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M1575 Are Non-Invasive Biochemical Markers Useful in the Assessment Liver Fibrosis in Patients with Chronic Hepatitis C?
M1575
than 98% exhibited autofluorescence typical of q-HSC. Freshly isolated HSC were harvested for mRNA analysis or cultured for up to 7 days in serum-supplemented media to induce MF activation. Expression of epithelial and mesenchymal markers, as well as Hh-target genes, was monitored during culture (days 0, 4, 7). Quantitative, real-time RT-PCR was performed to compare changes in gene expression over time. Results: As expected, expression of mesenchymal markers was neglible in freshly isolated q-HSC. Surprisingly, however, qHSC expressed several epithelial markers, including CK-7, CK-19, and desmoplakin. During culture, epithelial marker expression decreased dramatically. By day 7, expression of CK7, CK-19 and desmoplakin fell by >15-, 7- and 2-fold, respectively. Expression of BMP7, an inhibitor of EMT, also decreased by >4-fold. Coincidentally, expression of the following mesenchymal markers increased: αSMA (>70-fold), collagen 1αI (>100-fold), S100A4 (>29fold), fibronectin (>100-fold). Activation of the Hh pathway paralleled the process of EMT. Expression of Hedgehog interacting protein (Hip, a repressor of Hh signaling) was high in q-HSC and fell by 100-fold during culture, as expression of PDGFR and Hh target genes, including Gli2, increased by >100-fold and 33-fold, respectively. In Conclusion: Quiescent HSC express epithelial markers and Hip, an inhibitor of Hh signaling. Culture activation results in loss of Hip and epithelial markers, and concomitant induction of Hh-target genes and various mesenchymal markers. These results suggest that HSC activation may be a process of epithelial-to-mesenchymal transition driven by Hh signaling.
Are Non-Invasive Biochemical Markers Useful in the Assessment Liver Fibrosis in Patients with Chronic Hepatitis C? José Luis Castro Urda, Cristina Vicente, Elvira Quintanilla, Marta Morán, David Collado, Ivan Guerra, Luis R. Rabago, Francisco Gea Different tests using non-invasive biochemical markers to predict liver fibrosis have been described. The aim of this study was to assess an external validation of the APRI and Forn's fibrosis indexes in our population of HCV chronic hepatitis Patients and Methods: We performed a retrospective study in 251 patients with chronic HCV-hepatitis. All the patients were assessed by both liver biopsy and a complete set of liver function tests. Liver biopsies were graded by a single experienced observer using the METAVIR scoring system. Fibrosis was classified as mild (grade F0 - F1), intermediate (grade F2) and severe (F3 - F4). We considered significant fibrosis the intermediate and severe grades. Forn's fibrosis and APRI indexes were calculated using published mathematical formulae. For data analysis, receiver operating characteristic (ROC) curves and univariate and multivariate logistic regression analyses were used, using the SPSS 11.0 statistical package. Differences with P< 0.05 values were considered significant. Results: Mean age was 40.9 years (SD 0.56). 64% were females. The VHC genotype 1 was found in 169 out of 224 patients who were checked. Fibrosis found in liver biopsies was mild in 52.2%, intermediate in 27.1% and severe 20.7% of samples. Patients with mild fibrosis had a Forn's score of 4.17 (SD 1.52) vs 4.78 (SD 1.50) in pts with significant fibrosis. Difference of means was significant (95% CI: 0.23-0.98). Likewise the APRI score was 0.64 (25th- to 75th-percentile values 0.45-0.97) in mild fibrosis vs. 0.93 (P25-75 0.58-1.54) in significant fibrosis (p=0.00008). The Forn's and the APRI scores were able to predict mild fibrosis in 60.8% vs. 66.7%, and significant fibrosis in 50% vs. 49% of the cases, respectively. Diagnostic accuracy for prediction of fibrosis improved making use of both indexes simultaneously, 69.9% for mild fibrosis and 53.9% for significant fibrosis. However, using multivariate logistic regression, the predictive model was only able to explain 10.1% of the fibrosis. On the other hand we did not find any statistical difference in cholesterol levels of patients with different genotypes of HCV. Conclusion. Despite the fact that both indexes were related with the degree of fibrosis, in our experience they are not reliable enough to replace liver biopsy in the assessment of fibrosis grade.
