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Soluble epoxide hydrolase hepatic deficiency ameliorates alcohol-associated liver disease. Cellular and molecular gastroenterology and hepatology Mello, A., Hsu, M., Koike, S., Chu, B., Cheng, J., Yang, J., Morisseau, C., Torok, N. J., Hammock, B. D., Haj, F. G. 2020
Abstract

BACKGROUND & AIMS: Alcohol-associated liver disease (ALD) is a significant cause of liver-related morbidity and mortality worldwide and with limited therapies. Soluble epoxide hydrolase (sEH; Ephx2) is a largely cytosolic enzyme that is highly expressed in the liver and is implicated in hepatic function, but its role in ALD has heretofore remained uncharted.METHODS: To decipher the role of hepatic sEH in ALD, we generated mice with liver-specific sEH disruption (Alb-Cre; Ephx2fl/fl). Alb-Cre; Ephx2fl/fl and control (Ephx2fl/fl) mice were subjected to an ethanol challenge using the chronic plus binge model of ALD and hepatic injury, inflammation, and steatosis evaluated under pair- and ethanol-fed states. Additionally, we investigated the capacity of pharmacological inhibition of sEH in the chronic plus binge mouse model.RESULTS: We observed elevation of hepatic sEH in mice upon ethanol consumption, suggesting that dysregulated hepatic sEH expression might be involved in ALD. Alb-Cre; Ephx2fl/fl mice presented efficient deletion of hepatic sEH with the corresponding attenuation in the sEH activity and alteration in the lipid epoxide/diol ratio. Consistently, hepatic sEH deficiency ameliorated ethanol-induced hepatic injury, inflammation, and steatosis. Additionally, targeted metabolomics identified lipid mediators that were significantly impacted by hepatic sEH deficiency. Moreover, hepatic sEH deficiency was associated with a significant attenuation of ethanol-induced hepatic endoplasmic reticulum and oxidative stress. Notably, pharmacological inhibition of sEH recapitulated the effects of hepatic sEH deficiency and abrogated injury, inflammation, and steatosis caused by ethanol feeding.CONCLUSIONS: These findings elucidated a role for sEH in ALD and validated a pharmacological inhibitor of this enzyme in a preclinical mouse model as a potential therapeutic approach.

View details for DOI 10.1016/j.jcmgh.2020.10.002

View details for PubMedID 33068774
Non-phagocytic Activation of NOX2 is Implicated in Progressive Non-alcoholic Steatohepatitis During Aging. Hepatology (Baltimore, Md.) Jiang, J. X., Fish, S. R., Tomilov, A., Li, Y., Fan, W., Dehnad, A., Gae, D., Das, S., Mozes, G., Charville, G. W., Ramsey, J., Cortopassi, G., T�r�k, N. J. 2020
Abstract

Older patients with obesity/type II DM frequently present with advanced non-alcoholic steatohepatitis (NASH). Whether this is due to specific molecular pathways that accelerate fibrosis during aging, is unknown. Activation of the Src homology 2 domain containing collagen-related (Shc) proteins and redox stress have been recognized in aging, however their link to NASH has not been explored. Shc expression increased in livers of older patients with NASH, as assessed by RTqPCR or western blots. Fibrosis, Shc expression, markers of senescence and NADPH oxidases (NOXs) were studied in young/old mice on fast food diet (FFD). To inhibit Shc in old mice LV-shShc vs. control-LV were used during FFD. For hepatocyte-specific effects, fl/fl Shc mice on FFD were injected with AAV8-TBG-Cre vs. control. Fibrosis was accelerated in older mice on FFD, and Shc inhibition by LV in older mice, or hepatocyte-specific deletion resulted in significantly improved inflammation, reduction in senescence markers in older mice, lipid peroxidation and fibrosis. To study NOX2 activation, the interaction of p47phox (NOX2 regulatory subunit) and p52Shc was evaluated by proximity ligation, and co-IPs. Palmitate induced p52Shc binding to p47phox activating the NOX2 complex, more so at older age. Kinetics of binding were assessed in SH2 or PTB deletion mutants by biolayer interferometry, revealing the role of SH2 and the PTB domains. Lastly, an in silico model of p52Shc/p47phox interaction using RosettaDock was generated. Conclusion Accelerated fibrosis in the aged is modulated by p52Shc/NOX2. We show a novel pathway for direct activation of the phagocytic NOX2 in hepatocytes by p52Shc binding and activating the p47phox subunit that results in redox stress, and accelerated fibrosis in the aged.

View details for DOI 10.1002/hep.31118

View details for PubMedID 31950520
AGER1 downregulation associates with fibrosis in nonalcoholic steatohepatitis and type 2 diabetes. The Journal of clinical investigation Dehnad, A., Fan, W., Jiang, J. X., Fish, S. R., Li, Y., Das, S., Mozes, G., Wong, K. A., Olson, K. A., Charville, G. W., Ali, M., T�r�k, N. J. 2020
Abstract

Type 2 diabetes is clinically associated with progressive necroinflammation and fibrosis in nonalcoholic steatohepatitis (NASH). Advanced glycation end-products (AGEs) accumulate during prolonged hyperglycemia, but the mechanistic pathways that lead to accelerated liver fibrosis have not been well defined. In this study, we show that the AGEs clearance receptor AGER1 was downregulated in patients with NASH and diabetes and in our NASH models, whereas the proinflammatory receptor RAGE was induced. These findings were associated with necroinflammatory, fibrogenic, and pro-oxidant activity via the NADPH oxidase 4. Inhibition of AGEs or RAGE deletion in hepatocytes in vivo reversed these effects. We demonstrate that dysregulation of NRF2 by neddylation of cullin 3 was linked to AGER1 downregulation and that induction of NRF2 using an adeno-associated virus-mediated approach in hepatocytes in vivo reversed AGER1 downregulation, lowered the level of AGEs, and improved proinflammatory and fibrogenic responses in mice on a high AGEs diet. In patients with NASH and diabetes or insulin resistance, low AGER1 levels were associated with hepatocyte ballooning degeneration and ductular reaction. Collectively, prolonged exposure to AGEs in the liver promotes an AGER1/RAGE imbalance and consequent redox, inflammatory, and fibrogenic activity in NASH.

View details for DOI 10.1172/JCI133051

View details for PubMedID 32657776
Digoxin improves steatohepatitis with differential involvement of liver cell subsets in mice through inhibition of PKM2 transactivation. American journal of physiology. Gastrointestinal and liver physiology Zhao, P., Han, S., Arumugam, S., Yousaf, M. N., Qin, Y., Jiang, J. X., Torok, N. J., Chen, Y., Mankash, M. S., Liu, J., Li, J., Iwakiri, Y., Ouyang, X. 2019
Abstract

T The cardiac glycoside digoxin was identified as a potent suppressor of PKM2-HIF-1a pathway activation in liver injury mouse models via intraperitoneal injection. We have assessed the therapeutic effects of digoxin by the clinically relevant oral route to reduce non-alcoholic steatohepatitis (NASH) in mice and analyzed the cellular basis for this effect with differential involvement of liver cell subsets. C57BL/6J male mice were placed on a high fat diet (HFD) for 10 weeks and started concurrently with the gavage of digoxin (2.5, 0.5, 0.125 mg/kg twice a week) for 5 weeks. Digoxin significantly reduced HFD-induced hepatic damage, steatosis and liver inflammation across a wide dosage range. The lowest dose of digoxin (0.125 mg/kg) showed significant protective effects against liver injury and sterile inflammation. Consistently, digoxin attenuated HIF-1a sustained NLRP3 inflammasome activation in macrophages. We have reported for the first time that PKM2 is up-regulated in hepatocytes with hepatic steatosis and digoxin directly improved hepatocyte mitochondrial dysfunction and steatosis. Mechanistically, digoxin directly bound to PKM2 and inhibited PKM2 targeting HIF-1a transactivation without affecting PKM2 enzyme activation. Thus, oral digoxin showed potential to therapeutically inhibit liver injury in NASH through the regulation of PKM2-HIF1a pathway activation with involvement of multiple cell types. Due to the large clinical experience with oral digoxin this may have significant clinical applicability in human NASH.

View details for DOI 10.1152/ajpgi.00054.2019

View details for PubMedID 31411894
Digoxin Suppresses Pyruvate Kinase M2-Promoted HIF-1? Transactivation in Steatohepatitis. Cell metabolism Ouyang, X., Han, S. N., Zhang, J. Y., Dioletis, E., Nemeth, B. T., Pacher, P., Feng, D., Bataller, R., Cabezas, J., St�rkel, P., Caballeria, J., LePine Pongratz, R., Cai, S. Y., Schnabl, B., Hoque, R., Chen, Y., Yang, W. H., Garcia-Martinez, I., Wang, F. S., Gao, B., Torok, N. J., Kibbey, R. G., Mehal, W. Z. 2018; 27 (5): 1156
View details for DOI 10.1016/j.cmet.2018.04.007

View details for PubMedID 29719229
P300, A New Player in Mechanosensitivity and Activation of Cancer-Associated Fibroblasts. Gastroenterology Torok, N. J. 2018; 154 (8): 2025?26
View details for DOI 10.1053/j.gastro.2018.05.002

View details for PubMedID 29733834
Hepatocyte Nicotinamide Adenine Dinucleotide Phosphate Reduced Oxidase 4 Regulates Stress Signaling, Fibrosis, and Insulin Sensitivity During Development of Steatohepatitis in Mice GASTROENTEROLOGY Bettaieb, A., Jiang, J. X., Sasaki, Y., Chao, T., Kiss, Z., Chen, X., Tian, J., Katsuyama, M., Yabe-Nishimura, C., Xi, Y., Szyndralewiez, C., Schroeder, K., Shah, A., Brandes, R. P., Haj, F. G., Toeroek, N. J. 2015; 149 (2): 468-?
Abstract

Reactive oxidative species (ROS) are believed to be involved in the progression of nonalcoholic steatohepatitis (NASH). However, little is known about the sources of ROS in hepatocytes or their role in disease progression. We studied the effects of nicotinamide adenine dinucleotide phosphate reduced oxidase 4 (NOX4) in liver tissues from patients with NASH and mice with steatohepatitis.Liver biopsy samples were obtained from 5 patients with NASH, as well as 4 patients with simple steatosis and 5 patients without steatosis (controls) from the University of California, Davis Cancer Center Biorepository. Mice with hepatocyte-specific deletion of NOX4 (NOX4(hepKO)) and NOX4(floxp+/+) C57BL/6 mice (controls) were given fast-food diets (supplemented with high-fructose corn syrup) or choline-deficient l-amino acid defined diets to induce steatohepatitis, or control diets, for 20 weeks. A separate group of mice were given the NOX4 inhibitor (GKT137831). Liver tissues were collected and immunoblot analyses were performed determine levels of NOX4, markers of inflammation and fibrosis, double-stranded RNA-activated protein kinase, and phospho-eIF-2? kinase-mediated stress signaling pathways. We performed hyperinsulinemic-euglycemic clamp studies and immunoprecipitation analyses to determine the oxidation and phosphatase activity of PP1C.Levels of NOX4 were increased in patients with NASH compared with controls. Hepatocyte-specific deletion of NOX4 reduced oxidative stress, lipid peroxidation, and liver fibrosis in mice with diet-induced steatohepatitis. A small molecule inhibitor of NOX4 reduced liver inflammation and fibrosis and increased insulin sensitivity in mice with diet-induced steatohepatitis. In primary hepatocytes, NOX4 reduced the activity of the phosphatase PP1C, prolonging activation of double-stranded RNA-activated protein kinase and phosphorylation of extracellular signal-regulated kinase-mediated stress signaling. Mice with hepatocyte-specific deletion of NOX4 and mice given GKT137831 had increased insulin sensitivity.NOX4 regulates oxidative stress in the liver and its levels are increased in patients with NASH and mice with diet-induced steatohepatitis. Inhibitors of NOX4 reduce liver inflammation and fibrosis and increase insulin sensitivity, and might be developed for treatment of NASH.

