OxNASH Score Correlates With Histologic Features and Severity of Nonalcoholic Fatty Liver Disease

In WT mice, 22 week-CDAA diet feeding resulted in steatosis and inflammatory cell infiltration in the liver as assessed by HE staining. In agreement with HE staining, serum ALT levels, expressions of macrophage markers F4/80 and CD68 and proinflammatory cytokines TNFα and IL-1β were increased. The CDAA diet induced perisinusoidal fibrosis and an increase of fibrogenic genes collagens and TIMP-1 in the liver. In addition, the CDAA diet induced insulin resistance. In contrast, CCR2 deficient mice showed lesser degrees of steatosis and inflammatory cell infiltration after CDAA diet feeding. NAFLD activity score was significantly lower in CCR2 deficient mice. Increased macrophage infiltration, expression of proinflammatory cytokines and serum ALT levels were also suppressed in CCR2 deficient mice. Moreover, liver fibrosis and hepatic expression of fibrogenic genes were significantly suppressed in CCR2 deficient mice. We and others have reported that TLR2, TLR4, and TLR9 deficient mice have less steatohepatitis compared with WT mice. These TLR deficient mice had less expression of MCP-1 and CCR2, and less number of F4/80-positive cells in the liver. In Vitro study demonstrated that WT Kupffer cells and hepatic stellate cells express MCP-1 in response to TLR2 ligand Pam3CK4, and TLR4 ligand LPS. Finally, we examined the effect of CCR2 inhibitor in NASH. The CCR2 inhibitor ameliorated steatosis and inflammation in CDAA diet-induced NASH. The number of F4/80-positive cells in CCR2 inhibitor treated livers was significantly smaller than that in control livers. In addition, hepatic expressions of inflammatory cytokines and fibrogenic genes were suppressed by the CCR2 inhibitor treatment. (Conclusion) MCP-1 and CCR2 promote the progression of NASH, and CCR2 inhibitor will become a new therapeutic tool for NASH.

repressed at 6 hours associated with increase of caspase-3/7 activation in Bak/Bax double KO mice. Together with the facts that this liver injury was ameliorated neither with further cyclophilin D KO (cyclophilin d-/- Alb-Cre bak-/- baxflox/flox) nor administration of necrostatin1 but completely inhibited by administration of a caspase inhibitor even after activation of BH3-only protein Bid in the liver, delayed-onset cell death in Bak/Bax double KO mice was identified as apoptosis. Conclusion: Whereas either Bak or Bax is critically required for rapid execution of Fas-mediated massive apoptosis in the liver, delayed onset of mitochondriaindependent but caspase-dependent apoptosis still develops even in the absence of both. The present study unveils the extrinsic pathway of apoptosis like in type I cells lying under the mitochondrial pathway and serving as a backup system even in type II cells. 818

A wide variety of chemical toxicants and drugs induce acute liver injuries, which can lead to liver failure. Reactive metabolites resulting from these toxicants induce hepatocyte apoptosis/necrosis but our understanding of the mechanism of injury and the possible adaptive responses is still incomplete. The extracellular matrix (ECM) has long been recognized for its central role in tissue architecture and structural support. More recently, the importance of complex ECM structures in the context of cellular function has also been realized. There is tremendous interest directed at understanding how the ECM regulates a diverse set of biological processes including the development of diseased tissue microenvironments. Type XVIII collagen is a prominent liver ECM component. This member of the multiplexin family of collagens is highly expressed in liver, and levels have been shown to increase further during pro-fibrotic disease states including fibrosis, cirrhosis, and cancers of the liver. Here we demonstrate that when challenged with the hepatoxin, carbon tetrachloride (CCl4), mice deficient in type XVIII collagen (ColXVIII-/-) suffer severe acute liver dysfunction. Histological analysis of wild type mice revealed low/moderate levels of damage around the centrilobular venules. In contrast, substantial levels of lipid accumulation in hepatocytes of ColXVIII-/- mice with ballooning degeneration that extended into the mid and periportal zones was observed. TUNEL staining and quantification in ColXVIII-/- mice revealed significant cell death when compared to control mice. Through In Vitro cell adhesion assays, colocalization via immuno-fluorescence, and genetic ablation, we identify that the α1β1 integrin serves as a receptor for type XVIII collagen and this interaction is essential for hepatocyte survival. We utilized mice deficient in the α1 integrin subunit (α1INTG-/-) mice to test whether α1β1 integrin plays a role in hepatocyte survival during CCl4 injury. The α1INTG-/mice were moribund within 5 days post CCl4 injection, in a similar fashion as the ColXVIII-/mice. Apoptosis was significantly increased in α1INTG-/- mice treated with CCl4. Additional data suggests that the pro-survival signal generated by type XVIIII collagen is mediated by integrin-linked kinase/Akt signaling pathway. These findings demonstrate that type XVIII collagen is an important functional component of the liver matrix microenvironment and critical for hepatocyte survival during injury and point to a novel interaction between the α1β1 integrin with type XVIII collagen that provides essential survival cues for hepatocytes during the acute injury response.

