- Email: [email protected]
Outcome of Liver Transplantation in HCV Infected Patients and Implications for Antiviral Treatment Decisions
C (HCV) in the setting of previously treated HCC has raised a concern for increased relapse of HCC. Aims: Determine the effect of DAA on the recurrence rate (RR) of HCC and survival after liver transplantation (LT). Methods: 155 consecutive patients with HCV and HCC underwent LT between 2008 and 2015 in a tertiary care center (mean age 59±5 years, 82% males). Treatment of HCV post LT was noted. Time-to-event analysis was used to assess HCC recurrence. Follow-up was defined as months from transplant to the diagnosis of HCC recurrence or last follow-up visit. Results: After LT, 58 (37.4%) patients received no HCV treatment (NoTT group), 23 (14.8%) received IFN-based therapy (IFN group) and 74 (47.7%) received DAA without interferon (DAA group). Recurrent HCC developed in 21 patients; 12 (20.7%) occurred in NoTT group, 3 (13%) in IFN group and 6 (8%) in DAA group (figure 1). Median time to recurrence after LT was 11.1 months [IQR 5.9-24.3] in the noTT, 47.8 months [IQR 13.5-54.5] in IFN group, and 33.7 months [IQR 16.7-49.1] in DAA group. Only one patient had recurrence 5 years after LT (in DAA group). After adjusting for Milan criteria, subjects in NoTT group had higher risk of HCC recurrence as compared to DAA group; HR 3 [95% CI 1.01-8.8, p=0.04]. There was no difference in HCC recurrence between IFN treated patients and DAA group; HR 1.8 [95% CI 0.42-7.4, p=0.44]. A total of 35 (22.6%) subjects died after LT. Time of death was known in 23 subjects; 18 (31%) in NoTT group, 3 (13%) in IFN group and 2 (3%) in DAA group (figure 2). After adjusting for Milan criteria, there was an increased mortality in patients in NoTT group as compared to DAA group; HR 27.6 [95% CI 3.6-208.4, p<0.01]. IFN treated patients had a trend toward increased post-LT mortality when compared to DAA group; HR 9 [95% CI 0.94, 86.8, P=0.06]. Conclusion: Treatment of HCV after LT improved survival after liver transplantation in HCC patients and was associated with a lower HCC recurrence rate.
AASLD Abstracts
HEV cases (n=3) and other unavailable cases (n=9). We prospectively followed up 94-postLT patients every 4 months for 1 year for the clinical evidence of hepatitis, HEV serology and serum/feces HEV RNA. At 8 months, 65-post-LT patients had completed the follow up protocol. All patient samples (-70C storage) were evaluated simultaneously. HEV RNA in serum and feces were detected by real-time (in-house) RT-PCR technique (lowest level of detection=10 IU/mL). Result: The HEV seroprevalence was 53.8% (57/106). Of the 65 postLT patients, 35 cases had HEV IgG(+) (53%) and 30 cases of IgG(-) (46.2%). There were no significant differences in baseline characteristics in between groups, regarding pre-LT liver diseases, types and doses of immunosuppressive drugs. In HEV IgG(+) group (Table 1), serum HEV RNA was detected in 9/35 cases (25.7%) with 1 persistent case. One case had both serum and feces RNA (+). Persistent HEV IgM(+) without RNA detection was observed in 1 case. HEV IgG became negative during 8-month follow-up in 3/35 (2.9%). In HEV IgG(-) group (Table 1), serum HEV RNA was detected in 4/30 cases (13.3%) with 1 persistent case. The HEV seroconversion [IgG(+)] without RNA detection was observed in 4/30 cases (13.3%). Feces HEV RNA was identified 1 and 2 cases with and without simultaneous serum HEV RNA(+), respectively. Of those 17 cases whose IgM(+) or serum/ feces HEV RNA(+), there were 2 cases having abnormal liver tests, one with mild transient hepatitis and one with cholestatic hepatitis from bile duct stricture (Table 2). Conclusion: In this high-prevalence area, subclinical transient or chronic HEV infection in post-LT patients were demonstrated without significant or obvious clinical hepatitis. However, clinical significance of these subclinical cases required longer follow-up data. Not only serum, but also feces HEV RNA, even a few cases detected and quite troublesome for the patients, might add on clinical value for early detection when no other tests give a clue of the infection. Table 1: Number of cases with serum HEV serology and RNA detection in serum and feces at baseline and 8-month follow up period. [a, b, d,…] represents anonymous patients' name.
Figure 1. HCC Recurrence Table 2: Characteristics of 17 patients who had HEV IgM(+) or serum/feces HEV RNA(+) during the follow up period
Figure 2. Post-LT Survival
Sa1546 OUTCOME OF LIVER TRANSPLANTATION IN HCV INFECTED PATIENTS AND IMPLICATIONS FOR ANTIVIRAL TREATMENT DECISIONS Thomas Greuter, Holger Seidl, Benjamin Misselwitz, Philipp Dutkowski, Beat Müllhaupt, Thomas Kuntzen
*Patient g had hepaticojejunostomy stricture with intrahepatic duct stone. **Patient h had mild hepatitis at the time when the 3rd test was performed.