View details for DOI 10.1053/j.gastro.2015.04.009

View details for Web of Science ID 000358429600039

View details for PubMedID 25888330

View details for PubMedCentralID PMC4516583
Strategies and endpoints of antifibrotic drug trials: Summary and recommendations from the AASLD Emerging Trends Conference, Chicago, June 2014 HEPATOLOGY Torok, N. J., Dranoff, J. A., Schuppan, D., Friedman, S. L. 2015; 62 (2): 627-634
Abstract

There is an urgent need to develop antifibrotic therapies for chronic liver disease, and clarify which endpoints in antifibrotic trials will be acceptable to regulatory agencies. The American Association for the Study of Liver Diseases sponsored an endpoints conference to help accelerate the efficient testing of antifibrotic agents and develop recommendations on clinical trial design for liver fibrosis. In this review, we summarize the salient and novel elements of this conference and provide directions for future clinical trial design. The article follows the structure of the conference and is organized into five areas: (1) antifibrotic trial design; (2) preclinical proof-of-concept studies; (3) pharmacological targets, including rationale and lessons to learn; (4) rational drug design and development; and (5) consensus and recommendations on design of clinical trials in liver fibrosis. Expert overviews and collaborative discussions helped to summarize the key unmet needs and directions for the future, including: (1) greater clarification of at-risk populations and study groups; (2) standardization of all elements of drug discovery and testing; (3) standardization of clinical trial approaches; (4) accelerated development of improved noninvasive markers; and (5) need for exploration of potential off-target toxicities of future antifibrotic drugs.

View details for DOI 10.1002/hep.27720

View details for Web of Science ID 000358453700033

View details for PubMedID 25626988

View details for PubMedCentralID PMC4515973
THE INCIDENCE OF HEPATOCELLULAR CARCINOMA IN CHRONIC HEPATITIS B VIRUS INFECTION SUBJECTS NOT MEETING CRITERIA FOR ANTIVIRAL THERAPY Alshuwaykh, O., Goel, A., Daugherty, T., Cheung, A., Kim, W., Kwong, A. J., Ahmed, A., Ghaziani, T., Torok, N. J., Nguyen, M. H., Dronamraju, D., Dhanasekaran, R., Kumari, R., Kwo, P. Y. WILEY. 2020: 472A?473A
View details for Web of Science ID 000574027001230
SHC INHIBITORY DRUG IDEBENONE PROTECTS MICE FROM DIET-INDUCED NASH Jiang, J., Tomilov, A., Torok, N. J., Cortopassi, G. WILEY. 2020: 1036?37
View details for Web of Science ID 000574027004153
Leptin/adiponectin ratio correlates with hepatic steatosis but not arterial stiffness in nonalcoholic fatty liver disease in Japanese population. Cytokine Mikami, K., Endo, T., Sawada, N., Igarashi, G., Kimura, M., Hasegawa, T., Iino, C., Tomita, H., Sawada, K., Nakaji, S., Matsuzaka, M., Torok, N. J., Fukuda, S. 2019; 126: 154927
Abstract

BACKGROUND AND AIMS: Cardiovascular disease (CVD) is a leading cause of mortality in nonalcoholic fatty liver disease (NAFLD). The aim of this study was to investigate the relationship of leptin-to-adiponectin (L/A) ratio with hepatic steatosis and arterial stiffness in NAFLD.METHODS: The subjects were 871 Japanese adults who participated in a health survey. Dietary intake, body composition, lipid profile, serum interleukin-6 (IL-6), leptin, and adiponectin were analyzed. NAFLD was defined as fatty liver on ultrasonography in the absence of other causes of steatosis. Arterial stiffness was evaluated by the brachial-ankle pulse wave velocity (baPWV).RESULTS: The subjects with NAFLD had a greater body mass index (BMI) and body fat percentage (BFP); a higher intake of daily energy (kcal) and carbohydrates; and a higher prevalence of hypertension, diabetes, and hyperlipidemia. The subjects with NAFLD had higher serum leptin and lower serum adiponectin concentrations and a higher L/A ratio than subjects without NAFLD. The L/A ratio increased with increasing severity of steatosis. The L/A ratio showed positive correlations with BMI and BFP, and a negative correlation with age. Women had higher L/A ratio and BFP levels than men regardless of the presence or absence of NAFLD. There was a weak positive correlation between baPWV and severity of steatosis. BaPWV was strongly correlated with age, while no relation was found between baPWV and L/A ratio. IL-6 level was correlated with baPVW and age, while the correlation between Il and 6 level and L/A ratio was very weak. The L/A ratio was correlated with triglycerides and the ratio of total cholesterol to high-density lipoprotein-cholesterol.CONCLUSION: L/A ratio and arterial stiffness were associated with the severity of steatosis, whereas there was no correlation between L/A ratio and arterial stiffness in NAFLD. These findings suggest that not only leptin and adiponectin but also other factors might be involved in the pathogenesis for atherosclerosis in NAFLD.

View details for DOI 10.1016/j.cyto.2019.154927

View details for PubMedID 31756645
SHC LINKS OXIDATIVE AND INFLAMMATORY SIGNALS IN ALCOHOLIC LIVER DISEASE (ALD) Gupta, P., Dehnad, A., Das, S., Fish, S., Jiang, J., Cortopassi, G., Torok, N. J. WILEY. 2019: 826A?827A
View details for Web of Science ID 000488653503050
Patterns and co-occurrence of risk factors for hepatocellular carcinoma in four Asian American communities: a cross-sectional study. BMJ open Stewart, S. L., Dang, J. H., Torok, N. J., Chen, M. S. 2019; 9 (6): e026409
Abstract

OBJECTIVES: To investigate risk factor patterns and the simultaneous occurrence of multiple risk factors in the viral, metabolic and lifestyle domains among Asian Americans, who have had the highest mortality rates from hepatocellular carcinoma (HCC).SETTING: Sacramento County, California, USA.PARTICIPANTS: Eligible participants were county residents ages 18 and older who had not been screened for chronic hepatitis B virus (HBV) and were born in a CDC-defined endemic area or whose parent was born in that area. Of 1004 enrolled, 917 were foreign-born Chinese (130 women, 94 men), Hmong (133 women, 75 men), Korean (178 women, 90 men) or Vietnamese (136 women, 81 men) with complete risk factor data.PRIMARY AND SECONDARY OUTCOME MEASURES: We tested participants for HBV and chronic hepatitis C virus (HCV); measured haemoglobin A1c and waist circumference; and recorded self-reported history of diabetes, hypertension, alcohol use and smoking status. We identified risk factor patterns using cluster analysis and estimated gender-specific age-standardised prevalence rates.RESULTS: We identified four patterns: (1) viral (chronic HBV or HCV); (2) lifestyle (current smoker or alcohol user, no viral); (3) metabolic (?2metabolic, no lifestyle or viral); and (4) lower risk (?1metabolic, no lifestyle or viral). Vietnamese men (16.3%, 95%CI 7.4% to 25.3%) and Hmong women (15.1%, 95%CI 7.8% to 22.5%) had the highest viral pattern prevalence. Hmong women had the highest metabolic (37.8%, 95%CI 29.8% to 45.9%), and Vietnamese men the highest lifestyle (70.4%, 95%CI 59.1% to 81.7%) pattern prevalence. In multiple domains, Hmong men and women were most likely to have viral+metabolicrisk factors (men: 14.4%, 95%CI 6.0% to 22.7%; women: 11.9%, 95%CI 5.6% to 18.3%); Vietnamese men were most likely to have lifestyle+viral (10.7%, 95%CI 2.7% to 18.8%), and lifestyle+metabolicbut not viral (46.4%, 95%CI 34.4% to 58.5%) risk factors.CONCLUSIONS: Efforts to reduce HCC must comprehensively address multiple risk factors.TRIAL REGISTRATION NUMBER: NCT02596438.

View details for DOI 10.1136/bmjopen-2018-026409

View details for PubMedID 31256022
SHC MODULATES OXIDATIVE AND INFLAMMATORY SIGNALS IN ALCOHOLIC LIVER DISEASE Dehnad, A., Das, S., Fish, S. R., Jiang, J. X., Torok, N. J. WILEY. 2019: 46A
View details for Web of Science ID 000468963101076
Macrophage Nourishment in NASH: A Novel Role for Ketone Bodies. Hepatology (Baltimore, Md.) Torok, N. J. 2019
Abstract

Since the seminal paper by Krebs in 1966 on the regulation of hepatic ketone body production and release (1), there has been a recent resurrection of interest relating to ketogenesis and its role in tissue homeostasis. In physiological states, the liver relies on glucose for its primary fuel, while the production and export of ketone bodies provides other organs with nutrients needed to meet higher energy requirements. During increased calorie intake, hepatocytes are exposed to enhanced lipid influx, and they deal with this by increasing the tricarboxylic acid (TCA) cycle load but at the expense of enhanced redox stress. This article is protected by copyright. All rights reserved.

View details for DOI 10.1002/hep.30777

View details for PubMedID 31107977
Neutrophil-Hepatic Stellate Cell Interactions Promote Fibrosis in�Experimental Steatohepatitis. Cellular and molecular gastroenterology and hepatology Zhou, Z., Xu, M. J., Cai, Y., Wang, W., Jiang, J. X., Varga, Z. V., Feng, D., Pacher, P., Kunos, G., Torok, N. J., Gao, B. 2018; 5 (3): 399?413
Abstract

Hepatic infiltration of neutrophils is a hallmark of steatohepatitis; however, the role of neutrophils in the progression of steatohepatitis remains unknown.A clinically relevant mouse model of steatohepatitis induced by high-fat diet (HFD) plus binge ethanol feeding was used. Liver fibrosis was examined. In�vitro cell culture was used to analyze the interaction of hepatic stellate cells (HSCs) and neutrophils.HFD plus one binge ethanol (HFD+1B) feeding induced significant hepatic neutrophil infiltration, liver injury, and fibrosis. HFD plus multiple binges of ethanol (HFD+mB) caused more pronounced liver fibrosis. Microarray analyses showed that the most highly activated signaling pathway in this HFD+1B model was related to liver fibrosis and HSC activation. Blockade of chemokine (C-X-C motif) ligand 1 or intercellular adhesion molecule-1 expression reduced hepatic neutrophil infiltration and ameliorated liver injury and fibrosis. Disruption of the p47 phox gene (also called neutrophil cytosolic factor 1), a critical component of reactive oxygen species producing nicotinamide adenine dinucleotide phosphate-oxidase in neutrophils, diminished HFD+1B-induced liver injury and fibrosis. Co-culture of HSCs with neutrophils, but not with neutrophil apoptotic bodies, induced HSC activation and prolonged neutrophil survival. Mechanistic studies showed that activated HSCs produce granulocyte-macrophage colony-stimulating factor and interleukin-15�to prolong the survival of neutrophils, which may serve as a positive forward loop to promote liver damage and fibrosis.The current data from a mouse model of HFD plus binge ethanol feeding suggest that obesity and binge drinking synergize to promote liver fibrosis, which is partially mediated via the interaction of neutrophils and HSCs. Microarray data in this article have been uploaded to NCBI's Gene Expression Omnibus (GEO accession number: GSE98153).