738 OxNASH Score Correlates With Histologic Features and Severity of Nonalcoholic Fatty Liver Disease Naim Alkhouri, Michael P. Berk, Rocio Lopez, Tarek Abu-Rajab Tamimi, Lisa Yerian, Yoon-Mi Chung, Renliang Zhang, Thomas M. McIntyre, Stanley L. Hazen, Ariel E. Feldstein Nonalcoholic steatohepatitis (NASH), the advanced form of nonalcoholic fatty liver disease (NAFLD), is a serious condition that can progress to cirrhosis. NASH is characterized by lipid accumulation in the liver in addition to hepatocyte injury, inflammation and a variable degree of fibrosis. Oxidative stress plays a significant role in disease progression to NASH. In a previous study, we identified specific fatty acid oxidation products as novel noninvasive markers for NASH. We developed the oxNASH score using the ratio of 13- hydroxyoctadecadienoic acid (13-HODE) to linoleic acid, age, body mass index, and aspartate transaminases. The aims of this study were to assess the correlation between oxNASH and individual histologic features of NASH (steatosis, ballooning, inflammation, and fibrosis) and to assess the correlation with the NAFLD activity score (NAS). Methods: Our cohort consisted of 122 patients undergoing liver biopsy for clinical suspicion of NAFLD. Liver histology was assessed by an experienced hepatopathologist blinded to clinical and laboratory data. The grade of steatosis, hepatocyte ballooning, and inflammatory activity was measured using a 0 to 8 scale (steatosis 0-3, ballooning 0-2, and lobular inflammation 0-3). NAS was calculated for each patient. The stage of fibrosis was measured using a 4-point scale. Blood was collected from each patient at the time of liver biopsy. Levels of fatty acid oxidation products were quantified using tandem mass spectrometry and OxNASH was calculated as previously described. Analysis of covariance was performed to study the association between histology and oxNASH. A p value < 0.05 was considered statistically significant. Results: The mean age was 49.3 (± 11.6) years and the mean BMI was 31.5 (± 4.8) kg/m2. The majority of patients were Caucasian (82%) and 48% were female. OxNASH scores were significantly higher in females (64 ± 24 vs. 52 ± 30; p=0.017) and subjects with dyslipidemia (63 ± 27 vs. 52 ±27; p=0.024) or hypertension (65 ± 22 vs. 53 ± 30; p=0.013). OxNASH correlated with NAS and its individual histologic features (steatosis, inflammation, and ballooning. P <0.05) with the strongest association being with inflammation [rho (95% CI) = 0.40 (0.23-0.57), p < 0.001]. Furthermore, there was a correlation between the stage of fibrosis and oxNASH (p = 0.001). These associations remained statistically significant after adjusting for multiple confounders including gender, dyslipidemia and hypertension. Conclusion: In adult patients with NAFLD, oxNASH correlates with histologic features of NASH especially inflammation. OxNASH appears to be a promising noninvasive marker for NASH.