M1578
Background: Adenosine is increased during cellular apoptosis and inflammation. Adenosine inhibits HSC chemotaxis via the A2a receptor, but the mechanisms for this effect are not known. Activation of the Rho pathway is known to be central to organization of actin stress fibers, chemotaxis and contraction. We determined if adenosine can induce actin reorganization, identified the signaling pathways involved, and tested effects on HSC contraction. Hypothesis: Adenosine inhibits the Rho pathway, induces loss of actin stress fibers and inhibits HSC contraction. Methods: The HSC cell line LX-2 was treated with and without adenosine (10µM), and actin stress fibers imaged by confocal microscopy. Specific antagonists were used to identify receptor subtype requirements. DPCPX (A1 antagonist), ZM-241835 (A2a antagonist), MRS-1706 (A2b antagonist), MRS-1523 (A3 antagonist) were used. The role of cAMP was investigated using Forskolin (10µM). Following inhibitors were used to identify the requirement for the known signaling molecules downstream of adenosine receptors, ST-H31 (PKA inhibitor 25µM), rac-1 (Calbiochem 20µM), SB-203580 (P38 inhibitor 20µM). P38 phosphorylation was assayed by western blot. Rho activation was assayed by pull down assay, and HSC contraction was assessed by the collagen gel contraction assay. Results: Adenosine caused cytoskeletal reorganization with loss of actin stress fibers. These Cytoskeletal changes were mimicked by Forskolin, and were blocked by antagonist to the A2a receptor, PKA, Rac1 and P38. In response to adenosine there was p38 phosphorylation and reduction in Rho GTP. Adenosine did not induce any gel contraction, but significantly inhibited contraction mediated by endothelin-1. Conclusions: Signaling via the A2a receptor induces actin reorganization, with loss of actin stress fibers. This occurs via an increase in cAMP, PKA activation resulting in rac activation and inhibition of Rho GTP formation. Consistent with inhibition of rho and loss of actin stress fibers adenosine inhibits endothlein1 mediated contraction.
M1576 Curcumin Suppresses Insulin-Induced Hepatic Stellate Cell Activation By Interrupting Insulin Signaling and Attenuating Oxidative Stress Jianguo Lin, Anping Chen BACKGROUND: Patients with obesity and/or type II diabetes mellitus are often associated with hyperinsulinemia and are at risk to develop nonalcoholic fatty liver disease and hepatic fibrosis. Hepatic stellate cells (HSC) are the major effector cells during hepatic fibrogenesis. It remains largely unknown the roles and mechanisms of hyperinsulinemia in the promotion of HSC activation and hepatic fibrogenesis. We previously demonstrated that the antioxidant curcumin induced gene expression of PPARγ and enhanced its activity, resulting in the suppression of HSC activation and hepatic fibrogenesis In Vitro and In Vivo. AIM AND HYPOTHESIS: This study was aimed at evaluating effects of insulin on HSC activation, assessing the capability of curcumin in the attenuation of the effects, and further elucidating the underlying mechanisms. We hypothesized that insulin might stimulate HSC activation, which could be abrogated by curcumin by interrupting the insulin signaling pathway and attenuating oxidative stress. RESULTS: Insulin was shown to activate HSC In Vitro by stimulating cell proliferation and inducing expression of type I collagen gene, as well as genes relevant to HSC activation. These stimulatory effects of insulin were dose-dependently abrogated by curcumin. To elucidate the underlying mechanisms, curcumin was demonstrated to interrupt the insulin signaling pathways by reducing the level of phosphorylation of insulin receptor (InsR) and its downstream signaling cascades, including ERK1/2, JNK1/ 2, JAK2 and STAT3. In addition, curcumin suppressed InsR gene expression in activated HSC. Insulin was found to induce HSC activation by elevating cellular oxidative stress. Curcumin stimulated de novo synthesis of glutathione, the major intracellular antioxidant, by inducing gene expression of the rate-limiting enzyme glutamate-cysteine ligase, leading to the attenuation of the insulin-induced oxidative stress in HSC. It was of interest to observe that the pretreatment of HSC with the PPARγ antagonist PD68235 dramatically diminished these inhibitory effects of curcumin on the insulin-induced HSC activation, suggesting a critical role of PPARγ activation in the process. CONCLUSION: Curcumin inhibits the stimulatory effects of insulin on HSC activation, at least, by interrupting the insulin signaling pathway and attenuating oxidative stress, which is mainly mediated by the activation of PPARγ. The results in this report suggest that curcumin might be a therapeutic anti-fibrotic agent for the treatment of hepatic fibrogenesis associated with hyperinsulinemia. [The work was supported by the grant DK 47995 from NIH/NIDDK to A. Chen].