View details for DOI 10.1016/j.jcmgh.2018.01.003

View details for PubMedID 29552626

View details for PubMedCentralID PMC5852390
The NOX1 isoform of NADPH oxidase is involved in dysfunction of liver sinusoids in nonalcoholic fatty liver disease. Free radical biology & medicine Matsumoto, M., Zhang, J., Zhang, X., Liu, J., Jiang, J. X., Yamaguchi, K., Taruno, A., Katsuyama, M., Iwata, K., Ibi, M., Cui, W., Matsuno, K., Marunaka, Y., Itoh, Y., Torok, N. J., Yabe-Nishimura, C. 2018; 115: 412?20
Abstract

The increased production of reactive oxygen species (ROS) has been postulated to play a key role in the progression of nonalcoholic fatty liver disease (NAFLD). However, the source of ROS and mechanisms underlying the development of NAFLD have yet to be established. We observed a significant up-regulation of a minor isoform of NADPH oxidase, NOX1, in the liver of nonalcoholic steatohepatitis (NASH) patients as well as of mice fed a high-fat and high-cholesterol (HFC) diet for 8 weeks. In mice deficient in Nox1 (Nox1KO), increased levels of serum alanine aminotransferase and hepatic cleaved caspase-3 demonstrated in HFC diet-fed wild-type mice (WT) were significantly attenuated. Concomitantly, increased protein nitrotyrosine adducts, a marker of peroxynitrite-induced injury detected in hepatic sinusoids of WT, were significantly suppressed in Nox1KO. The expression of NOX1 mRNA was much higher in the fractions of enriched liver sinusoidal endothelial cells (LSECs) than in those of hepatocytes. In primary cultured LSECs, palmitic acid (PA) up-regulated the mRNA level of NOX1, but not of NOX2 or NOX4. The production of nitric oxide by LSECs was significantly attenuated by PA-treatment in WT but not in Nox1KO. When the in vitro relaxation of TWNT1, a cell line that originated from hepatic stellate cells, was assessed by the gel contraction assay, the relaxation of stellate cells induced by LSECs was attenuated by PA treatment. In contrast, the relaxation effect of LSECs was preserved in cells isolated from Nox1KO. Taken together, the up-regulation of NOX1 in LSECs may elicit peroxynitrite-mediated cellular injury and impaired hepatic microcirculation through the reduced bioavailability of nitric oxide. ROS derived from NOX1 may therefore constitute a critical component in the progression of NAFLD.

View details for DOI 10.1016/j.freeradbiomed.2017.12.019

View details for PubMedID 29274380

View details for PubMedCentralID PMC5969997
AGER1/RAGE imbalance and accumulation of AGEs result in inflammation, expansion of ductular cells and fibrosis in NASH Dehnad, A., Wong, K. A., Fish, S., Jiang, J., Alzofon, N., Olson, K., Gui, D., Matsukuma, K., Abdi, R., Ali, M., Singh, S., Stewart, S., Angel, P., Torok, N. J. WILEY. 2017: 210A
View details for Web of Science ID 000412089800376
Activation of NOX2 in older mice by the aging protein p52Shc leads to accelerated fibrosis in NASH Fish, S., Tomilov, A., Dehnad, A., Jiang, J., Alzofon, N., Das, S., Ramsey, J., Cortopassi, G., Torok, N. J. WILEY. 2017: 208A
View details for Web of Science ID 000412089800373
TRIF as a Novel Modulator of Liver Inflammation and Fibrosis. Cellular and molecular gastroenterology and hepatology T�r�k, N. J. 2017; 3 (3): 299-300
View details for DOI 10.1016/j.jcmgh.2017.03.002

View details for PubMedID 28462370

View details for PubMedCentralID PMC5404095
Galectin-3 regulates inflammasome activation in cholestatic liver injury FASEB JOURNAL Tian, J., Yang, G., Chen, H., Hsu, D. K., Tomilov, A., Olson, K. A., Dehnad, A., Fish, S. R., Cortopassi, G., Zhao, B., Liu, F., Gershwin, M. E., Torok, N. J., Jiang, J. X. 2016; 30 (12): 4202-4213
Abstract

Macrophage activation is an important feature of primary biliary cholangitis (PBC) pathogenesis and other cholestatic liver diseases. Galectin-3 (Gal3), a pleiotropic lectin, is produced by monocytic cells and macrophages. However, its role in PBC has not been addressed. We hypothesized that Gal3 is a key to induce NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome in macrophages and in turn to propagate proinflammatory IL-17 signaling. In liver tissues from patients with PBC and dnTGF-?RII mice, a model of autoimmune cholangitis, the expression of Gal3, NLRP3, and the adaptor protein adaptor apoptosis-associated speck-like protein was induced, with the downstream activation of caspase-1 and IL-1?. In wild-type hepatic macrophages, deoxycholic acid induced the association of Gal3 and NLRP3 with direct activation of the inflammasome, resulting in an increase in IL-1?. Downstream retinoid-related orphan receptor C mRNA, IL-17A, and IL-17F were induced. In Gal3-/- macrophages, no inflammasome activation was detected. To confirm the key role of Gal3 in the pathogenesis of cholestatic liver injury, we generated dnTGF-?RII/galectin-3-/- (dn/Gal3-/-) mice, which showed impaired inflammasome activation along with significantly improved inflammation and fibrosis. Taken together, our data point to a novel role of Gal3 as an initiator of inflammatory signaling in autoimmune cholangitis, mediating the activation of NLRP3 inflammasome and inducing IL-17 proinflammatory cascades. These studies provide a rationale to target Gal3 in autoimmune cholangitis and potentially other cholestatic diseases.-Tian, J., Yang, G., Chen, H.-Y., Hsu, D. K., Tomilov, A., Olson, K. A., Dehnad, A., Fish, S. R., Cortopassi, G., Zhao, B., Liu, F.-T., Gershwin, M. E., T�r�k, N. J., Jiang, J. X. Galectin-3 regulates inflammasome activation in cholestatic liver injury.

View details for DOI 10.1096/fj.201600392RR

View details for Web of Science ID 000388201500026

View details for PubMedID 27630169

View details for PubMedCentralID PMC5102125
Ductular reaction-on-a-chip: Microfluidic co-cultures to study stem cell fate selection during liver injury SCIENTIFIC REPORTS Haque, A., Gheibi, P., Stybayeva, G., Gao, Y., Torok, N., Revzin, A. 2016; 6
Abstract

Liver injury modulates local microenvironment, triggering production of signals that instruct stem cell fate choices. In this study, we employed a microfluidic co-culture system to recreate important interactions in the liver stem cell niche, those between adult hepatocytes and liver progenitor cells (LPCs). We demonstrate that pluripotent stem cell-derived LPCs choose hepatic fate when cultured next to healthy hepatocytes but begin biliary differentiation program when co-cultured with injured hepatocytes. We connect this fate selection to skewing in production of hepatocyte growth factor (HGF) and transforming growth factor (TGF)-?1 caused by injury. Significantly, biliary fate selection of LPCs was not observed in the absence of hepatocytes nor did it happen in the presence of TGF-? inhibitors. Our study demonstrates that microfluidic culture systems may offer an interesting new tool for dissecting cellular interactions leading to aberrant stem cell differentiation during injury.

View details for DOI 10.1038/srep36077

View details for Web of Science ID 000386562400001

View details for PubMedID 27796316

View details for PubMedCentralID PMC5086854
Dysregulation of redox pathways in liver fibrosis AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY Torok, N. J. 2016; 311 (4): G667-G674
Abstract

Reactive oxygen species are implicated in physiological signaling and cell fate decisions. In chronic liver diseases persistent and increased production of oxidative radicals drives a fibrogenic response that is a common feature of disease progression. Despite our understanding the biology of the main prooxidant enzymes, their targets, and antioxidant mechanisms in the liver, there is still lack of knowledge concerning their precise role in the pathogenesis of fibrosis. This review will examine the role of physiological redox signaling in the liver, provide an overview on recent advances in prooxidant and antioxidant pathways that are dysregulated during fibrosis, and highlight possible novel treatment targets.

View details for DOI 10.1152/ajpgi.00050.2016

View details for Web of Science ID 000387910800009

View details for PubMedID 27562057

View details for PubMedCentralID PMC5142204
Aged mice exhibit a more proinflammatory and fibrogenic NASH phenotype linked to the induction of Shc and NADPH oxidase 2 Fish, S., Dehnad, A., Jiang, J., Tomilov, A., Ramsey, J., Cortopassi, G., Torok, N. J. WILEY. 2016: 825A
View details for Web of Science ID 000385493804030
Advanced glycation end products and dysregulated RAGE/AGER1 induce proinflammatory and fibrogenic signaling in NASH via NOX4 Dehnad, A., Fish, S., Jiang, J., Torok, N. J. WILEY. 2016: 830A
View details for Web of Science ID 000385493804041
Metabolic Syndrome Post-Liver Transplant: Can We Predict? METABOLIC SYNDROME AND RELATED DISORDERS Marsano, J. G., Torok, N. J. 2016; 14 (6): 289-290
View details for DOI 10.1089/met.2016.29007.tor

View details for Web of Science ID 000380807400002

View details for PubMedID 27304753
Extracellular vesicles and ceramide: new mediators for macrophage chemotaxis? JOURNAL OF LIPID RESEARCH Toeroek, N. J. 2016; 57 (2): 157-158
View details for DOI 10.1194/jlr.C066191

View details for Web of Science ID 000369091600001

View details for PubMedID 26729816

View details for PubMedCentralID PMC4727415
Update on Alcoholic Hepatitis. Biomolecules Torok, N. J. 2015; 5 (4): 2978-2986
Abstract

Alcoholic liver disease is one of the most prevalent liver diseases worldwide, and a major cause of morbidity and mortality. Alcoholic hepatitis is a severe form of liver injury in patients with alcohol abuse, can present as an acute on chronic liver failure associated with a rapid decline in liver synthetic function, and consequent increase in mortality. Despite therapy, about 30%-50% of patients with severe alcoholic hepatitis eventually die. The pathogenic pathways that lead to the development of alcoholic hepatitis are complex and involve oxidative stress, gut dysbiosis, and dysregulation of the innate and adaptive immune system with injury to the parenchymal cells and activation of hepatic stellate cells. As accepted treatment approaches are currently limited, a better understanding of the pathophysiology would be required to generate new approaches that improve outcomes. This review focuses on recent advances in the diagnosis, pathogenesis of alcoholic hepatitis and novel treatment strategies.

View details for DOI 10.3390/biom5042978

View details for PubMedID 26540078

View details for PubMedCentralID PMC4693265
Vascular adhesion protein 1 in nonalcoholic steatohepatitis: A novel biomarker? HEPATOLOGY Torok, N. J. 2015; 62 (4): 1313-1315
View details for DOI 10.1002/hep.27942

View details for Web of Science ID 000362214800033

View details for PubMedID 26084585

View details for PubMedCentralID PMC4589450
Galectin 3 regulates HCC cell invasion by RhoA and MLCK activation LABORATORY INVESTIGATION Serizawa, N., Tian, J., Fukada, H., Baghy, K., Scott, F., Chen, X., Kiss, Z., Olson, K., Hsu, D., Liu, F., Toeroek, N. J., Zhao, B., Jiang, J. X. 2015; 95 (10): 1145-1156
Abstract

Hepatocellular carcinoma (HCC) carries a poor prognosis with no effective treatment available other than liver transplantation for selected patients. Vascular invasion of HCC is one of the most important negative predictor of survival. As the regulation of invasion of HCC cells is not well understood, our aim was to study the mechanisms by which galectin 3, a ?-galactosidase-binding lectin mediates HCC cell migration. HCC was induced by N-diethylnitrosamine in wild-type and galectin 3(-/-) mice, and tumor formation, histology, and tumor cell invasion were assessed. The galectin 3(-/-) mice developed significantly smaller tumor burden with a less invasive phenotype than the wild-type animals. Galectin 3 was upregulated in the wild-type HCC tumor tissue, but not in the surrounding parenchyma. Galectin 3 expression in HCC was induced by NF-?B transactivation as determined by chromatin immunoprecipitation assays. In vitro studies assessed the pro-migratory effects of galectin 3. The migration of hepatoma cells was significantly decreased after transfection by the galectin 3 siRNA and also after using the Rho kinase inhibitor Y-27632. The reorganization of the actin cytoskeleton, RhoA GTPase activity and the phosphorylation of MLC2 (myosin light chain 2) were decreased in the galectin 3 siRNA-transfected cells. In addition, in vitro and in vivo evidence showed that galectin 3 deficiency reduced hepatoma cell proliferation and increased their apoptosis rate. In conclusion, galectin 3 is an important lectin that is induced in HCC cells, and promotes hepatoma cell motility and invasion by an autocrine pathway. Targeting galectin 3 therefore could be an important novel treatment strategy to halt disease progression.