Background and Aims: The indication and optimal time point for anti-HCV treatment before and after orthotopic liver transplantation (OLT) continues to be a matter of debate in the era of directly acting antivirals (DAA) due to side effects and cost considerations still requiring individualized treatment decisions, for which this study aims to contribute to a better basis of knowledge. Methods: In 448 patients who underwent liver transplantation at the University Hospital Zurich between 1995 and 2013 we retrospectively studied fibrosis progression as well as patient and graft survival in HCV infected patients with and without antiviral treatment before and after liver transplantation compared to patients transplanted for other disease entities. Results: In eighteen patients interferon-based antiviral therapy - in two cases including first generation DAAs - was carried out on the transplant waiting list. Cure was achieved in 14 (78%) patients, notably in 13/14 (93%) who achieved at least 27 days of pre-OLT aviremia. After transplantation, 124/159 patients with a history of HCV infection experienced HCV recurrence, which was treated in 67 cases up to three times, eventually achieving SVR
Sa1545 TREATMENT OF HCV IN HCC PATIENTS AFTER LIVER TRANSPLANTATION IMPROVES SURVIVAL AND DECREASES RISK OF HCC RECURRENCE Fadi Niyazi, Vedha Sanghi, Baraa Abduljawad, Mohammad Maysara Asfari, Rocio Lopez, KV Narayanan Menon, Carlos Romero-Marrero Background: The impact of Direct Acting Antivirals (DAA) treatment for hepatitis C on liver transplant (LT) outcomes and post-transplant recurrence of Hepatocellular carcinoma (HCC) is yet to be determined. Recent assessment of DAA for the treatment of Chronic Hepatitis
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AASLD Abstracts
(≈30%↓), and expression of profibrogenic genes col1α1 (≈50%↓), αSMA (≈50%↓), TIMP1 (≈50%↓). Markers of aPFs like Fibulin2 were also downregulated by 50% in Msln-/- mice. Conclusion: In both MDR2-/- and DDC diet models, the major source of myofibroblasts are aPFs. In MDR2-/- mice, a heterogeneous population of aPFs that are Thy-1+ and Thy1- are a major source of myofibroblasts, while Thy-1+ aPFs are the main source of myofibroblasts in BDL. Msln is also working as a profibrogenic molecule in an additional model of cholestatic liver injury.
in 26/67 (39%) patients. Data on fibrosis progression was available in 94, 79, 54 and 36 patients at years one, 2-3, 4-5 and 6-9 after OLT. Here, the proportion of liver fibrosis ≥ Metavir F2 rose from 27% (25/94) in the first year to 75% (27/36) in years 6-9. Likewise, cirrhosis developed in 2.1% (2/94) by year 1 and in 41.7% (15/36) of patients by years 69. Graft survival was reduced compared to HCV negative patients at 1 year (85% versus 92%) and 5 years (76% versus 87%) after OLT mainly due to HCV induced graft failure (17/159, 11%). Upon antiviral treatment liver fibrosis remained unchanged (mean Metavir fibrosis score 1.8 versus 1.9) between the pre- and post-treatment evaluation time points in 16 sustained responders, but rose from a mean Metavir score of 1.9 to 3.3 (p<0.001) in 25 non-responders. Conclusions: At least 27 days of aviremia appear sufficient to achieve SVR in the immediate pre-OLT setting for most patients. Using a watch and wait strategy only 25% of patients remained below the Metavir F2 threshold triggering antiviral treatment 6-9 years after OLT, but a significant number of patients was put at risk of HCV induced complications, suggesting antiviral treatment should be strongly considered for patients after OLT irrespective of the early development of liver fibrosis in the DAA era.