Collagen 1 Gel Assay A: Endothelin-1 B: Adenosine + Endothelin-1 M1579 Insulin-Mediated Signaling Events in the Activation of Hepatic Stellate Cells Cindy X. Cai, Rosemary O'Neill, Robert Britton, Bruce R. Bacon
M1577
Insulin resistance is a hallmark feature for non-alcoholic steatohepatitis (NASH), and NASH can result in liver fibrosis and cancer. Hyperinsulinemia and increased adipocytokines have been shown to contribute to the pathology of NASH and its related fibrosis. However, the molecular mechanisms underlying how hepatic stellate cells (HSC) are activated in NASH remain unclear. Previous studies have shown controversial results in the activation of cultured HSC by insulin. p70-S6K, a 70-kDa ribosomal S6 kinase can stimulate HSC proliferation and collagen I production. It is activated by mitogens, growth factors and hormones including insulin through the phosphatidylinositol 3 kinase (PI3K-Akt-mTOR1) pathway. We propose that insulin and adipocytokines may play a role in HSC activation by stimulating p70-S6K via the PI3K-Akt pathway. Aims: The aims of the current study were to investigate the effect of insulin and leptin on the phosphorylation of Akt and p70-S6K, and their potential roles in HSC activation. Methods: HSC were isolated from rat livers by sequential pronase and collagenase digestion, and separated by density gradient centrifugation. Primary and passaged cells were serum-starved for 24 hrs and exposed to different concentrations of insulin for 30 min with and without leptin (50 ng/ml) followed by immunoblot analysis with antibodies specifically against phosphorylated Akt and p70-S6K. Cell proliferation, αsmooth muscle actin (SMA) and type I collagen expression were also assessed. Some HSC
Culture Activation of Murine Hepatic Stellate Cells Involves Hedgehog Pathway Activation and Epithelial-to-Mesenchymal Transitions Steve S. Choi, Alessia Omenetti, Liu Yang, Rafal P. Witek, Kevin D. Brown, Mercedes Vazquez-Chantada, Jiawen Huang, Youngmi Jung, Wing-Kin Syn, Jason K. Sicklick, Anna Mae Diehl Background & Aims: Liver injury induces regenerative responses, including activation of quiescent hepatic stellate cells (q-HSC) to an activated, mesenchymal phenotype that leads to scar formation and liver dysfunction. Of the known HSC mitogens, platelet-derived growth factor (PDGF) is the most potent. We previously reported that PDGF receptor signaling activates the Hedgehog (Hh) pathway in HSC. Hh signaling promotes epithelial-to-mesenchymal transitions (EMT) during development and in adult cancers. In many tissues, EMT and mesenchymal-epithelial transitions (MET) are involved in repair. Cultured adult hepatocytes can undergo EMT. Whether or not HSC are capable of MET is less clear. Our Aim was to assess the relationship between Hh pathway activation and EMT/MET during HSC activation. Methods: Q-HSC were isolated from C57Bl6 mice by density gradient centrifugation. Greater
A-797
AASLD Abstracts
AASLD Abstracts
Adenosine Induces Loss of Actin Stress Fibers and Inhibits Contraction in Hepatic Stellate Cells Via Rho Inhibition Muhammad A. Sohail, Azuma Watanabe, Shamail Mahmood, Ardeshir Z. Hashmi, Wyel Hakim, Wajahat Z. Mehal