819 Compromised Energetics and Redox Cycling via GAPDH Underlie Genetic Predisposition to Hepatocyte Protein Aggregation During Liver Injury Natasha T. Snider, Amika Singla, Sujith Weerasinghe, Shinichiro Hanada, Xiankui Sun, Philip C. Andrews, Bishr Omary Background and aims: Mallory-Denk bodies (MDBs) are protein aggregates commonly found in hepatocytes of patients with alcoholic liver disease and nonalcoholic fatty liver disease (NAFLD), where their presence correlates with worse outcomes. Understanding the pathogenesis of MDBs may help uncover therapeutic strategies for MDB-related diseases. There is a genetic component to MDB formation in mouse models and in humans. For example, MDB induction in mice, via chronic 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) administration, is highly strain dependent. Also, MDBs are seen more frequently in Hispanics versus other groups who have NAFLD. The aim of this study is to identify genetic factors that contribute to MDB formation in mice, and to correlate these findings to human liver disease. Methods: 2-dimensional differential in-gel electrophoresis (2D-DIGE) proteomic comparison was done on normal and DDC-injured livers of MDB-prone (C57BL) and MDB-resistant (C3H) mice. 2D-DIGE results were validated at the mRNA and protein levels then subjected to mechanistic studies in hepatocytes ex vivo, and in mouse and human livers using molecular, biochemical, and cell-biological approaches. Results: In hepatocytes and livers of MDBprone mice, DDC upregulated reactive oxygen species (ROS)-generating enzymes (Cyp2e1, Cyp3a), increased ROS levels, and induced the expression of antioxidant enzymes (glutathione S-transferase, carbonyl reductase). In contrast to MDB-resistant mice, MDB-prone mice had significant impairments in basal and post-injury liver expression and activity of nucleoside diphosphate kinase (NDPK), which functions to generate energy and protect against oxidative stress-induced damage. Notably, there was also significant downregulation, aggregation and nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in hepatocytes and livers of MDB-prone mice, which was reversed by the antihyperglycemic agent pioglitazone. Knockdown of GAPDH in primary hepatocytes by siRNA caused a DDC-like effect by markedly downregulating bioenergetic and antioxidant enzymes, including NDPK, peroxiredoxin 6, and carbonic anhydrase 3. Furthermore, cytoplasmic and nuclear GAPDH aggregates were observed in alcoholic cirrhosis, but not normal human livers. Conclusions: Genetic susceptibility to MDBs likely results from basal deficiencies in energy metabolism that are intensified by elevated oxidative stress, ultimately resulting in compromised energy-dependent protein folding/degradation responses. We propose a central role for GAPDH and its oxidative stress-induced aggregation in injury-induced hepatocyte inclusion formation. Correction of GAPDH function may be one mechanism whereby pioglitazone confers hepatoprotection in NAFLD patients. Our findings have implications for other inclusion-associated diseases.

817 Delayed Onset of Caspase-Dependent Liver Failure Induced by Fas Stimulation in Bak and BAX Double Knockout Mice Hayato Hikita, Takahiro Kodama, Satoshi Shimizu, Minoru Shigekawa, Atsushi Hosui, Takuya Miyagi, Hisashi Ishida, Tomohide Tatsumi, Tatsuya Kanto, Naoki Hiramatsu, Norio Hayashi, Tetsuo Takehara Background and aim: Fas (CD95) stimulation activates caspase-8 which in turn truncates Bid leading to activation of Bak/Bax in hepatocytes. Activated Bak/Bax releases cytochrome c from mitochondria followed by caspase-9 activation leading to execute apoptosis by caspase3/7 activations. Since in type II cells like hepatocytes, various apoptotic stimuli bundles to Bak/Bax activation, Bak/Bax is considered to be an essential gateway in apoptosis. However the significance of Bak and/or Bax in the liver has not been clearly understood especially In Vivo. In this study we examined the significance of Bak and/or Bax in Fas-induced hepatocellular apoptosis by using knockout mice. Methods and results: To address this issue, we administrated Jo2 agonistic anti-Fas antibody (1.5 mg/kg) into Bak knockout (KO) (bak-/-), Bax KO (bax-/-) and hepatocyte-specific Bak and Bax double KO mice (Alb-Cre bak-/- baxflox/flox). Fas-induced rapid fulminant hepatocyte apoptosis was partially ameliorated in Bak KO mice but not in Bax KO mice, and completely abolished in double KO mice at 3 hours after Jo2 injection. Importantly, at 6 hours, double KO mice also displayed severe liver injury associated with moderate activation of caspase-3/7 and oligonucleosomal DNA breaks in the liver, without activation of caspase-9. Electron microscopic studies revealed that hepatocytes with chromatin condensation did not show mitochondrial alterations, such as disruption of outer membrane and herniation of the matrix, in Bak/Bax double KO mice at 6 hours, which are in marked contrast to those in control Bak KO littermates. Interestingly, while the liver expression levels of cIAP1 and cIAP2 did not change, XIAP was upregulated at 3hours in Bak/Bax double KO mice as well as control Bak KO littermates and then

S-905

AASLD Abstracts

AASLD Abstracts

Type XVIII Collagen Induced Signaling via α1β1 Integrin in Hepatocytes is Essential for Surviving Liver Injury Michael Duncan, Raghu Kalluri