentation/condensation, and activation of caspase 3. Insulin dose-dependently inhibited MG132 and TRAIL-induced apoptosis, while inducing phosphorylation of Akt and p70S6K. Insulin-mediated inhibition of MG132-induced HSC apoptosis also involves attenuation of IκBα and enhanced nuclear translocation of NFκB. Furthermore, insulin potentiated cell proliferation and collagen I secretion. AG1024, LY294002 and rapamycin all inhibited insulin-induced cell proliferation, collagen I secretion and inhibition of HSC apoptosis. Conclusions: Insulin is a profibrogenic factor for HSC by enhancing cell proliferation, collagen I secretion and inhibition of apoptosis. One of the anti-apoptotic effects of insulin involves activation of the NFκB pathway. Importantly, the insulin-mediated antiapoptotic effect is AKt and p70S6K dependent. Thus, an insulin-mediated PI3K-Akt-p70S6K pathway is involved not only in the initiation and perpetuation phase but also in the resolution phase of HSC activation. This suggests potential therapeutic targets for inhibiting hepatic fibrogenesis in NASH.
846 Is the Primary Immunosuppressive Drug (Cyclosporin a or Tacrolimus) Playing a Role on the Response to Antiviral Treatment for Post-Transplant HCV Recurrence? Vittoria Vero, Senzolo Marco, Luisa Pasulo, Francesca Romana Ponziani, Raffaella Viganò, Maria Marino, Maria F. Donato, Maria Rendina, Pierluigi Toniutto, Matteo Cescon, Patrizia Burra, Lucia Miglioresi, Daniele Di Paolo, Valerio Giannelli, Antonio Gasbarrini, Stefano Fagiuoli Background: HCV-related cirrhosis is the most common indication for liver transplantation. Standard immunosuppression, based on Calcineurin inhibitors (CNI) may also affect HCV replication and response to antiviral therapy. Aim: to evaluate the impact of CNI on SVR in a population of HCV transplanted patients undergoing antiviral therapy for HCV recurrence. Patients and methods: A multicenter database of 12 Italian Centres was set up to carry on a retrospective analysis of 464 liver transplant recipients, treated for HCV recurrence, from 1992 to 2008. Patients were considered eligible for combination interferon plus ribavirinbased therapy according to defined criteria .Antiviral treatment was aimed for 48 weeks regardless of viral genotype (73,9% genotype 1); median follow up was 87± 45 months. Immunosuppressive therapy was based on cyclosporine in 39% of cases, on tacrolimus in 56,9%. Results: SVR rate was 34,1%. EOT was significantly higher in the Cyclosporine group (64%) compared with the Tacrolimus (54,5%) (p= 0,04): A longer interval between OLT and starting of antiviral therapy (32,7 vs 19,2 months), higher daily dose of Ribavirin (659,9 versus 561,9 mg) were associated with virological response in the Cyclosporin group. Acute and chronic rejection rate (p=0,536 and p=0,585 respectively) and pre-treatment staging score, were no different between the two groups. No difference in SVR rate and in patients survival was observed (88% survival in Cyclosporin group vs 87%). At multivariate analysis Cyclosporine was confirmed as an independent significant predictor of EOT (p=
AASLD Abstracts
S-934
0,04) , regardless of viremia, donor and recipient features, genotype, distance from OLT, olt-recurrence interval, fibrosis stage and treatment dose and duration. Conclusions: EOT response to antiviral treatment for post-OLT HCV recurrence is significantly higher among Cyclosporin treated recipients, however no differences in SVR and patient survival was observed. 926 Bid and Bim are Essential Regulators Involving the Intrinsic Pathway of Apoptosis in Hepatocytes in the Absence of Anti-Apoptotic BCL-2 Family Proteins Takahiro Kodama, Hayato Hikita, Tsukasa Kawaguchi, Satoshi Tanaka, Hinako Tsunematsu, Kumiko Nishio, Takatoshi Nawa, Minoru Shigekawa, Satoshi Shimizu, Wei Li, Takuya Miyagi, Atsushi Hosui, Tomohide Tatsumi, Tatsuya Kanto, Naoki Hiramatsu, Tetsuo Takehara
845
Hepatocyte apoptosis via the intrinsic pathway, also known as the mitochondrial pathway, is involved in the pathophysiology of various liver diseases and regulated by a fine balance between anti- and pro-apoptotic core Bcl-2 family proteins. We previously reported that hepatocyte-specific disruption of anti-apoptotic Bcl-2 family members, Bcl-xL or Mcl-1, caused spontaneous hepatocyte apoptosis, which was completely prevented in the additional deficiency of Bak and Bax, downstream apoptosis executioners (Hikita H, Kodama T, et al., Hepatology 2009). Meanwhile, BH3-only proteins are known to function as a pro-apoptotic sensor and precede this intrinsic apoptosis, but their actual involvement and requisites for hepatocyte intrinsic apoptosis machinery