View details for DOI 10.1038/labinvest.2015.77

View details for Web of Science ID 000361909200005

View details for PubMedID 26146960

View details for PubMedCentralID PMC4586310
Calciphylaxis in a Patient With Alcoholic Cirrhosis. ACG case reports journal Akhtar, E., Parikh, D. A., Torok, N. J. 2015; 2 (4): 209-210
View details for DOI 10.14309/crj.2015.60

View details for PubMedID 26203440

View details for PubMedCentralID PMC4508942
Role of intestinal myofibroblasts in HIV-associated intestinal collagen deposition and immune reconstitution following combination antiretroviral therapy AIDS Asmuth, D. M., Pinchuk, I. V., Wu, J., Vargas, G., Chen, X., Mann, S., Albanese, A., Ma, Z., Saroufeem, R., Melcher, G. P., Troia-Cancio, P., Torok, N. J., Miller, C. J., Powell, D. W. 2015; 29 (8): 877-888
Abstract

To investigate the potential role of mucosal intestinal myofibroblasts (IMFs) in HIV and associated fibrosis in gut-associated lymphoid tissue.Profibrotic changes within the secondary lymphoid organs and mucosa have been implicated in failed immune reconstitution following effective combination antiretroviral therapy (cART). Microbial translocation is believed to be sustaining these systemic inflammatory pathways. IMFs are nonprofessional antigen-presenting cells with both immunoregulatory and mesenchymal functions that are ideally positioned to respond to translocating microbial antigen.Duodenal biopsies, obtained from patients naive to cART, underwent trichrome staining and were examined for tissue growth factor-beta (TGF-?) expression. Combined immunostaining and second harmonic generation analysis were used to determine IMF activation and collagen deposition. Confocal microscopy was performed to examine IMF activation and Toll-like receptor (TLR)4 expression. Finally, primary IMF cultures were stimulated with lipopolysaccharide to demonstrate the expression of the inflammatory biomarkers.The expression of the fibrosis-promoting molecule, TGF-?1, is significantly increased in duodenal biopsies from HIV patients na�ve to cART, and negatively correlated with subsequent peripheral CD4(+) recovery. The increase in TGF-?1 coincided with an increase in collagen deposition in the duodenal mucosa in the tissue area adjacent to the IMFs. We also observed that IMFs expressed TLR4 and had an activated phenotype since they were positive for fibroblast activation protein. Finally, stimulation of IMFs from HIV patients with TLR4 resulted in significantly increased expression of profibrotic molecules, TGF-?1, and interleukin-6.Our data support the hypothesis that activated IMFs may be among the major cells contributing to the profibrotic changes, and thus, the establishment and maintenance of systemic inflammation interfering with immune reconstitution in HIV patients.

View details for DOI 10.1097/QAD.0000000000000636

View details for PubMedID 25784439
MLK3 as a regulator of disease progression in Non-alcoholic steatohepatitis LIVER INTERNATIONAL Jiang, J. X., Toeroek, N. J. 2014; 34 (8): 1131-1132
View details for DOI 10.1111/liv.12556

View details for Web of Science ID 000342578100001

View details for PubMedID 24690035

View details for PubMedCentralID PMC4392882
Advanced glycation endproducts induce fibrogenic activity in nonalcoholic steatohepatitis by modulating TNF-a-converting enzyme activity in mice. Hepatology Jiang, J. X., Chen, X., Fukada, H., Serizawa, N., Devaraj, S., T�r�k, N. J. 2013; 58 (4): 1339-1348
Abstract

Advanced glycation endproducts (AGEs) accumulate in patients with diabetes, yet the link between AGEs and inflammatory and fibrogenic activity in nonalcoholic steatohepatitis (NASH) has not been explored. Tumor necrosis factor alpha (TNF-?)-converting enzyme (TACE) is at the center of inflammatory processes. Because the main natural regulator of TACE activity is the tissue inhibitor of metalloproteinase 3 (Timp3), we hypothesized that AGEs induce TACE through nicotinamide adenine dinucleotide phosphate reduced oxidase 2 (NOX2); and the down-regulation of Sirtuin 1 (Sirt1)/Timp3 pathways mediate fibrogenic activity in NASH. The role of NOX2, Sirt1, Timp3, and TACE was evaluated in choline-deficient L-amino acid defined (CDAA) or Western diet (WD)-fed wild-type (WT) and NOX2(-/-) mice. To restore Timp3, mice were injected with adenovirus (Ad)-Timp3. Sirt1 and Timp3 expressions were studied in livers from NASH patients, and we found that their levels were significantly lower than in healthy controls. In WT mice on the CDAA or WD, Sirt1 and Timp3 expressions were lower, whereas production of reactive oxidative species and TACE activity significantly increased with an increase in active TNF-? production as well as induction of fibrogenic transcripts. Ad-Timp3 injection resulted in a significant decline in TACE activity, procollagen ?1 (I), alpha smooth muscle actin (?-SMA) and transforming growth factor beta (TGF-?) expression. NOX2(-/-) mice on the CDAA or WD had no significant change in Sirt1, Timp3, and TACE activity or the fibrosis markers assessed. In vitro, AGE exposure decreased Sirt1 and Timp3 in hepatic stellate cells by a NOX2-dependent pathway, and TACE was induced after exposure to AGEs.TACE activation is central to the pathogenesis of NASH and is mediated by AGEs through NOX2 induction and down-regulation of Sirt1/Timp3 pathways.

View details for DOI 10.1002/hep.26491

View details for PubMedID 23703665

View details for PubMedCentralID PMC3897213
Calciphylaxis in a Patient with Alcoholic Cirrhosis Akhtar, E., Parikh, D., Torok, N. NATURE PUBLISHING GROUP. 2013: S337
View details for Web of Science ID 000330178101290
Hepatocyte NOX4 plays an important role in modulating stress response-mediated fibrogenic injury during NASH chao, T., Jiang, X., Fukada, H., Haj, F., Bettaieb, A., Torok, N. WILEY-BLACKWELL. 2013: 221A
View details for Web of Science ID 000330252201029
Liver Injury and the Activation of the Hepatic Myofibroblasts. Current pathobiology reports Jiang, J. X., T�r�k, N. J. 2013; 1 (3): 215-223
Abstract

Liver fibrosis is a wound healing process, the end result of chronic liver injury elicited by different noxious stimuli. Activated hepatic stellate cells or myofibroblasts and portal myofibroblasts are considered as the main producers of the extracellular matrix in the liver. Upon liver injury the quiescent stellate cells transdifferentiate into myofibroblasts a process highlighted by the loss of vitamin A stores, upregulation of interstitial type collagens, smooth muscle ? actin, matrix metalloproteinases, proteoglycans, and the induction of cell survival pathways. Activation of hepatic stellate cells is a result of a complex interplay between the parenchymal cells, immune cells, extracellular matrix mechanics and extrahepatic milieu such as the gut microbiome. In this review we will focus on the pathomechanism of stellate cell activation following chronic liver injury; with the aim of identifying possible treatment targets for anti-fibrogenic agents.

View details for PubMedID 23977452

View details for PubMedCentralID PMC3748972
Wilson's Disease: Changes in Methionine Metabolism and Inflammation Affect Global DNA Methylation in Early Liver Disease HEPATOLOGY Medici, V., Shibata, N. M., Kharbanda, K. K., LaSalle, J. M., Woods, R., Liu, S., Engelberg, J. A., Devaraj, S., Toeroek, N. J., Jiang, J. X., Havel, P. J., Loennerdal, B., Kim, K., Halsted, C. H. 2013; 57 (2): 555-565
Abstract

Hepatic methionine metabolism may play an essential role in regulating methylation status and liver injury in Wilson's disease (WD) through the inhibition of S-adenosylhomocysteine hydrolase (SAHH) by copper (Cu) and the consequent accumulation of S-adenosylhomocysteine (SAH). We studied the transcript levels of selected genes related to liver injury, levels of SAHH, SAH, DNA methyltransferases genes (Dnmt1, Dnmt3a, Dnmt3b), and global DNA methylation in the tx-j mouse (tx-j), an animal model of WD. Findings were compared to those in control C3H mice, and in response to Cu chelation by penicillamine (PCA) and dietary supplementation of the methyl donor betaine to modulate inflammatory and methylation status. Transcript levels of selected genes related to endoplasmic reticulum stress, lipid synthesis, and fatty acid oxidation were down-regulated at baseline in tx-j mice, further down-regulated in response to PCA, and showed little to no response to betaine. Hepatic Sahh transcript and protein levels were reduced in tx-j mice with consequent increase of SAH levels. Hepatic Cu accumulation was associated with inflammation, as indicated by histopathology and elevated serum alanine aminotransferase (ALT) and liver tumor necrosis factor alpha (Tnf-?) levels. Dnmt3b was down-regulated in tx-j mice together with global DNA hypomethylation. PCA treatment of tx-j mice reduced Tnf-? and ALT levels, betaine treatment increased S-adenosylmethionine and up-regulated Dnmt3b levels, and both treatments restored global DNA methylation levels.Reduced hepatic Sahh expression was associated with increased liver SAH levels in the tx-j model of WD, with consequent global DNA hypomethylation. Increased global DNA methylation was achieved by reducing inflammation by Cu chelation or by providing methyl groups. We propose that increased SAH levels and inflammation affect widespread epigenetic regulation of gene expression in WD.

View details for DOI 10.1002/hep.26047

View details for Web of Science ID 000315643400016

View details for PubMedID 22945834

View details for PubMedCentralID PMC3566330
Role of FNA and Core Biopsy of Primary and Metastatic Liver Disease. International journal of hepatology McGahan, J. P., Bishop, J., Webb, J., Howell, L., Torok, N., Lamba, R., Corwin, M. T. 2013; 2013: 174103-?
Abstract

Objective. To examine our experience with cytology and histology biopsy of the liver and to define methods for improvement of diagnosis of primary liver tumors. Methods. This include retrospective study of 189 biopsies of 185 liver masses for cytological or histological analysis. Patients were subdivided into two groups. Group 1 consisted of 124 suspected metastasis. Group 2 consisted of 61 suspected primary neoplasms. Biopsies were considered positive or equivocal. In equivocal cases, special stains were performed. In Group 2, cases were classified by contrast CT or MRI as to (I) classic HCC, (II) infiltrated HCC, or (Ill) equivocal. Results. Definitive diagnosis was obtained in 117/124 masses (94%) in Group 1, 48/61 masses (79%) in Group 2, and (Ill) equivocal 13 cases in Group II. In two equivocal cases in which special stains were performed, they were reclassified as HCC. In 8/13 cases, CT findings were consistent with HCC. Conclusion. Liver biopsies are useful in obtaining a definitive diagnosis of suspected metastatic liver disease. Biopsy results are less reliable in patients with suspected primary liver tumors. In these situations, strategies can include basing treatment on imaging criteria or use of newer special pathological stains. Advances in Knowledge. Use of newer special immunological stains improves accuracy in definitive diagnosis of primary liver tumors.