Sa1551 MRTF-A/SRF INHIBITION AMELIORATES LIVER FIBROSIS VIA INHIBITION OF TYPE I COLLAGEN EXPRESSION IN HEPATIC STELLATE CELLS Zengdun Shi, Don C. Rockey Background: Liver fibrosis is characterized by excessive extracellular matrix deposition. Hepatic stellate cells (HSCs) have been recognized as a primary source of matrix synthesis. Our goal has been to understand mechanisms underlying this process. In this study, we hypothesized that the actin cytoskeleton is critical specifically in the regulation of type I collagen expression. We have focused on myocardin related transcription factor-A (MRTFA), an actin binding protein and SRF cotransactivator that supports the actin cytoskeleton. Methods: HSCs were isolated from normal rat liver and exposed to CCG-203971 (20 mM) (DMSO was used as control), which blocks MRTF's interaction with serum respnse factor (SRF), and thus impairs expression of actin. Immunoblotting and immunocytochemistry were performed to examine protein expression, signal pathways and actin cytoskeleton. The effect of CCG-203971 was evaluated in vivo by inducing fibrosis with carbon tetrachloride (CCl4) (6 doses over 6 weeks); type 1 collagen expression was determined by RT-PCR and picrosirius red histomorphometry. Results: HSC activation was associated with prominent MRTF-A nuclear accumulation as well as a 2.4-fold increase in expression (P < 0.05 for activated HSCs vs. quiescent HSCs). When HSCs were exposed to CCG-203971, the actin cytoskeleton was disrupted and disorganized, which led to alterations in cell shape and size. A variety of HSC functions were impaired by CCG-203971, including cell contraction, migration and proliferation, all of which were decreased. Additionally, type 1 collagen expression was decreased by 60% (P < 0.05 for HSCs exposed to CCG-203971 vs. control). Exogenous expression of MRTF-A partially restored type 1 collagen expression levels in HSCs exposed to CCG-203971. We also found that the Smad and Erk signaling pathways were interrupted by CCG-203971. CCG-203971 inhibited both type 1a1 and 1a2 collagen promoter activities (n=4 for each, 10.5 and 9.3-fold reduction, respectively; each P < 0.01 for HSCs treated with CCG-203971 vs. control), consistent with interruption of SRF function. Finally, CCG-203971 had prominent effects on in vivo fibrogenesis, leading to reduced CCl4induced liver fibrosis (31% decease in morphometric collagen vs. control (n=8, P < 0.05); and 35% decrease in whole liver type 1 collagen mRNA expression vs. control (n=8, P < 0.05)). Conclusions: The actin cytoskeleton plays a critical role in HSC function and type I collagen synthesis, suggesting that targeting actin cytoskeletal dynamics by interruption of MRTF-A signaling could be a highly novel approach for treatment of liver fibrosis.
Sa1549
AASLD Abstracts
HEPATOCYTE VERSUS STELLATE CELL SPECIFIC DELETION OF LIVER FATTY ACID BINDING PROTEIN (L-FABP) REVEALS CELL- TYPE SPECIFIC ROLES IN RETINOL METABOLISM, STEATOSIS AND FIBROSIS Elizabeth P. Newberry, Roberto Solis, Susan Kennedy, Seung-Ah Lee, William S. Blaner, Anping Chen, Nicholas O. Davidson Background: Hepatic lipotoxic injury promotes inflammation and fibrosis but the mechanisms and modulators are poorly understood. Liver fatty acid binding protein (L-Fabp) facilitates intracellular fatty acid (FA) trafficking and germline L-Fabp deletion (KO) attenuates dietinduced obesity (DIO), hepatic steatosis and fibrosis. Methods: We examined cell type specific [hepatocyte, stellate cell (HSC)] functions of L-Fabp in FA trafficking and injury. We generated conditional L-Fabp deletor lines in HSC (GFAP-CreTg, HSC-KO) and hepatocytes (Alb-CreTg, Liv KO) and examined FA uptake, incorporation and lipotoxic injury. Results: On a low fat chow diet, hepatic triglyceride (TG) was reduced in Liv-KO mice vs both f/f mice and HSC-KO (Liv-KO, 26 ± 9 µg TG/mg protein; f/f, 107 ± 12; HSC-KO, 79 ± 12, p=0.04). In response to 48h fasting, Liv-KO mice exhibited attenuated steatosis compared to both HSC-KO and f/f mice (Liv-KO, 81 ± 15 µg TG/mg protein; HSC-KO, 161 ± 12, f/f, 190 ± 43). These data suggest that Liv-KO hepatocytes are unable to incorporate exogenous FA mobilized during prolonged fasting (as observed in KO), findings confirmed by in-vitro FA uptake. We next asked if cell-specific L-Fabp deletion alters DIO, steatosis or fibrosis. Mice were fed either high saturated fat or high trans-fat fructose (TFF) diet. LivKO mice had decreased steatosis (Sat FA: Liv-KO, 248 ± 38 µg TG/mg; f/f, 493 ± 98; TFF: Liv-KO, 210 ± 62; f/f, 556 ± 123; HSC KO, 597 ± 120) and TFF fed Liv-KO mice had reduced fibrotic injury (% fibrotic area: f/f, 0.7 ± 0.08; Liv-KO, 0.5 ± 0.03; HSC-KO, 0.8 ± 0.08) and reduced fibrogenic gene expression. These data show that exogenous FA trafficking is impaired in Liv KO mice, and that diet induced (ie lipotoxic) fibrotic injury is attenuated with reduced steatosis. In contrast, carbon tetrachloride (CCl4) treated mice revealed worse injury (increased ALT, HSC activation) in both germline KO as well as HSCKO mice compared to both f/f or Liv-KO, and independent of hepatocyte lipid content. We further examined HSC retinol and retinol ester (RE) content. HSC-KO stellate cells showed slightly decreased retinol and RE content, with significant decreases in distinct RE species (16:0, 18:1) suggesting altered FA trafficking into HSC RE. Total retinol and RE levels were also decreased ~50% in Liv-KO HSC compared to f/f, with reduced abundance of all RE species, raising the question of how hepatocyte lipid content modulates HSC retinol metabolism. Consistent with this, HSC RE levels were also reduced in germline KO mice. Conclusions: These data suggest a cell type specific role of L-Fabp in distinct models of liver injury, with attenuated lipid mediated injury in Liv-KO but augmented fibrogenic injury in HSCKO. In addition, L-Fabp is involved in RE synthesis in hepatocytes and RE storage in stellate cells.