remained unclear. In the present study, we examined whether hepatocyte apoptosis via the intrinsic pathway can be induced by just a lack of Bcl-xL or/and Mcl-1, or requires the BH3-only proteins In Vivo. Hepatocyte apoptosis in the absence of Bcl-xL was significantly ameliorated by an additional deficiency of Bid or Bim and completely prevented by both as evidenced by TUNEL staining of liver sections and serum alanine transaminase (ALT) levels (Bcl-xL-/-, 537 ± 184 IU/L; Bcl-xL-/-Bid-/-, 64 ± 31 IU/L; Bcl-xL-/-Bim-/-, 114 ± 61 IU/L; Bcl-xL-/-Bid-/-Bim-/-, 27 ± 3 IU/L). In consistent with these findings, Bid/Bim double knockout mice did not show any hepatocyte apoptosis compared to Bim knockout mice upon administration of ABT-737 (100 mg/kg), which could inhibit Bcl-xL (Serum ALT levels: Bim-/-, 83 ± 33 IU/L; Bid-/-Bim-/-, 31 ± 15 IU/L). In Vivo administration of mcl-1 siRNA decreased Mcl-1 expression in the liver but did not cause hepatocyte apoptosis in the Bid/Bim double knockout mice. In addition, Bid/Bim/Bcl-xL triple knockout mice could survive even upon siRNA-mediated In Vivo knockdown of Mcl1 in the liver, in sharp contrast to our previous report that inhibition of both Bcl-xL and Mcl-1 led to lethal hepatocyte apoptosis In Vivo (Hikita H, Kodama T, et al., Hepatology 2010). These findings indicated that the BH3-only proteins, Bid and Bim, are essential for hepatocyte apoptosis via the intrinsic pathway in the absence of anti-apoptotic Bcl-2 family proteins, suggesting clinical implication that inhibition of these BH3-only proteins may be useful for preventing unfavorable hepatocyte apoptosis involved in a variety of liver diseases.
AASLD Abstracts
Use of Telaprevir Plus PEG Interferon/Ribavirin for Null Responders Post OLT With Advanced Fibrosis/Cholestatic Hepatitis C Paul Y. Kwo, Marwan Ghabril, Marco A. Lacerda, Rakesh Vinayek, Jonathan A. Fridell, A. Joseph Tector, Rodrigo M. Vianna Hepatitis C is the most common indication for OLT worldwide and aggressive recurrence of hepatitis C remains problematic with low SVR rates when therapy with PEGIFN/RBV (PR) is administered. To date, there is limited data on treatment of G1 HCV infection with DAA therapy in combination with PR and there are substantial drug-drug interactions with telaprevir(TVR)/calcineurin inhibitors (CNIs). Our AIM was to review our experience with telaprevir addition to PR in treatment of aggressive hepatitis C in null responders to PR post OLT. Methods: Adult patients who developed recurrent HCV infection following OLT with null response to PR for at least 12 weeks (< 2 log reduction) post-OLT received 4 week lead-in PEG-IFN alfa-2b (1.0 μg/kg/wk) plus RBV (600-1000 mg/day) followed by addition of telaprevir 750 q8. All patients who were not on cyclosporine modified (CYA) were converted to CYA from tacrolimus prior to initiation of HCV therapy. On the first day of TVR, patients received 25 mg CYA with trough levels titrated to 100 ng/ml . HCV RNA measured every 4 weeks by Cobas TaqMan HCV Test. Results. Seven patients (3 M, 4 F), mean age 56 years, have been treated thus far, 4 patients have received 12 weeks of TVR after lead-in. Three were < 1 year post-OLT, 5 had cirrhosis and two bridging fibrosis.Treatment paramenters are described in table. All patients required ribavirin dose reduction, EPO, ¾ GSF, mean packed-red-blood-cell requirement was 10 U per pt during TVR treatment. One patient who reached futility at week 4 of TVR therapy has continued PR and has viral level of HCV RNA <25 IU/ml, undetectable at week 24.Mild-moderate rash noted in all pts with 1 patient having perianal pain requiring narcotics. No supra/sub-therapeutic CYA levels encountered, though 2 patients were hospitalized for anemia and one for decompensated cirrhosis. Conclusions. Conversion to CYA followed by four week PR lead-in with addition of telaprevir for 12 weeks can lead to significant clearance rates in null responders with advanced fibrosis though high rates of anemia/RBV dose reduction, growth factor, and transfusion requirements were noted. CYA Interactions were easily managed by CYA dose adjustment. Twelve week TVR treatment data will be available for 7 patients in 5/2012. HCV RNA, RBV, CYA, Hgb weeks 0-16