than 98% exhibited autofluorescence typical of q-HSC. Freshly isolated HSC were harvested for mRNA analysis or cultured for up to 7 days in serum-supplemented media to induce MF activation. Expression of epithelial and mesenchymal markers, as well as Hh-target genes, was monitored during culture (days 0, 4, 7). Quantitative, real-time RT-PCR was performed to compare changes in gene expression over time. Results: As expected, expression of mesenchymal markers was neglible in freshly isolated q-HSC. Surprisingly, however, qHSC expressed several epithelial markers, including CK-7, CK-19, and desmoplakin. During culture, epithelial marker expression decreased dramatically. By day 7, expression of CK7, CK-19 and desmoplakin fell by >15-, 7- and 2-fold, respectively. Expression of BMP7, an inhibitor of EMT, also decreased by >4-fold. Coincidentally, expression of the following mesenchymal markers increased: αSMA (>70-fold), collagen 1αI (>100-fold), S100A4 (>29fold), fibronectin (>100-fold). Activation of the Hh pathway paralleled the process of EMT. Expression of Hedgehog interacting protein (Hip, a repressor of Hh signaling) was high in q-HSC and fell by 100-fold during culture, as expression of PDGFR and Hh target genes, including Gli2, increased by >100-fold and 33-fold, respectively. In Conclusion: Quiescent HSC express epithelial markers and Hip, an inhibitor of Hh signaling. Culture activation results in loss of Hip and epithelial markers, and concomitant induction of Hh-target genes and various mesenchymal markers. These results suggest that HSC activation may be a process of epithelial-to-mesenchymal transition driven by Hh signaling.
Are Non-Invasive Biochemical Markers Useful in the Assessment Liver Fibrosis in Patients with Chronic Hepatitis C? José Luis Castro Urda, Cristina Vicente, Elvira Quintanilla, Marta Morán, David Collado, Ivan Guerra, Luis R. Rabago, Francisco Gea Different tests using non-invasive biochemical markers to predict liver fibrosis have been described. The aim of this study was to assess an external validation of the APRI and Forn's fibrosis indexes in our population of HCV chronic hepatitis Patients and Methods: We performed a retrospective study in 251 patients with chronic HCV-hepatitis. All the patients were assessed by both liver biopsy and a complete set of liver function tests. Liver biopsies were graded by a single experienced observer using the METAVIR scoring system. Fibrosis was classified as mild (grade F0 - F1), intermediate (grade F2) and severe (F3 - F4). We considered significant fibrosis the intermediate and severe grades. Forn's fibrosis and APRI indexes were calculated using published mathematical formulae. For data analysis, receiver operating characteristic (ROC) curves and univariate and multivariate logistic regression analyses were used, using the SPSS 11.0 statistical package. Differences with P< 0.05 values were considered significant. Results: Mean age was 40.9 years (SD 0.56). 64% were females. The VHC genotype 1 was found in 169 out of 224 patients who were checked. Fibrosis found in liver biopsies was mild in 52.2%, intermediate in 27.1% and severe 20.7% of samples. Patients with mild fibrosis had a Forn's score of 4.17 (SD 1.52) vs 4.78 (SD 1.50) in pts with significant fibrosis. Difference of means was significant (95% CI: 0.23-0.98). Likewise the APRI score was 0.64 (25th- to 75th-percentile values 0.45-0.97) in mild fibrosis vs. 0.93 (P25-75 0.58-1.54) in significant fibrosis (p=0.00008). The Forn's and the APRI scores were able to predict mild fibrosis in 60.8% vs. 66.7%, and significant fibrosis in 50% vs. 49% of the cases, respectively. Diagnostic accuracy for prediction of fibrosis improved making use of both indexes simultaneously, 69.9% for mild fibrosis and 53.9% for significant fibrosis. However, using multivariate logistic regression, the predictive model was only able to explain 10.1% of the fibrosis. On the other hand we did not find any statistical difference in cholesterol levels of patients with different genotypes of HCV. Conclusion. Despite the fact that both indexes were related with the degree of fibrosis, in our experience they are not reliable enough to replace liver biopsy in the assessment of fibrosis grade.