Sa1552 RASAL1 IS A POTENT REGULATOR OF HEPATIC STELLATE CELL ACTIVITIES AND LIVER FIBROSIS Motoyuki Otsuka, Akemi Takata, Takahiro Kishikawa, Motoko Ohno, Mari Yamagami, Rei Ishibashi, Kazuma Sekiba, Takeshi Yoshikawa, Kazuhiko Koike Liver fibrosis, leading to cirrhosis and liver failure, is observed after chronic liver injury. The transition of hepatic stellate cells (HSCs) from quiescent cells to proliferative and fibrogenic cells is a central event in liver fibrosis. Here, we show that RAS protein activator like-1 (RASAL1), a member of the RAS-GTPase-activating proteins that switch off RAS activity, is significantly decreased during mouse primary HSC activation, and that HSC activation can be antagonized by the forced expression of RASAL1 protein. We demonstrate that RASAL1 suppresses HSC proliferation by regulating the RasMAPK pathway, and that RASAL1 suppresses HSC fibrogenic activity through regulating the PKA- LKB1-AMPK-SRF pathway by interacting with angiotensin II receptor, type 1 (AGTR1). We also show that RASAL1-deficient mice are more prone to liver fibrosis. These studies demonstrate that deregulated RASAL1 expression levels and its subsequent downstream intracellular signaling are central mediators of perpetuated HSC activation and fibrogenesis in the liver.
Sa1550 THE CHARACTERISTICS OF MYOFIBROBLASTS IN VARIOUS CHOLESTATIC LIVER INJURY MODELS IN MICE Yukinori Koyama, Ronglin Hu, Jun Xu, Xiao Liu, Hsiao-Yen Ma, Shuang Liang, David A. Brenner, Tatiana Kisseleva Introduction: Cholestatic liver diseases include primary biliary cholangitis, biliary atresia, and primary sclerosing cholangitis, all of which lead to liver cirrhosis. By using bile duct ligation (BDL) model, we have revealed the following findings of cholestatic liver fibrosis: 1) In BDL model, bone marrow (BM) derived fibrocytes minimally contribute to myofibroblasts; 2) Activated portal fibroblasts (aPFs) are a major contributor of myofibroblasts, especially in its early stage of pathogenesis; 3) aPFs express several markers such as Thy1, Msln and Muc16, which are related to proliferation and activation of aPFs. However, BDL does not fully mimic the cholestatic liver diseases described above. To elucidate the pathogenesis of cholestatic fibrosis, we need to know what is common in cholestatic liver fibrosis and what are disease (model) specific findings. In this study, based on our previous findings in BDL, we analyzed the origin of myofibroblasts and the role of Msln, a profibrogenic molecule in BDL, in MDR2-/- mouse model and Diethoxycarbonyl dihydrocollidine (DDC) diet model. Methods: To analyze the contribution of BM derived cells in MDR2-/- mice we transplanted BM from collagen 1α1-GFP reporter mice into irradiated MDR2-/- mice. The contribution of aPFs in MDR2-/- (8 weeks old) and DDC diet model was analyzed by immunohistochemistry and flow cytometry. In order to analyze the role of Msln, which works as a profibrotic molecule in BDL model, DDC diet was given to wt and Msln-/- mice for 3 weeks and liver fibrosis was evaluated. Results: In MDR2-/- mice, the contribution of BM derived cells to myofibroblasts was less than 1%. Flow cytometry revealed that in MDR2-/- and DDC model, 50-70% of myofibroblasts were vitamin A negative aPFs, which is comparable to BDL model. In contrast to BDL, in which almost all aPFs are characterized by the expression of Thy-1, Thy-1+ aPFs were limited to 50-60% of total vitamin A negative myofibroblasts in MDR2-/- mice. In DDC diet model, the expression of Msln was upregulated in the liver. Liver fibrosis was attenuated in Msln-/- mice, as evaluated by Sirius red staining
AASLD Abstracts