View details for DOI 10.1155/2013/174103

View details for PubMedID 24369506

View details for PubMedCentralID PMC3857922
Nox4 plays an important role in alcohol-induced hepatic oxidative stress Chen, X., Jiang, J., Fukada, H., Schroder, K., Brandes, R. P., Torok, N. WILEY-BLACKWELL. 2012: 1124A
View details for Web of Science ID 000310955603601
NADPH oxidase 4 plays a key role in hepatocyte injury and HSC activation leading to NASH progression Jiang, J., Chen, X., Fukada, H., Schroder, K., Brandes, R. P., Devaraj, S., Torok, N. WILEY-BLACKWELL. 2012: 258A
View details for Web of Science ID 000310955601130
NOX4 IS A KEY ENZYME IN ALCOHOLIC LIVER DISEASE Chen, X., Fukada, H., Jiang, X., Torok, N. J. WILEY-BLACKWELL. 2012: 76A
View details for Web of Science ID 000308396700256
Liver fibrosis and hepatocyte apoptosis are attenuated by GKT137831, a novel NOX4/NOX1 inhibitor in vivo FREE RADICAL BIOLOGY AND MEDICINE Jiang, J. X., Chen, X., Serizawa, N., Szyndralewiez, C., Page, P., Schroeder, K., Brandes, R. P., Devaraj, S., Toeroek, N. J. 2012; 53 (2): 289-296
Abstract

Reactive oxygen species (ROS) play a key role in chronic liver injury and fibrosis. Homologs of NADPH oxidases (NOXs) are major sources of ROS, but the exact role of the individual homologs in liver disease is unknown. Our goal was to determine the role of NOX4 in liver fibrosis induced by bile duct ligation (BDL) with the aid of the pharmacological inhibitor GKT137831, and genetic deletion of NOX4 in mice. GKT137831 was either applied for the full term of BDL (preventive arm) or started at 10 day postoperatively (therapeutic arm). Primary hepatic stellate cells (HSC) from control mice with and without BDL were analyzed and the effect of NOX4 inhibition on HSC activation was also studied. FasL or TNF?/actinomycin D-induced apoptosis was studied in wild-type and NOX4(-/-) hepatocytes. NOX4 was upregulated by a TGF-?/Smad3-dependent mechanism in HSC. Downregulation of NOX4 decreased ROS production and the activation of NOX4(-/-) HSC was attenuated. NOX4(-/-) hepatocytes were more resistant to FasL or TNF?/actinomycin D-induced apoptosis. Similarly, after pharmacological NOX4 inhibition, ROS production, the expression of fibrogenic markers, and hepatocyte apoptosis were reduced. NOX4 was expressed in human livers with stage 2-3 autoimmune hepatitis. Fibrosis was attenuated by the genetic deletion of NOX4. BDL mice gavaged with GKT137831 in the preventive or the therapeutic arm displayed less ROS production, significantly attenuated fibrosis, and decreased hepatocyte apoptosis. In conclusion, NOX4 plays a key role in liver fibrosis. GKT137831 is a potent inhibitor of fibrosis and hepatocyte apoptosis; therefore, it is a promising therapeutic agent for future translational studies.

View details for DOI 10.1016/j.freeradbiomed.2012.05.007

View details for Web of Science ID 000306677500012

View details for PubMedID 22618020

View details for PubMedCentralID PMC3392471
Human ESC self-renewal promoting microRNAs induce epithelial-mesenchymal transition in hepatocytes by controlling the PTEN and TGF� tumor suppressor signaling pathways. Molecular cancer research Jung, C. J., Iyengar, S., Blahnik, K. R., Jiang, J. X., Tahimic, C., Torok, N. J., de Vere White, R. W., Farnham, P. J., Zern, M. 2012; 10 (7): 979-991
Abstract

The self-renewal capacity ascribed to embryonic stem cells (ESC) is reminiscent of cancer cell proliferation, raising speculation that a common network of genes may regulate these traits. A search for general regulators of these traits yielded a set of microRNAs for which expression is highly enriched in human ESCs and liver cancer cells (HCC) but attenuated in differentiated quiescent hepatocytes. Here, we show that these microRNAs promote hESC self-renewal, as well as HCC proliferation, and when overexpressed in normally quiescent hepatocytes, induce proliferation and activate cancer signaling pathways. Proliferation in hepatocytes is mediated through translational repression of Pten, Tgfbr2, Klf11, and Cdkn1a, which collectively dysregulates the PI3K/AKT/mTOR and TGF? tumor suppressor signaling pathways. Furthermore, aberrant expression of these miRNAs is observed in human liver tumor tissues and induces epithelial-mesenchymal transition in hepatocytes. These findings suggest that microRNAs that are essential in normal development as promoters of ESC self-renewal are frequently upregulated in human liver tumors and harbor neoplastic transformation potential when they escape silencing in quiescent human hepatocytes.

View details for DOI 10.1158/1541-7786.MCR-11-0421

View details for PubMedID 22622027

View details for PubMedCentralID PMC4166560
Galectin-3 modulates phagocytosis-induced stellate cell activation and liver fibrosis in vivo AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY Jiang, J. X., Chen, X., Hsu, D. K., Baghy, K., Serizawa, N., Scott, F., Takada, Y., Takada, Y., Fukada, H., Chen, J., Devaraj, S., Adamson, R., Liu, F., Toeroek, N. J. 2012; 302 (4): G439-G446
Abstract

Hepatic stellate cells (HSC), the key fibrogenic cells of the liver, transdifferentiate into myofibroblasts upon phagocytosis of apoptotic hepatocytes. Galectin-3, a ?-galactoside-binding lectin, is a regulator of the phagocytic process. In this study, our aim was to study the mechanism by which extracellular galectin-3 modulates HSC phagocytosis and activation. The role of galectin-3 in engulfment was evaluated by phagocytosis and integrin binding assays in primary HSC. Galectin-3 expression was studied by real-time PCR and enzyme-linked immunosorbent assay, and in vivo studies were done in wild-type and galectin-3(-/-) mice. We found that HSC from galectin-3(-/-) mice displayed decreased phagocytic activity, expression of transforming growth factor-?1, and procollagen ?1(I). Recombinant galectin-3 reversed this defect, suggesting that extracellular galectin-3 is required for HSC activation. Galectin-3 facilitated the ?(v)?(3) heterodimer-dependent binding, indicating that galectin-3 modulates HSC phagocytosis via cross-linking this integrin and enhancing the tethering of apoptotic cells. Blocking integrin ?(v)?(3) resulted in decreased phagocytosis. Galectin-3 expression and release were induced in active HSC engulfing apoptotic cells, and this was mediated by the nuclear factor-?B signaling. The upregulation of galectin-3 in active HSC was further confirmed in vivo in bile duct-ligated (BDL) rats. Galectin-3(-/-) mice displayed significantly decreased fibrosis, with reduced expression of ?-smooth muscle actin and procollagen ?1(I) following BDL. In summary, extracellular galectin-3 plays a key role in liver fibrosis by mediating HSC phagocytosis, activation, and subsequent autocrine and paracrine signaling by a feedforward mechanism.

View details for DOI 10.1152/ajpgi.00257.2011

View details for Web of Science ID 000300318000004

View details for PubMedID 22159281

View details for PubMedCentralID PMC3287392
NOX4 INDUCTION IS ATTENUATED IN NOX2-/- MICE DURING LIVER FIBROSIS Chen, X., Jiang, X., Serizawa, N., Torok, N. WILEY-BLACKWELL. 2011: 739A?740A
View details for Web of Science ID 000295578003028
ADVANCED GLYCATION END PRODUCTS INDUCE INFLAMMATORY AND FIBROGENIC ACTIVITY IN NASH BY REGULATING TIMP3/TACE ACTIVITY Jiang, X., Chen, X., Serizawa, N., Torok, N. WILEY-BLACKWELL. 2011: 735A
View details for Web of Science ID 000295578003018
TREATMENT WITH A NOVEL NOX4 INHIBITOR ATTENUATES LIVER FIBROSIS Chen, X., Jiang, X., Serizawa, N., Szyndralewiez, C., Page, P., Torok, N. WILEY-BLACKWELL. 2011: 740A
View details for Web of Science ID 000295578003029
NOX1/Nicotinamide Adenine Dinucleotide Phosphate, Reduced Form (NADPH) Oxidase Promotes Proliferation of Stellate Cells and Aggravates Liver Fibrosis Induced by Bile Duct Ligation HEPATOLOGY Cui, W., Matsuno, K., Iwata, K., Ibi, M., Matsumoto, M., Zhang, J., Zhu, K., Katsuyama, M., Torok, N. J., Yabe-Nishimura, C. 2011; 54 (3): 949-958
Abstract

Among multiple isoforms of nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase expressed in the liver, the phagocytic NOX2 isoform in hepatic stellate cells (HSCs) has been demonstrated to play a key role in liver fibrogenesis. The aim of this study was to clarify the role of NOX1, a nonphagocytic form of NADPH oxidase, in the development of fibrosis using Nox1-deficient mice (Nox1KO). Liver injury and fibrosis were induced by bile duct ligation (BDL) and carbon tetrachloride in Nox1KO and wildtype littermate mice (WT). Primary HSCs were isolated to characterize the NOX1-induced signaling cascade involved in liver fibrogenesis. Following BDL, a time-dependent increase in NOX1 messenger RNA (mRNA) was demonstrated in WT liver. Compared with those in WT, levels of collagen-1? mRNA and hydroxyproline were significantly suppressed in Nox1KO with a reduced number of activated HSCs and less severe fibrotic lesions. The expression levels of ?-smooth muscle actin, a marker of HSCs activation, were similar in cultured HSCs isolated from both genotypes. However, cell proliferation was significantly attenuated in HSCs isolated from Nox1KO. In these cells, the expression of p27(kip1) , a cell cycle suppressor, was significantly up-regulated. Concomitantly, a significant reduction in phosphorylated forms of Akt and forkhead box O (FOXO) 4, a downstream effector of Akt that regulates the transcription of p27(kip1) gene, was demonstrated in Nox1KO. Finally, the level of the oxidized inactivated form of phosphatase and tensin homolog (PTEN), a negative regulator of PI3K/Akt pathway, was significantly attenuated in HSCs of Nox1KO.These findings indicate that reactive oxygen species derived from NOX1/NADPH oxidase oxidize and inactivate PTEN to positively regulate the Akt/FOXO4/p27(kip1) signaling pathway. NOX1 may thus promote proliferation of HSCs and accelerate the development of fibrosis following BDL-induced liver injury.