M1578
Background: Adenosine is increased during cellular apoptosis and inflammation. Adenosine inhibits HSC chemotaxis via the A2a receptor, but the mechanisms for this effect are not known. Activation of the Rho pathway is known to be central to organization of actin stress fibers, chemotaxis and contraction. We determined if adenosine can induce actin reorganization, identified the signaling pathways involved, and tested effects on HSC contraction. Hypothesis: Adenosine inhibits the Rho pathway, induces loss of actin stress fibers and inhibits HSC contraction. Methods: The HSC cell line LX-2 was treated with and without adenosine (10µM), and actin stress fibers imaged by confocal microscopy. Specific antagonists were used to identify receptor subtype requirements. DPCPX (A1 antagonist), ZM-241835 (A2a antagonist), MRS-1706 (A2b antagonist), MRS-1523 (A3 antagonist) were used. The role of cAMP was investigated using Forskolin (10µM). Following inhibitors were used to identify the requirement for the known signaling molecules downstream of adenosine receptors, ST-H31 (PKA inhibitor 25µM), rac-1 (Calbiochem 20µM), SB-203580 (P38 inhibitor 20µM). P38 phosphorylation was assayed by western blot. Rho activation was assayed by pull down assay, and HSC contraction was assessed by the collagen gel contraction assay. Results: Adenosine caused cytoskeletal reorganization with loss of actin stress fibers. These Cytoskeletal changes were mimicked by Forskolin, and were blocked by antagonist to the A2a receptor, PKA, Rac1 and P38. In response to adenosine there was p38 phosphorylation and reduction in Rho GTP. Adenosine did not induce any gel contraction, but significantly inhibited contraction mediated by endothelin-1. Conclusions: Signaling via the A2a receptor induces actin reorganization, with loss of actin stress fibers. This occurs via an increase in cAMP, PKA activation resulting in rac activation and inhibition of Rho GTP formation. Consistent with inhibition of rho and loss of actin stress fibers adenosine inhibits endothlein1 mediated contraction.
M1576 Curcumin Suppresses Insulin-Induced Hepatic Stellate Cell Activation By Interrupting Insulin Signaling and Attenuating Oxidative Stress Jianguo Lin, Anping Chen BACKGROUND: Patients with obesity and/or type II diabetes mellitus are often associated with hyperinsulinemia and are at risk to develop nonalcoholic fatty liver disease and hepatic fibrosis. Hepatic stellate cells (HSC) are the major effector cells during hepatic fibrogenesis. It remains largely unknown the roles and mechanisms of hyperinsulinemia in the promotion of HSC activation and hepatic fibrogenesis. We previously demonstrated that the antioxidant curcumin induced gene expression of PPARγ and enhanced its activity, resulting in the suppression of HSC activation and hepatic fibrogenesis In Vitro and In Vivo. AIM AND HYPOTHESIS: This study was aimed at evaluating effects of insulin on HSC activation, assessing the capability of curcumin in the attenuation of the effects, and further elucidating the underlying mechanisms. We hypothesized that insulin might stimulate HSC activation, which could be abrogated by curcumin by interrupting the insulin signaling pathway and attenuating oxidative stress. RESULTS: Insulin was shown to activate HSC In Vitro by stimulating cell proliferation and inducing expression of type I collagen gene, as well as genes relevant to HSC activation. These stimulatory effects of insulin were dose-dependently abrogated by curcumin. To elucidate the underlying mechanisms, curcumin was demonstrated to interrupt the insulin signaling pathways by reducing the level of phosphorylation of insulin receptor (InsR) and its downstream signaling cascades, including ERK1/2, JNK1/ 2, JAK2 and STAT3. In addition, curcumin suppressed InsR gene expression in activated HSC. Insulin was found to induce HSC activation by elevating cellular oxidative stress. Curcumin stimulated de novo synthesis of glutathione, the major intracellular antioxidant, by inducing gene expression of the rate-limiting enzyme glutamate-cysteine ligase, leading to the attenuation of the insulin-induced oxidative stress in HSC. It was of interest to observe that the pretreatment of HSC with the PPARγ antagonist PD68235 dramatically diminished these inhibitory effects of curcumin on the insulin-induced HSC activation, suggesting a critical role of PPARγ activation in the process. CONCLUSION: Curcumin inhibits the stimulatory effects of insulin on HSC activation, at least, by interrupting the insulin signaling pathway and attenuating oxidative stress, which is mainly mediated by the activation of PPARγ. The results in this report suggest that curcumin might be a therapeutic anti-fibrotic agent for the treatment of hepatic fibrogenesis associated with hyperinsulinemia. [The work was supported by the grant DK 47995 from NIH/NIDDK to A. Chen].