Sa1553 HIGH-MOBILITY GROUP BOX 1(HMGB1) ACTIVATES CASPASE-1 AND INDUCES THE ACTIVATION OF HSCS Wei Yan, Yixing Luo, Ping Han, Yu Fu Background: The activation of hepatic stellate cells (HSCs) plays an important role in the development of liver fibrosis and it always begins with inflammation. The exact mechanisms driving inflammation induced activation of HSCs remain elusive. HMGB1, a pro-inflammatory cytokine, has recently been showed to be closely associated with the activation of HSCs. The inflammasome, a caspase-1 activation platform, regulates immune responses to diverse stimuli that appear during infection or after tissue damage. We hypothesize that HMGB1 activation of the inflammasome plays a key role in the activation of HSCs and fibrogenesis. Methods: Hepatic stellate cell lines were treated with recombinant HMGB1(10 ng/ml or 100 ng/ml) for 24 hours. The migration of HSCs was detected by transwell cell migration assay and scratch assay, the collagen synthesis in HSCs was tested by Westernblot and immunofluorescent staining. The expression of caspase-1 in HSCs was detected by western blot and Colorimetric Assay. Z-YVAD-FMK (caspase-1 inhibitor, 2µM) was used to inhibit caspase-1 after recombinant HMGB1(100 ng/ml) treatment in HSCs. The rat fibrosis models were built by TAA-intraperitoneal injection (dissolved in PBS, 150 mg/kg B.W.). After TAAintraperitoneal injection for 4 weeks, the rats were treated with intraperitoneal injection of
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AASLD Abstracts
HEV cases (n=3) and other unavailable cases (n=9). We prospectively followed up 94-postLT patients every 4 months for 1 year for the clinical evidence of hepatitis, HEV serology and serum/feces HEV RNA. At 8 months, 65-post-LT patients had completed the follow up protocol. All patient samples (-70C storage) were evaluated simultaneously. HEV RNA in serum and feces were detected by real-time (in-house) RT-PCR technique (lowest level of detection=10 IU/mL). Result: The HEV seroprevalence was 53.8% (57/106). Of the 65 postLT patients, 35 cases had HEV IgG(+) (53%) and 30 cases of IgG(-) (46.2%). There were no significant differences in baseline characteristics in between groups, regarding pre-LT liver diseases, types and doses of immunosuppressive drugs. In HEV IgG(+) group (Table 1), serum HEV RNA was detected in 9/35 cases (25.7%) with 1 persistent case. One case had both serum and feces RNA (+). Persistent HEV IgM(+) without RNA detection was observed in 1 case. HEV IgG became negative during 8-month follow-up in 3/35 (2.9%). In HEV IgG(-) group (Table 1), serum HEV RNA was detected in 4/30 cases (13.3%) with 1 persistent case. The HEV seroconversion [IgG(+)] without RNA detection was observed in 4/30 cases (13.3%). Feces HEV RNA was identified 1 and 2 cases with and without simultaneous serum HEV RNA(+), respectively. Of those 17 cases whose IgM(+) or serum/ feces HEV RNA(+), there were 2 cases having abnormal liver tests, one with mild transient hepatitis and one with cholestatic hepatitis from bile duct stricture (Table 2). Conclusion: In this high-prevalence area, subclinical transient or chronic HEV infection in post-LT patients were demonstrated without significant or obvious clinical hepatitis. However, clinical significance of these subclinical cases required longer follow-up data. Not only serum, but also feces HEV RNA, even a few cases detected and quite troublesome for the patients, might add on clinical value for early detection when no other tests give a clue of the infection. Table 1: Number of cases with serum HEV serology and RNA detection in serum and feces at baseline and 8-month follow up period. [a, b, d,…] represents anonymous patients' name.
Figure 1. HCC Recurrence Table 2: Characteristics of 17 patients who had HEV IgM(+) or serum/feces HEV RNA(+) during the follow up period
Figure 2. Post-LT Survival
Sa1546 OUTCOME OF LIVER TRANSPLANTATION IN HCV INFECTED PATIENTS AND IMPLICATIONS FOR ANTIVIRAL TREATMENT DECISIONS Thomas Greuter, Holger Seidl, Benjamin Misselwitz, Philipp Dutkowski, Beat Müllhaupt, Thomas Kuntzen
*Patient g had hepaticojejunostomy stricture with intrahepatic duct stone. **Patient h had mild hepatitis at the time when the 3rd test was performed.