View details for DOI 10.1002/hep.24465

View details for Web of Science ID 000294738300023

View details for PubMedID 21618578
Molecular Characterization of Stool Microbiota in HIV-Infected Subjects by Panbacterial and Order-Level 16S Ribosomal DNA (rDNA) Quantification and Correlations With Immune Activation JAIDS-JOURNAL OF ACQUIRED IMMUNE DEFICIENCY SYNDROMES Ellis, C. L., Ma, Z., Mann, S. K., Li, C., Wu, J., Knight, T. H., Yotter, T., Hayes, T. L., Maniar, A. H., Troia-Cancio, P. V., Overman, H. A., Torok, N. J., Albanese, A., Rutledge, J. C., Miller, C. J., Pollard, R. B., Asmuth, D. M. 2011; 57 (5): 363-370
Abstract

The relationship between gut microbial community composition at the higher-taxonomic order level and local and systemic immunologic abnormalities in HIV disease may provide insight into how bacterial translocation impacts HIV disease.Antiretroviral-naive patients with HIV underwent upper endoscopy before and 9 months after starting antiretroviral treatment. Duodenal tissue was paraffin-embedded for immunohistochemical analysis and digested for fluorescence activated cell sorting for T-cell subsets and immune activation (CD38+/HLA-DR+) enumeration. Stool samples were provided from patients and control subjects for comparison. Metagenomic microbial DNA was extracted from feces for optimized 16S ribosomal RNA gene (rDNA) real-time quantitative polymerase chain reaction assays designed to quantify panbacterial loads and the relative abundances of proinflammatory Enterobacteriales order and the dominant Bacteroidales and Clostridiales orders.Samples from 10 HIV subjects before initiating and from six subjects receiving antiretroviral treatment were available for analysis. There was a trend for a greater proportion of Enterobacteriales in HIV-positive subjects compared with control subjects (P = 0.099). There were significant negative correlations between total bacterial load and duodenal CD4 and CD8 T-cell activation levels (r = -0.74, P = 0.004 and r = -0.67, P = 0.013, respectively). The proportions of Enterobacteriales and Bacteroidales were significantly correlated with duodenal CD4 T-cell depletion and peripheral CD8 T-cell activation, respectively.These data represent the first report of quantitative molecular and cellular correlations between total/universal and order-level gut bacterial populations and gastrointestinal-associated lymphoid tissue levels of immune activation in HIV-infected subjects. The correlations between lower overall 16S rDNA levels and tissue immune activation suggest that the gut microbiome may contribute to immune activation and influence HIV progression.

View details for DOI 10.1097/QAI.0b013e31821a603c

View details for Web of Science ID 000293381300010

View details for PubMedID 21436711

View details for PubMedCentralID PMC3153564
Leptin: The missing link between obesity and heart disease? ATHEROSCLEROSIS Devaraj, S., Torok, N. 2011; 217 (2): 322-323
View details for DOI 10.1016/j.atherosclerosis.2011.04.012

View details for Web of Science ID 000293212400003

View details for PubMedID 21663911

View details for PubMedCentralID PMC4823138
Increased Soluble Leptin Receptor Levels in Morbidly Obese Patients With Insulin Resistance and Nonalcoholic Fatty Liver Disease OBESITY Medici, V., Ali, M. R., Seo, S., Aoki, C. A., Rossaro, L., Kim, K., Fuller, W. D., Vidovszky, T. J., Smith, W., Jiang, J. X., Maganti, K., Havel, P. J., Kamboj, A., Ramsamooj, R., Toeroek, N. J. 2010; 18 (12): 2268-2273
Abstract

The adipocyte hormone, leptin has been demonstrated to have profibrogenic actions in vitro and in animal models. However, no correlation was found between plasma leptin levels and fibrosis stage in humans. Thus, our aim was to study whether soluble leptin receptor (SLR) or free leptin index (FLI; calculated as the ratio of leptin to SLR), may correlate better with the features of metabolic syndrome and with the histological grade and stage of nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH). We studied a population (n = 104) of morbidly obese patients undergoing bariatric surgery. Data including BMI, type 2 diabetes mellitus, hypertension, and hyperlipidemia were obtained. Plasma fasting leptin and SLR, fasting glucose and insulin were measured, and homeostasis model of assessment insulin resistance (HOMA(IR)) index and FLI were calculated. All patients had intraoperative liver biopsies. Leptin levels correlated with the BMI. The multiple regression analysis indicated that increasing HOMA and decreasing FLI were predictors of steatosis in the liver (P < 0.0003). SLR levels were positively correlated with the presence of diabetes mellitus and the stage of fibrosis. In conclusion, increased SLR levels in morbidly obese patients with diabetes are correlated with the stage of liver fibrosis, and may reflect progressive liver disease.

View details for DOI 10.1038/oby.2010.95

View details for Web of Science ID 000284524700005

View details for PubMedID 20448542

View details for PubMedCentralID PMC4820322
Reduced Nicotinamide Adenine Dinucleotide Phosphate Oxidase 2 Plays a Key Role in Stellate Cell Activation and Liver Fibrogenesis In Vivo GASTROENTEROLOGY Jiang, J. X., Venugopal, S., Serizawa, N., Chen, X., Scott, F., Li, Y., Adamson, R., Devaraj, S., Shah, V., Gershwin, M. E., Friedman, S. L., Toeroek, N. J. 2010; 139 (4): 1375-?
Abstract

Hepatocyte apoptosis and activation of hepatic stellate cells (HSC) are critical events in fibrogenesis. We previously demonstrated that phagocytosis of apoptotic hepatocytes by HSC is profibrogenic. Based on this, as well as the observation that reduced nicotinamide adenine dinucleotide phosphate oxidase (NADPH) oxidase induction is central to fibrogenesis, our aim was to study the phagocytic NADPH oxidase NOX2.An in vivo phagocytosis model was developed by injecting wild type (wt) or NOX2(-/-) mice with lentiviral-green fluorescence protein (GFP) containing a hepatocyte-specific promoter, and adeno-tumor necrosis factor-related apoptosis-inducing ligand (ad-TRAIL). Fibrosis was evaluated in bile duct ligated (BDL) wt and NOX2(-/-) mice with or without gadolinium treatment. NOX2 expression was studied in human liver samples and in HSC isolated from fibrotic livers. The fibrogenic activity of NOX2 was assessed by collagen reporter assays.In the phagocytosis model, engulfment of GFP-labeled apoptotic bodies was seen, and the expression of ?-smooth muscle actin (?-SMA) and collagen I increased significantly in the wt but not in the NOX2(-/-) mice. Inhibiting apoptosis decreased the profibrogenic response. NOX2(-/-) animals exhibited significantly less fibrosis following BDL. Inactivating macrophages in wt BDL mice did not lower collagen production to the level observed in NOX2(-/-) mice, suggesting that NOX2-expressing HSC are important in fibrogenesis. NOX2 was up-regulated in HSC from fibrotic livers, and phagocytosis-induced NOX2 expression and activity were demonstrated. Based on reporter assays, production of NOX2-mediated reactive oxygen species directly induced collagen promoter activity in HSC.Apoptosis and phagocytosis of hepatocytes directly induce HSC activation and initiation of fibrosis. NOX2, the phagocytic NADPH oxidase, plays a key role in this process and in liver fibrogenesis in vivo.

View details for DOI 10.1053/j.gastro.2010.05.074

View details for Web of Science ID 000282372700047

View details for PubMedID 20685364

View details for PubMedCentralID PMC2949521
GALECTIN 3 INDUCES HCC INVASIVENESS BY A RHOA AND MLCK MEDIATED PATHWAY Serizawa, N., Jiang, J., Chen, X., Torok, N. WILEY-BLACKWELL. 2010: 952A?953A
View details for Web of Science ID 000288775601639
PHAGOCYTOSIS OF APOPTOTIC CELLS BY HEPATIC STELLATE CELLS INDUCES CD8+PROLIFERATION AND ACTIVATION IN VITRO AND IN VIVO Jiang, J., Serizawa, N., Chen, X., Murphy, W. J., Ames, E., Ma, W., Torok, N. WILEY-BLACKWELL. 2010: 1263A
View details for Web of Science ID 000288775602601
Genomic variants associated with primary biliary cirrhosis. Genome medicine Selmi, C., Torok, N. J., Affronti, A., Gershwin, M. E. 2010; 2 (1): 5-?
Abstract

Primary biliary cirrhosis (PBC) is an autoimmune hepatobiliary disease characterized by immune-mediated injury of small and medium-sized bile ducts, eventually leading to liver cirrhosis. Several studies have addressed PBC immunopathology, and the data support an immune activation leading to autoantibodies and autoreactive T cells acting against the lipoylated 2-oxoacid dehydrogenase complexes. The causes of the disease remain unknown, but environmental factors and genetic susceptibility both contribute to its onset. Over the past two decades several association studies have addressed the role of genetic polymorphisms in PBC pathogenesis and have reported multiple associations. However, only a few studies had sufficient statistical power, and in most cases results were not independently validated. A genome-wide association study has recently been reported, but this too awaits independent confirmation. The aim of this present work is to critically review the numerous studies dedicated to revealing genetic associations in PBC, and to predict the potential for future studies based on these data.

View details for DOI 10.1186/gm126

View details for PubMedID 20193050

View details for PubMedCentralID PMC2829930
Liver fibrosis causes downregulation of miRNA-150 and miRNA-194 in hepatic stellate cells, and their overexpression causes decreased stellate cell activation AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY Venugopal, S. K., Jiang, J., Kim, T., Li, Y., Wang, S., Torok, N. J., Wu, J., Zern, M. A. 2010; 298 (1): G101-G106
Abstract

Activation of hepatic stellate cells (HSC) results in their proliferation and in the secretion of extracellular matrix (ECM) proteins, which leads to hepatic fibrosis. microRNAs (miRNAs) have been shown to regulate various cell functions, such as proliferation, differentiation, and apoptosis. Hence, we have analyzed the miRNAs that were differentially expressed in HSC isolated from sham-operated and bile duct-ligated rats. Expression of two miRNAs, miRNA-150 and miRNA-194, was reduced in HSC isolated from fibrotic rats compared with sham-operated animals. These two miRNAs were overexpressed in LX-2 cells, and their ability to inhibit cell proliferation, the expression of smooth muscle alpha-actin (SMA), a marker for activation, and collagen type I, a marker for ECM secretion, was determined. Overexpression of these two miRNAs resulted in a significant inhibition of proliferation (P < 0.05) and reduced SMA and collagen I levels compared with either untreated cells or nonspecific miRNA-expressing cells. Next, the protein targets of these two miRNAs were found using bioinformatics approaches. C-myb was found to be a target for miRNA-150, and rac 1 was found to be one of the targets for miRNA-194. Therefore, we studied the expression of these two proteins by overexpressing these two miRNAs in LX-2 cells and found that overexpression of miRNA-150 and miRNA-194 resulted in a significant inhibition of c-myb and rac 1 expression, respectively. We conclude that both miRNA-150 and miRNA-194 inhibit HSC activation and ECM production, at least in part, via inhibition of c-myb and rac 1 expression.

View details for DOI 10.1152/ajpgi.00220.2009

View details for Web of Science ID 000272926000013

View details for PubMedID 19892940

View details for PubMedCentralID PMC2806096
GALECTIN 3 IS A SURVIVAL PROTEIN AND PLAYS A ROLE IN THE DEVELOPMENT OF HCC IN MICE Jiang, J., Scott, F., Li, Y., Serizawa, N., Devaraj, S., Hsu, D. K., Liu, F., Torok, N. JOHN WILEY & SONS INC. 2009: 854A
View details for Web of Science ID 000270456001178
Apoptotic body engulfment by hepatic stellate cells promotes their survival by the JAK/STAT and Akt/NF-kappaB-dependent pathways. JOURNAL OF HEPATOLOGY Jiang, J. X., Mikami, K., Venugopal, S., Li, Y., Torok, N. J. 2009; 51 (1): 139-148
Abstract

We have previously shown that phagocytosis of apoptotic bodies (AB) by hepatic stellate cells (HSC) is profibrogenic. As HSC survival is central to the progression of liver fibrosis, our goal was to investigate if phagocytosis induces HSC survival.Apoptosis of phagocytosing HSC was studied in the presence of known apoptotic agents. The JAK/STAT- and PI3K/Akt-dependent pathways, NF-kappaB activation and expression of the anti-apoptotic proteins Mcl-1 and A1 were evaluated. Apoptosis was assessed after blocking A1 by an siRNA approach.Phagocytosing HSC were resistant to FasL/cycloheximide or TRAIL-induced apoptosis. Inhibition of the JAK/STAT or PI3K-mediated pathways induced apoptosis of HSC. Phagocytosis induced JAK1/STAT3 phosphorylation, and this was prevented by inhibiting JAK. Translocation of STAT3 to the nucleus was also blocked by JAK inhibition. Mcl-1 expression was upregulated in a JAK-dependent manner. PI3K-dependent phosphorylation of Akt depended on NADPH oxidase activity and superoxide production. NF-kappaB activation and subsequent upregulation of A1 was observed, and A1 inhibition induced apoptosis of HSC.Phagocytosis of AB promotes HSC survival by two pathways, of which the A1 dependent is more significant. This represents a new mechanism by which engulfment of AB contributes to the propagation of liver fibrosis.