Collagen 1 Gel Assay A: Endothelin-1 B: Adenosine + Endothelin-1 M1579 Insulin-Mediated Signaling Events in the Activation of Hepatic Stellate Cells Cindy X. Cai, Rosemary O'Neill, Robert Britton, Bruce R. Bacon
M1577
Insulin resistance is a hallmark feature for non-alcoholic steatohepatitis (NASH), and NASH can result in liver fibrosis and cancer. Hyperinsulinemia and increased adipocytokines have been shown to contribute to the pathology of NASH and its related fibrosis. However, the molecular mechanisms underlying how hepatic stellate cells (HSC) are activated in NASH remain unclear. Previous studies have shown controversial results in the activation of cultured HSC by insulin. p70-S6K, a 70-kDa ribosomal S6 kinase can stimulate HSC proliferation and collagen I production. It is activated by mitogens, growth factors and hormones including insulin through the phosphatidylinositol 3 kinase (PI3K-Akt-mTOR1) pathway. We propose that insulin and adipocytokines may play a role in HSC activation by stimulating p70-S6K via the PI3K-Akt pathway. Aims: The aims of the current study were to investigate the effect of insulin and leptin on the phosphorylation of Akt and p70-S6K, and their potential roles in HSC activation. Methods: HSC were isolated from rat livers by sequential pronase and collagenase digestion, and separated by density gradient centrifugation. Primary and passaged cells were serum-starved for 24 hrs and exposed to different concentrations of insulin for 30 min with and without leptin (50 ng/ml) followed by immunoblot analysis with antibodies specifically against phosphorylated Akt and p70-S6K. Cell proliferation, αsmooth muscle actin (SMA) and type I collagen expression were also assessed. Some HSC
Culture Activation of Murine Hepatic Stellate Cells Involves Hedgehog Pathway Activation and Epithelial-to-Mesenchymal Transitions Steve S. Choi, Alessia Omenetti, Liu Yang, Rafal P. Witek, Kevin D. Brown, Mercedes Vazquez-Chantada, Jiawen Huang, Youngmi Jung, Wing-Kin Syn, Jason K. Sicklick, Anna Mae Diehl Background & Aims: Liver injury induces regenerative responses, including activation of quiescent hepatic stellate cells (q-HSC) to an activated, mesenchymal phenotype that leads to scar formation and liver dysfunction. Of the known HSC mitogens, platelet-derived growth factor (PDGF) is the most potent. We previously reported that PDGF receptor signaling activates the Hedgehog (Hh) pathway in HSC. Hh signaling promotes epithelial-to-mesenchymal transitions (EMT) during development and in adult cancers. In many tissues, EMT and mesenchymal-epithelial transitions (MET) are involved in repair. Cultured adult hepatocytes can undergo EMT. Whether or not HSC are capable of MET is less clear. Our Aim was to assess the relationship between Hh pathway activation and EMT/MET during HSC activation. Methods: Q-HSC were isolated from C57Bl6 mice by density gradient centrifugation. Greater
A-797
AASLD Abstracts
AASLD Abstracts
Adenosine Induces Loss of Actin Stress Fibers and Inhibits Contraction in Hepatic Stellate Cells Via Rho Inhibition Muhammad A. Sohail, Azuma Watanabe, Shamail Mahmood, Ardeshir Z. Hashmi, Wyel Hakim, Wajahat Z. Mehal