Background and Aims: The indication and optimal time point for anti-HCV treatment before and after orthotopic liver transplantation (OLT) continues to be a matter of debate in the era of directly acting antivirals (DAA) due to side effects and cost considerations still requiring individualized treatment decisions, for which this study aims to contribute to a better basis of knowledge. Methods: In 448 patients who underwent liver transplantation at the University Hospital Zurich between 1995 and 2013 we retrospectively studied fibrosis progression as well as patient and graft survival in HCV infected patients with and without antiviral treatment before and after liver transplantation compared to patients transplanted for other disease entities. Results: In eighteen patients interferon-based antiviral therapy - in two cases including first generation DAAs - was carried out on the transplant waiting list. Cure was achieved in 14 (78%) patients, notably in 13/14 (93%) who achieved at least 27 days of pre-OLT aviremia. After transplantation, 124/159 patients with a history of HCV infection experienced HCV recurrence, which was treated in 67 cases up to three times, eventually achieving SVR
Sa1545 TREATMENT OF HCV IN HCC PATIENTS AFTER LIVER TRANSPLANTATION IMPROVES SURVIVAL AND DECREASES RISK OF HCC RECURRENCE Fadi Niyazi, Vedha Sanghi, Baraa Abduljawad, Mohammad Maysara Asfari, Rocio Lopez, KV Narayanan Menon, Carlos Romero-Marrero Background: The impact of Direct Acting Antivirals (DAA) treatment for hepatitis C on liver transplant (LT) outcomes and post-transplant recurrence of Hepatocellular carcinoma (HCC) is yet to be determined. Recent assessment of DAA for the treatment of Chronic Hepatitis
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AASLD Abstracts
(≈30%↓), and expression of profibrogenic genes col1α1 (≈50%↓), αSMA (≈50%↓), TIMP1 (≈50%↓). Markers of aPFs like Fibulin2 were also downregulated by 50% in Msln-/- mice. Conclusion: In both MDR2-/- and DDC diet models, the major source of myofibroblasts are aPFs. In MDR2-/- mice, a heterogeneous population of aPFs that are Thy-1+ and Thy1- are a major source of myofibroblasts, while Thy-1+ aPFs are the main source of myofibroblasts in BDL. Msln is also working as a profibrogenic molecule in an additional model of cholestatic liver injury.
in 26/67 (39%) patients. Data on fibrosis progression was available in 94, 79, 54 and 36 patients at years one, 2-3, 4-5 and 6-9 after OLT. Here, the proportion of liver fibrosis ≥ Metavir F2 rose from 27% (25/94) in the first year to 75% (27/36) in years 6-9. Likewise, cirrhosis developed in 2.1% (2/94) by year 1 and in 41.7% (15/36) of patients by years 69. Graft survival was reduced compared to HCV negative patients at 1 year (85% versus 92%) and 5 years (76% versus 87%) after OLT mainly due to HCV induced graft failure (17/159, 11%). Upon antiviral treatment liver fibrosis remained unchanged (mean Metavir fibrosis score 1.8 versus 1.9) between the pre- and post-treatment evaluation time points in 16 sustained responders, but rose from a mean Metavir score of 1.9 to 3.3 (p<0.001) in 25 non-responders. Conclusions: At least 27 days of aviremia appear sufficient to achieve SVR in the immediate pre-OLT setting for most patients. Using a watch and wait strategy only 25% of patients remained below the Metavir F2 threshold triggering antiviral treatment 6-9 years after OLT, but a significant number of patients was put at risk of HCV induced complications, suggesting antiviral treatment should be strongly considered for patients after OLT irrespective of the early development of liver fibrosis in the DAA era.
Sa1551 MRTF-A/SRF INHIBITION AMELIORATES LIVER FIBROSIS VIA INHIBITION OF TYPE I COLLAGEN EXPRESSION IN HEPATIC STELLATE CELLS Zengdun Shi, Don C. Rockey Background: Liver fibrosis is characterized by excessive extracellular matrix deposition. Hepatic stellate cells (HSCs) have been recognized as a primary source of matrix synthesis. Our goal has been to understand mechanisms underlying this process. In this study, we hypothesized that the actin cytoskeleton is critical specifically in the regulation of type I collagen expression. We have focused on myocardin related transcription factor-A (MRTFA), an actin binding protein and SRF cotransactivator that supports the actin cytoskeleton. Methods: HSCs were isolated from normal rat liver and exposed to CCG-203971 (20 mM) (DMSO was used as control), which blocks MRTF's interaction with serum respnse factor (SRF), and thus impairs expression of actin. Immunoblotting and immunocytochemistry were performed to examine protein expression, signal pathways and actin cytoskeleton. The effect of CCG-203971 was evaluated in vivo by inducing fibrosis with carbon tetrachloride (CCl4) (6 doses over 6 weeks); type 1 collagen expression was determined by RT-PCR and picrosirius red histomorphometry. Results: HSC activation was associated with prominent MRTF-A nuclear accumulation as well as a 2.4-fold increase in expression (P < 0.05 for activated HSCs vs. quiescent HSCs). When HSCs were exposed to CCG-203971, the actin cytoskeleton was disrupted and disorganized, which led to alterations in cell shape and size. A variety of HSC functions were impaired by CCG-203971, including cell contraction, migration and proliferation, all of which were decreased. Additionally, type 1 collagen expression was decreased by 60% (P < 0.05 for HSCs exposed to CCG-203971 vs. control). Exogenous expression of MRTF-A partially restored type 1 collagen expression levels in HSCs exposed to CCG-203971. We also found that the Smad and Erk signaling pathways were interrupted by CCG-203971. CCG-203971 inhibited both type 1a1 and 1a2 collagen promoter activities (n=4 for each, 10.5 and 9.3-fold reduction, respectively; each P < 0.01 for HSCs treated with CCG-203971 vs. control), consistent with interruption of SRF function. Finally, CCG-203971 had prominent effects on in vivo fibrogenesis, leading to reduced CCl4induced liver fibrosis (31% decease in morphometric collagen vs. control (n=8, P < 0.05); and 35% decrease in whole liver type 1 collagen mRNA expression vs. control (n=8, P < 0.05)). Conclusions: The actin cytoskeleton plays a critical role in HSC function and type I collagen synthesis, suggesting that targeting actin cytoskeletal dynamics by interruption of MRTF-A signaling could be a highly novel approach for treatment of liver fibrosis.