View details for DOI 10.1016/j.jhep.2009.03.024

View details for Web of Science ID 000267713400016

View details for PubMedID 19457567

View details for PubMedCentralID PMC2765371
Minocycline in the Treatment of Patients With Primary Sclerosing Cholangitis: Results of a Pilot Study AMERICAN JOURNAL OF GASTROENTEROLOGY Silveira, M. G., Torok, N. J., Gossard, A. A., Keach, J. C., Jorgensen, R. A., Petz, J. L., Lindor, K. D. 2009; 104 (1): 83-88
Abstract

Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease of young adults that is associated with significant morbidity and mortality. No effective medical therapy is available. Minocycline has been found to exert biological effects independent of its antimicrobial properties, including anti-inflammatory activities such as inhibition of inducible nitric oxide synthase, upregulation of interleukin 10, and direct suppressive effect on B- and T-cell function. Minocycline may also inhibit cell death pathways by reducing both proapoptotic and proinflammatory enzyme activation. We sought to investigate the safety and efficacy of minocycline among patients with PSC.We evaluated the efficacy of minocycline in patients with PSC in a pilot study. Sixteen patients with PSC were enrolled. Minocycline, 100 mg orally twice daily, was given for 1 year.A statistically significant improvement in serum alkaline phosphatase activity (330 U/l vs. 265 U/l, P=0.04) and Mayo risk score (0.55 vs. 0.02, P=0.05) occurred with treatment. Serum bilirubin and albumin remained essentially unchanged while on treatment.The results of this pilot study indicate that minocycline is reasonably well tolerated and potentially effective in patients with PSC. These findings might be explained by the anti-inflammatory and antiapoptotic properties of minocycline. Though the data presented are too preliminary to support the clinical use of minocycline in the treatment of PSC at this time, its use should be further investigated.

View details for DOI 10.1038/ajg.2008.14

View details for Web of Science ID 000262265800016

View details for PubMedID 19098854
Adenosine Induces Loss of Actin Stress Fibers and Inhibits Contraction in Hepatic Stellate Cells via Rho Inhibition HEPATOLOGY Sohail, M. A., Hashmi, A. Z., Hakim, W., Watanabe, A., Zipprich, A., Groszmann, R. J., Dranoff, J. A., Torok, N. J., Mehal, W. Z. 2009; 49 (1): 185-194
Abstract

The Rho/ROCK pathway is activated in differentiated hepatic stellate cells (HSCs) and is necessary for assembly of actin stress fibers, contractility, and chemotaxis. Despite the importance of this pathway in HSC biology, physiological inhibitors of the Rho/ROCK pathway in HSCs are not known. We demonstrate that adenosine induces loss of actin stress fibers in the LX-2 cell line and primary HSCs in a manner indistinguishable from Rho/ROCK inhibition. Loss of actin stress fibers occurs via the A2a receptor at adenosine concentrations above 10 muM, which are present during tissue injury. We further demonstrate that loss of actin stress fibers is due to a cyclic adenosine monophosphate, protein kinase A-mediated pathway that results in Rho inhibition. Furthermore, a constitutively active Rho construct can inhibit the ability of adenosine to induce loss of actin stress fibers. Actin stress fibers are required for HSC contraction, and we demonstrate that adenosine inhibits endothelin-1 and lysophosphatidic acid-mediated HSC contraction. We propose that adenosine is a physiological inhibitor of the Rho pathway in HSCs with functional consequences, including loss of HSC contraction.

View details for DOI 10.1002/hep.22589

View details for Web of Science ID 000262127400023

View details for PubMedID 18844235

View details for PubMedCentralID PMC3129263
Leptin Induces Phagocytosis of Apoptotic Bodies by Hepatic Stellate Cells via a Rho Guanosine Triphosphatase-Dependent Mechanism HEPATOLOGY Jiang, J. X., Mikami, K., Shah, V. H., Torok, N. J. 2008; 48 (5): 1497?1505
Abstract

Leptin, a profibrogenic cytokine, plays an important role in the development of non-alcoholic steatohepatitis. Leptin also regulates immune responses, including macrophage phagocytic activity. Stellate cells are key elements in liver fibrogenesis, and previously we have demonstrated that phagocytosis of apoptotic bodies by stellate cells is profibrogenic. To study the effects of leptin on the phagocytic activity of hepatic stellate cells, we exposed both LX-2 cells and primary stellate cells to leptin, and we have observed increased phagocytic activity. In stellate cells isolated from Zucker (fa/fa) rats, the rate of phagocytosis was significantly decreased. To investigate the mechanism by which leptin induces phagocytosis, we focused on the role of Rho-guanosine triphosphate (GTP)-ases. We found that leptin induced the PI3K-dependent activation of Rac1, and that nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase activation was also implicated in this process. Leptin also induced RhoA activation and translocation to the phagosomes. Expression of the constitutive active Rac1 and RhoA both increased the phagocytic rate, whereas inhibition of the Rho-dependent kinase decreased the phagocytic activity.We describe a novel role of leptin in the fibrogenic process, the induction of phagocytosis of apoptotic bodies by hepatic stellate cells. The data provide strong evidence of a Rho-GTPase-mediated regulation of the cytoskeleton during stellate cell phagocytosis. Leptin-mediated phagocytic activity of stellate cells therefore could be an important mechanism responsible for progression of fibrosis in non-alcoholic steatohepatitis.

View details for DOI 10.1002/hep.22515

View details for Web of Science ID 000260596600019

View details for PubMedID 18925608

View details for PubMedCentralID PMC2596754
SOLUBLE LEPTIN RECEPTOR CORRELATES WITH THE STATE OF INSULIN RESISTANCE AND ADVANCED FIBROSIS IN NAFLD/NASH Medici, V., Aoki, C., Ali, M., Jiang, J., Havel, P. J., Seo, S., Ramsamooj, R., Torok, N. JOHN WILEY & SONS INC. 2008: 520A?521A
View details for Web of Science ID 000259757400470
Adiponectin decreases C-reactive protein synthesis and secretion from endothelial cells - Evidence for an adipose tissue-vascular loop ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY Devaraj, S., Torok, N., Dasu, M. R., Samols, D., Jialal, I. 2008; 28 (7): 1368?74
Abstract

Inflammation is pivotal in atherosclerosis. C-reactive protein (CRP), in addition to being a cardiovascular risk marker, may also be proatherogenic. We have previously shown that in addition to the liver, human aortic endothelial cells (HAECs) synthesize and secrete CRP. Whereas CRP levels are increased in obesity, metabolic syndrome, and diabetes, levels of adiponectin are reduced in these conditions. We tested the hypothesis that adiponectin reduces CRP synthesis and secretion in HAECs under normoglycemic (5.5 mmol/L glucose) and hyperglycemic conditions (15 mmol/L glucose).Adiponectin dose-dependently reduced CRP mRNA and protein from HAECs. Adiponectin treatment of HAECs significantly decreased IkappaB phosphorylation and NFkappaB binding activity. There was no effect of adiponectin on STAT or C/EBP transcriptional activity. Adiponectin also activated AMP kinase resulting in decreased NFkappaB activity and decreased CRP mRNA and protein. These effects of adiponectin were mimicked by AICAR, an activator of AMPK, and reversed by inhibition of AMPK. Thus, adiponectin reduces CRP synthesis and secretion from HAECs under hyperglycemia via upregulation of AMP kinase and downregulation of NFkappaB. Similar findings were observed in rat primary hepatocytes.Thus, in obesity and diabetes, the hypoadiponectinemia could exacerbate the proinflammatory state by inducing CRP production.

View details for DOI 10.1161/ATVBAHA.108.163303

View details for Web of Science ID 000256890400029

View details for PubMedID 18451326

View details for PubMedCentralID PMC2771588
Nonalcoholic steatohepatitis and the metabolic syndrome. Metabolic syndrome and related disorders Jiang, J., Torok, N. 2008; 6 (1): 1-7
Abstract

Nonalcoholic fatty liver disease (NAFLD) is a major form of chronic liver disease in adults and children. It is one of the consequences of the current obesity epidemic, and can progress to nonalcoholic steatohepatitis (NASH), characterized by steatosis, inflammation, and progressive fibrosis, ultimately leading to cirrhosis and end-stage liver disease. The factors implicated in this progression are poorly understood. NASH is closely associated with obesity and the metabolic syndrome. Recent studies emphasize the role of insulin resistance, oxidative stress, lipid peroxidation, and cytokine release in the development of NASH. This review summarizes the current knowledge on the etiology and pathomechanism of NASH and the role of the metabolic syndrome in NASH development.

View details for DOI 10.1089/met.2007.0026

View details for PubMedID 18370830
Recent advances in the pathogenesis and diagnosis of liver fibrosis JOURNAL OF GASTROENTEROLOGY Torok, N. J. 2008; 43 (5): 315?21
View details for DOI 10.1007/s00535-008-2181-x

View details for Web of Science ID 000256313300001

View details for PubMedID 18592147
Apoptotic cell death takes its toll HEPATOLOGY Torok, N. J. 2007; 46 (5): 1323-1325
View details for DOI 10.1002/hep.21968

View details for Web of Science ID 000250701200003

View details for PubMedID 17969040
Phagocytosis of apoptotic bodies by hepatic stellate cells induces NADPH oxidase and is associated with liver fibrosis in vivo HEPATOLOGY Zhan, S. S., jiang, J. X., Wu, J., Halsted, C., Friedman, S. L., Zern, M. A., Torok, N. J. 2006; 43 (3): 435-443
Abstract

Hepatic stellate cell activation is a main feature of liver fibrogenesis. We have previously shown that phagocytosis of apoptotic bodies by stellate cells induces procollagen alpha1 (I) and transforming growth factor beta (TGF-beta) expression in vitro. Here we have further investigated the downstream effects of phagocytosis by studying NADPH oxidase activation and its link to procollagen alpha1 (I) and TGF-beta1 expression in an immortalized human stellate cell line and in several models of liver fibrosis. Phagocytosis of apoptotic bodies in LX-1 cells significantly increased superoxide production both in the extracellular and intracellular milieus. By confocal microscopy of LX-1 cells, increased intracellular reactive oxygen species (ROS) were detected in the cells with intracellular apoptotic bodies, and immunohistochemistry documented translocation of the NADPH oxidase p47phox subunit to the membrane. NADPH oxidase activation resulted in upregulation of procollagen alpha1 (I); in contrast, TGF-beta1 expression was independent of NADPH oxidase activation. This was also confirmed by using siRNA to inhibit TGF-beta1 production. In addition, with EM studies we showed that phagocytosis of apoptotic bodies by stellate cells occurs in vivo. In conclusion, these data provide a mechanistic link between phagocytosis of apoptotic bodies, production of oxidative radicals, and the activation of hepatic stellate cells.