927 Antiapoptotic Effect of Insulin on Activated Hepatic Stellate Cells is Dependent on AKT and P70s6k Cindy X. Cai, Hema Buddha, Amara J. Nidimusili, Vanita T. Talkad, Bruce R. Bacon, Brent A. Tetri Background: Hyperinsulinemia is a hallmark feature of nonalcoholic steatohepatitis (NASH). Insulin might play a role in NASH-related hepatic fibrosis and cirrhosis. Hepatic stellate cells (HSC) are central to the development and maintenance of fibrosis. We previously showed that insulin has differential profibrogenic effects in the initiation and perpetuation phase of HSC activation via the insulin-activated PI3K-Akt-p70S6K pathway. We investigated whether insulin may also be involved in regulating the resolution phase of activated HSC, mainly HSC apoptosis, a potential target for antifibrotic therapy. Aims: To investigate the molecular mechanisms of insulin-mediated antiapoptotic effect on activated HSC. Methods: Primary rat HSCs and the human immortalized HSC cell line, LX-2 were used in this study. Primary HSCs were isolated by sequential pronase and collagenase digestion, separated by density gradient centrifugation. Apoptosis was induced by proteasome inhibitor MG132 and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Cells were treated with MG132 or TRAIL in the absence or presence of insulin (20 μU/ml, 100 μU/ml, 1 mU/ml) followed by analysis of Annexin V FITC labeling, nuclear fragmentation/condensation, and immunoblotting for caspase 3, IκBα, phosphorylated Akt and p70S6K. Nuclear translocation of NFκB was visualized by immunofluorescence. Cell proliferation was measured by BrdU incorporation and collagen I was measured by ELISA. Some cells were pretreated with the insulin receptor tyrosine kinase inhibitor AG1024, PI3K inhibitor LY294002 or the mammalian target of rapamycin 1 (mTOR1) inhibitor rapamycin. Results: MG132 and TRAIL induced apoptosis in both human and rat HSCs, as detected by increased Annexin V labeling, nuclear fragmentation/condensation, and activation of caspase 3. Insulin dose-dependently inhibited MG132 and TRAIL-induced apoptosis, while inducing phosphorylation of Akt and p70S6K. Insulin-mediated inhibition of MG132-induced HSC apoptosis also involves attenuation of IκBα and enhanced nuclear translocation of NFκB. Furthermore, insulin potentiated cell proliferation and collagen I secretion. AG1024, LY294002 and rapamycin all inhibited insulin-induced cell proliferation, collagen I secretion and inhibition of HSC apoptosis. Conclusions: Insulin is a profibrogenic factor for HSC by enhancing cell proliferation, collagen I secretion and inhibition of apoptosis. One of the anti-apoptotic effects of insulin involves activation of the NFκB pathway. Importantly, the insulin-mediated antiapoptotic effect is AKt and p70S6K dependent. Thus, an insulin-mediated PI3K-Akt-p70S6K pathway is involved not only in the initiation and perpetuation phase but also in the resolution phase of HSC activation. This suggests potential therapeutic targets for inhibiting hepatic fibrogenesis in NASH.
846 Is the Primary Immunosuppressive Drug (Cyclosporin a or Tacrolimus) Playing a Role on the Response to Antiviral Treatment for Post-Transplant HCV Recurrence? Vittoria Vero, Senzolo Marco, Luisa Pasulo, Francesca Romana Ponziani, Raffaella Viganò, Maria Marino, Maria F. Donato, Maria Rendina, Pierluigi Toniutto, Matteo Cescon, Patrizia Burra, Lucia Miglioresi, Daniele Di Paolo, Valerio Giannelli, Antonio Gasbarrini, Stefano Fagiuoli Background: HCV-related cirrhosis is the most common indication for liver transplantation. Standard immunosuppression, based on Calcineurin inhibitors (CNI) may also affect HCV replication and response to antiviral therapy. Aim: to evaluate the impact of CNI on SVR in a population of HCV transplanted patients undergoing antiviral therapy for HCV recurrence. Patients and methods: A multicenter database of 12 Italian Centres was set up to carry on a retrospective analysis of 464 liver transplant recipients, treated for HCV recurrence, from 1992 to 2008. Patients were considered eligible for combination interferon plus ribavirinbased therapy according to defined criteria .Antiviral treatment was aimed for 48 weeks regardless of viral genotype (73,9% genotype 1); median follow up was 87± 45 months. Immunosuppressive therapy was based on cyclosporine in 39% of cases, on tacrolimus in 56,9%. Results: SVR rate was 34,1%. EOT was significantly higher in the Cyclosporine group (64%) compared with the Tacrolimus (54,5%) (p= 0,04): A longer interval between OLT and starting of antiviral therapy (32,7 vs 19,2 months), higher daily dose of Ribavirin (659,9 versus 561,9 mg) were associated with virological response in the Cyclosporin group. Acute and chronic rejection rate (p=0,536 and p=0,585 respectively) and pre-treatment staging score, were no different between the two groups. No difference in SVR rate and in patients survival was observed (88% survival in Cyclosporin group vs 87%). At multivariate analysis Cyclosporine was confirmed as an independent significant predictor of EOT (p=
AASLD Abstracts
S-934