Sa1549
AASLD Abstracts
HEPATOCYTE VERSUS STELLATE CELL SPECIFIC DELETION OF LIVER FATTY ACID BINDING PROTEIN (L-FABP) REVEALS CELL- TYPE SPECIFIC ROLES IN RETINOL METABOLISM, STEATOSIS AND FIBROSIS Elizabeth P. Newberry, Roberto Solis, Susan Kennedy, Seung-Ah Lee, William S. Blaner, Anping Chen, Nicholas O. Davidson Background: Hepatic lipotoxic injury promotes inflammation and fibrosis but the mechanisms and modulators are poorly understood. Liver fatty acid binding protein (L-Fabp) facilitates intracellular fatty acid (FA) trafficking and germline L-Fabp deletion (KO) attenuates dietinduced obesity (DIO), hepatic steatosis and fibrosis. Methods: We examined cell type specific [hepatocyte, stellate cell (HSC)] functions of L-Fabp in FA trafficking and injury. We generated conditional L-Fabp deletor lines in HSC (GFAP-CreTg, HSC-KO) and hepatocytes (Alb-CreTg, Liv KO) and examined FA uptake, incorporation and lipotoxic injury. Results: On a low fat chow diet, hepatic triglyceride (TG) was reduced in Liv-KO mice vs both f/f mice and HSC-KO (Liv-KO, 26 ± 9 µg TG/mg protein; f/f, 107 ± 12; HSC-KO, 79 ± 12, p=0.04). In response to 48h fasting, Liv-KO mice exhibited attenuated steatosis compared to both HSC-KO and f/f mice (Liv-KO, 81 ± 15 µg TG/mg protein; HSC-KO, 161 ± 12, f/f, 190 ± 43). These data suggest that Liv-KO hepatocytes are unable to incorporate exogenous FA mobilized during prolonged fasting (as observed in KO), findings confirmed by in-vitro FA uptake. We next asked if cell-specific L-Fabp deletion alters DIO, steatosis or fibrosis. Mice were fed either high saturated fat or high trans-fat fructose (TFF) diet. LivKO mice had decreased steatosis (Sat FA: Liv-KO, 248 ± 38 µg TG/mg; f/f, 493 ± 98; TFF: Liv-KO, 210 ± 62; f/f, 556 ± 123; HSC KO, 597 ± 120) and TFF fed Liv-KO mice had reduced fibrotic injury (% fibrotic area: f/f, 0.7 ± 0.08; Liv-KO, 0.5 ± 0.03; HSC-KO, 0.8 ± 0.08) and reduced fibrogenic gene expression. These data show that exogenous FA trafficking is impaired in Liv KO mice, and that diet induced (ie lipotoxic) fibrotic injury is attenuated with reduced steatosis. In contrast, carbon tetrachloride (CCl4) treated mice revealed worse injury (increased ALT, HSC activation) in both germline KO as well as HSCKO mice compared to both f/f or Liv-KO, and independent of hepatocyte lipid content. We further examined HSC retinol and retinol ester (RE) content. HSC-KO stellate cells showed slightly decreased retinol and RE content, with significant decreases in distinct RE species (16:0, 18:1) suggesting altered FA trafficking into HSC RE. Total retinol and RE levels were also decreased ~50% in Liv-KO HSC compared to f/f, with reduced abundance of all RE species, raising the question of how hepatocyte lipid content modulates HSC retinol metabolism. Consistent with this, HSC RE levels were also reduced in germline KO mice. Conclusions: These data suggest a cell type specific role of L-Fabp in distinct models of liver injury, with attenuated lipid mediated injury in Liv-KO but augmented fibrogenic injury in HSCKO. In addition, L-Fabp is involved in RE synthesis in hepatocytes and RE storage in stellate cells.