View details for DOI 10.1002/hep.21093

View details for Web of Science ID 000235911200009

View details for PubMedID 16496318
Apoptotic body engulfment by a human stellate cell line is profibrogenic LABORATORY INVESTIGATION Canbay, A., Taimr, P., Torok, N., Higuchi, H., Friedman, S., Gores, G. J. 2003; 83 (5): 655-663
Abstract

Hepatocyte apoptosis and stellate cell activation are both features of chronic liver diseases, but a relationship between these events has not been explored. In macrophages, engulfment of apoptotic bodies induces expression of transforming growth factor-beta (TGF-beta), a profibrogenic cytokine. We examined whether a similar response occurs in stellate cells. Fluorescently labeled hepatocyte apoptotic bodies were added to cultures of primary and immortalized human stellate cells. Stellate cells, but not hepatocytes, readily engulfed apoptotic bodies in a time-dependent manner as assessed by confocal microscopy. The activation of primary and immortalized human stellate cells after incubation with apoptotic bodies, as well as their fibrogenic activity, was indicated by an increase in alpha-smooth muscle actin (primary cells), TGF-beta1, and collagen alpha1(I) mRNA (primary and immortalized cells). The profibrogenic response was dependent upon apoptotic body engulfment, because nocodazole, a microtubule-inhibiting agent, blocked both the engulfment and the increase of TGF-beta1 and collagen alpha1(I) mRNA. As described in primary rodent stellate cells, up-regulation of collagen alpha1(I) mRNA was inhibited by a PI-3K inhibitor (LY294002) and a p38 mitogen-activated protein kinase inhibitor (SB203580) in LX-1 cells. In conclusion, these data support a model in which engulfment of hepatocyte apoptotic bodies by stellate cells leads to a fibrogenic response by eliciting a kinase-signaling pathway.

View details for DOI 10.1097/01.LAB.0000069036.63405.5C

View details for Web of Science ID 000183114700006

View details for PubMedID 12746475
Nitric oxide inhibits apoptosis downstream of cytochrome c release by nitrosylating caspase 9 CANCER RESEARCH Torok, N. J., Higuchi, H., Bronk, S., Gores, G. J. 2002; 62 (6): 1648-1653
Abstract

Inhibition of the mitochondrial pathway of apoptosis has been implicated as a mechanism contributing to carcinogenesis. Chronic inflammation, which is accompanied by activation of inducible nitric oxide synthase and generation of nitric oxide (NO), is associated with cancer development in a variety of gastrointestinal diseases, including cholangiocarcinoma. Therefore, we examined the effects of NO on the mitochondrial pathway of apoptosis in human cholangiocarcinoma cell lines. Transfection with inducible NO synthase inhibited etoposide-induced apoptosis. S-Nitroso-N-acetyl-D,L-penicillamine (SNAP), a pharmacological NO donor, did not prevent mitochondrial cytochrome c release as assessed by immunoblot analysis or cellular localization of cytochrome c-green fluorescent protein. In contrast, SNAP did prevent activation of caspase 9 in etoposide-treated cells. Furthermore, SNAP also blocked caspase 9 activation in a cell-free system and reversibly inhibited catalytic activity of human recombinant caspase 9. As assessed by the Saveille reaction, immunoprecipitated procaspase 9 from SNAP-treated cells released 6-fold more NO than untreated cells, confirming that cellular procaspase 9 is susceptible to nitrosylation. In conclusion, NO inhibits apoptosis downstream of cytochrome c release by directly blocking caspase 9 activation.

View details for Web of Science ID 000174560200012

View details for PubMedID 11912135
Alterations in vesicle transport and cell polarity in rat hepatocytes subjected to mechanical or chemical cholestasis GASTROENTEROLOGY Torok, N. J., Larusso, E. M., McNiven, M. A. 2001; 121 (5): 1176-1184
Abstract

The molecular mechanisms that contribute to the cholestatic condition in hepatocytes are poorly defined. It has been postulated that a disruption of normal vesicle-based protein trafficking may lead to alterations in hepatocyte polarity.To determine if vesicle motility is reduced by cholestasis, hepatocytes cultured from livers of bile duct ligation (BDL)- or ethinyl estradiol (EE)-injected rats, were viewed and recorded by high-resolution video microscopy. Cholestatic hepatocytes were analyzed by phalloidin staining and electron microscopy. Functional analysis was done by the sodium fluorescein sequestration assay.In cholestatic hepatocytes, there was a significant decrease in the number of motile cytoplasmic vesicles observed compared with control cells. Further examination of cells from BDL- or EE-treated livers revealed the presence of numerous large intracellular lumina. More than 24% of cells in BDL-treated livers and 19% of cells in EE-treated livers displayed these structures, compared with 1.1% found in control hepatocytes. Phalloidin staining of hepatocytes showed a prominent sheath of actin surrounding the lumina, reminiscent of those seen about bile canaliculi. Electron microscopy revealed that these structures were lined by actin-filled microvilli. Further, these pseudocanaliculi perform many of the functions exhibited by bona fide canaliculi, such as sequestering sodium fluorescein.Both mechanically and chemically induced cholestasis have substantial effects on vesicle-based transport, leading to marked disruption of hepatocellular polarity.

View details for Web of Science ID 000171890100022

View details for PubMedID 11677210
Cholangiocarcinoma. Seminars in gastrointestinal disease Torok, N., Gores, G. J. 2001; 12 (2): 125-132
Abstract

Chronic inflammatory disease of the biliary tract predispose to the development of cholangiocarcinoma. For example, the prevalence of cholangiocarcinoma is between 7% to 14% in patients with primary sclerosing cholangitis. The diagnosis of cholangiocarcinoma is challenging because it is difficult to distinguish benign from malignant strictures. Recently, several advances have helped in the diagnosis of cholangiocarcinoma. The serum Ca 19-9 value is a useful adjunct for the diagnosis of malignant stictures in the absence of bacterial cholangitis. The recent development of digital image analysis for assessing biopsy specimens and brush cytology is also useful for the diagnosis of malignant strictures. Positron emission tomography is a new imaging technique that uses 18F fluoro-2-deoxy-D-glucose to noninvasively assess metabolism in human tissues. Early studies suggest that this technique is sensitive in identifying small bile duct cancers. Thus, the combination of a Ca 19-9 value, digital image analysis, and positron emission tomography scanning have greatly helped in the differential diagnosis of benign from malignant strictures. Management of cholangiocarcinoma is also challenging with limited survival after surgical resection. Recently, we have shown that preoperative chemoirradiation followed by liver transplantation results in prolonged disease-free survival in highly selected patients with early bile duct cancers. Successful treatment outcome of these patients highlights the need for an early diagnosis of cholangiocarcinoma using the approaches described above.

View details for PubMedID 11352119
Vesicle movement in rat hepatocytes is reduced by ethanol exposure: Alterations in microtubule-based motor enzymes GASTROENTEROLOGY Torok, N., Marks, D., Hsiao, K., Oswald, B. J., McNiven, M. A. 1997; 113 (6): 1938-1948
Abstract

Ethanol is known to alter vesicle-mediated protein trafficking in hepatocytes by undefined mechanisms. In this study, the effects of long- and short-term ethanol exposure on vesicle movements were measured in isolated hepatocytes, and alterations in the function of the microtubule-associated motor enzymes dynamin, kinesin, and dynein, which are believed to support the transport and/or budding of vesicles along microtubules, were tested.Vesicular movements in isolated hepatocytes exposed to short- and long-term ethanol treatment were measured. Motor adenosine triphosphatase activities and their association with specific membrane organelles were assessed in response to long-term administration of ethanol in vivo or acetaldehyde in vitro.Hepatocytes exposed to short- or long-term ethanol treatment showed a significant reduction in the number of motile vesicles. No alterations in the levels of motor messenger RNA, protein, or enzymatic activity were observed. Interestingly, ethanol had no effect on the association of dynein and kinesin with membranes, whereas there was a significant increase in the amount of dynamin associated specifically with Golgi membranes.Long- and short-term ethanol exposure markedly reduces hepatocellular vesicle transport by a mechanism apparently independent of any alteration in the enzymatic activity of molecular motors, possibly involving a change in the function of dynamin.

View details for Web of Science ID A1997YH82900019

View details for PubMedID 9394734
Upregulation of molecular motor-encoding genes during hepatocyte growth factor- and epidermal growth factor-induced cell motility JOURNAL OF CELLULAR PHYSIOLOGY Torok, N., Urrutia, R., Nakamura, T., McNiven, M. A. 1996; 167 (3): 422-433
Abstract

Hepatocyte growth factor (HGF) and epidermal growth factor (EGF) are known to stimulate the locomotion of epithelial cells in culture. However, the molecular mechanisms which mediate these important changes are poorly understood. Here we have determined the effects of HGF and EGF on hepatocyte morphology, cytoskeletal organization, and the expression of molecular motor-encoding genes. Primary cultures of hepatocytes were treated with 10 ng/ml of HGF or EGF and observed with phase and fluorescence microscopy at 10, 24, and 48 h after treatment. We found that, over time, treated cells spread and became elongated after 24 h of treatment while forming long processes with dramatic alterations in the microtubule and actin cytoskeletons by 48 h. Quantitative Northern blot analysis was performed to measure expression of cytoskeletal-(beta-actin, alpha-tubulin) and molecular motor-(dynein, kinesin, and myosin I alpha and II) encoding genes which may contribute to this change in form. We observed the highest increase in levels of expression for myosin II (3.3-fold), kinesin (2.7-fold), myosin I alpha (2.2-fold), and alpha-tubulin (1.9-fold) after only 2 h of treatment with HGF. In contrast, EGF upregulated the expression of myosin I alpha (2.4-fold), kinesin (1.5-fold), and dynein (1.5-fold) at 10 h. The expression of the beta-actin gene remained constant in HGF-treated cells, while EGF induced a slight upregulation after 10 h of treatment. These results show for the first time that a selective upregulation of molecular motor-encoding genes correlates with alterations in cell shape and motility induced by HGF and EGF.

View details for Web of Science ID A1996UL40400006

View details for PubMedID 8655596
Keratin 19 as a biochemical marker of skin stem cells in vivo and in vitro: Keratin 19 expressing cells are differentially localized in function of anatomic sites, and their number varies with donor age and culture stage JOURNAL OF CELL SCIENCE Michel, M., Torok, N., Godbout, M. J., Lussier, M., GAUDREAU, P., Royal, A., GERMAIN, L. 1996; 109: 1017-1028
Abstract

This study was undertaken to evaluate keratin 19 (K19) as a biochemical marker for skin stem cells in order to address some long standing questions concerning these cells in the field of cutaneous biology. We first used the well-established mouse model enabling us to identify skin stem cells as [3H]thymidine-label-retaining cells. A site directed antibody was raised against a synthetic peptide of K19. It reacted specifically with a 40 kDa protein (K19) on immunoblotting. It labelled the bulge area of the outer root sheath on mouse skin by immunohistochemistry. Double-labelling revealed that K19-positive-cells were also [3H]thymidine-label-retaining cells, suggesting that K19 is a marker for skin stem cells of hair follicles. K19-expression was then used to investigate the variation in mouse and human skin stem cells as a function of body site, donor age and culture time. K19 was expressed in the hair follicle and absent from the interfollicular epidermis at hairy sites (except for some K18 coexpressing Merkel cells). In contrast, at glabrous sites, K19-positive-cells were in deep epidermal rete ridges. K19 expressing cells also contained high levels of alpha 3 beta 1 integrin. The proportion of K19-positive-cells was greater in newborn than older foreskins. This correlated with keratinocyte culture lifespan variation with donor age. Moreover, it could explain clinical observations that children heal faster than adults. In conclusion, K19 expression in skin provides an additional tool to allow further characterization of skin stem cells under normal and pathological conditions in situ and in vitro.

View details for Web of Science ID A1996UN10000013

View details for PubMedID 8743949
Retinitis pigmentosa with special reference to otologic, neurologic, and endocrine complications. Acta ophthalmologica KJERRUMGAARD, E. 1948; 26 (1): 55-65
View details for PubMedID 18861149

Natalie Torok

Professor of Medicine (Gastroenterology and Hepatology) at the Stanford University Medical Center

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