Sa1552 RASAL1 IS A POTENT REGULATOR OF HEPATIC STELLATE CELL ACTIVITIES AND LIVER FIBROSIS Motoyuki Otsuka, Akemi Takata, Takahiro Kishikawa, Motoko Ohno, Mari Yamagami, Rei Ishibashi, Kazuma Sekiba, Takeshi Yoshikawa, Kazuhiko Koike Liver fibrosis, leading to cirrhosis and liver failure, is observed after chronic liver injury. The transition of hepatic stellate cells (HSCs) from quiescent cells to proliferative and fibrogenic cells is a central event in liver fibrosis. Here, we show that RAS protein activator like-1 (RASAL1), a member of the RAS-GTPase-activating proteins that switch off RAS activity, is significantly decreased during mouse primary HSC activation, and that HSC activation can be antagonized by the forced expression of RASAL1 protein. We demonstrate that RASAL1 suppresses HSC proliferation by regulating the RasMAPK pathway, and that RASAL1 suppresses HSC fibrogenic activity through regulating the PKA- LKB1-AMPK-SRF pathway by interacting with angiotensin II receptor, type 1 (AGTR1). We also show that RASAL1-deficient mice are more prone to liver fibrosis. These studies demonstrate that deregulated RASAL1 expression levels and its subsequent downstream intracellular signaling are central mediators of perpetuated HSC activation and fibrogenesis in the liver.
Sa1550 THE CHARACTERISTICS OF MYOFIBROBLASTS IN VARIOUS CHOLESTATIC LIVER INJURY MODELS IN MICE Yukinori Koyama, Ronglin Hu, Jun Xu, Xiao Liu, Hsiao-Yen Ma, Shuang Liang, David A. Brenner, Tatiana Kisseleva Introduction: Cholestatic liver diseases include primary biliary cholangitis, biliary atresia, and primary sclerosing cholangitis, all of which lead to liver cirrhosis. By using bile duct ligation (BDL) model, we have revealed the following findings of cholestatic liver fibrosis: 1) In BDL model, bone marrow (BM) derived fibrocytes minimally contribute to myofibroblasts; 2) Activated portal fibroblasts (aPFs) are a major contributor of myofibroblasts, especially in its early stage of pathogenesis; 3) aPFs express several markers such as Thy1, Msln and Muc16, which are related to proliferation and activation of aPFs. However, BDL does not fully mimic the cholestatic liver diseases described above. To elucidate the pathogenesis of cholestatic fibrosis, we need to know what is common in cholestatic liver fibrosis and what are disease (model) specific findings. In this study, based on our previous findings in BDL, we analyzed the origin of myofibroblasts and the role of Msln, a profibrogenic molecule in BDL, in MDR2-/- mouse model and Diethoxycarbonyl dihydrocollidine (DDC) diet model. Methods: To analyze the contribution of BM derived cells in MDR2-/- mice we transplanted BM from collagen 1α1-GFP reporter mice into irradiated MDR2-/- mice. The contribution of aPFs in MDR2-/- (8 weeks old) and DDC diet model was analyzed by immunohistochemistry and flow cytometry. In order to analyze the role of Msln, which works as a profibrotic molecule in BDL model, DDC diet was given to wt and Msln-/- mice for 3 weeks and liver fibrosis was evaluated. Results: In MDR2-/- mice, the contribution of BM derived cells to myofibroblasts was less than 1%. Flow cytometry revealed that in MDR2-/- and DDC model, 50-70% of myofibroblasts were vitamin A negative aPFs, which is comparable to BDL model. In contrast to BDL, in which almost all aPFs are characterized by the expression of Thy-1, Thy-1+ aPFs were limited to 50-60% of total vitamin A negative myofibroblasts in MDR2-/- mice. In DDC diet model, the expression of Msln was upregulated in the liver. Liver fibrosis was attenuated in Msln-/- mice, as evaluated by Sirius red staining
AASLD Abstracts
Sa1553 HIGH-MOBILITY GROUP BOX 1(HMGB1) ACTIVATES CASPASE-1 AND INDUCES THE ACTIVATION OF HSCS Wei Yan, Yixing Luo, Ping Han, Yu Fu Background: The activation of hepatic stellate cells (HSCs) plays an important role in the development of liver fibrosis and it always begins with inflammation. The exact mechanisms driving inflammation induced activation of HSCs remain elusive. HMGB1, a pro-inflammatory cytokine, has recently been showed to be closely associated with the activation of HSCs. The inflammasome, a caspase-1 activation platform, regulates immune responses to diverse stimuli that appear during infection or after tissue damage. We hypothesize that HMGB1 activation of the inflammasome plays a key role in the activation of HSCs and fibrogenesis. Methods: Hepatic stellate cell lines were treated with recombinant HMGB1(10 ng/ml or 100 ng/ml) for 24 hours. The migration of HSCs was detected by transwell cell migration assay and scratch assay, the collagen synthesis in HSCs was tested by Westernblot and immunofluorescent staining. The expression of caspase-1 in HSCs was detected by western blot and Colorimetric Assay. Z-YVAD-FMK (caspase-1 inhibitor, 2µM) was used to inhibit caspase-1 after recombinant HMGB1(100 ng/ml) treatment in HSCs. The rat fibrosis models were built by TAA-intraperitoneal injection (dissolved in PBS, 150 mg/kg B.W.). After TAAintraperitoneal injection for 4 weeks, the rats were treated with intraperitoneal injection of
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