Tung Ming Leung

CYP2E1 and oxidant stress in alcoholic and non-alcoholic fatty liver disease

Alcoholic (ALD) and non-alcoholic fatty liver diseases (NAFLD) are clinical conditions leading to hepatocellular injury and inflammation resulting from alcohol consumption, high fat diet, obesity and diabetes, among others. Oxidant stress is a major contributing factor to the pathogenesis of ALD and NAFLD. Multiple studies have shown that generation of reactive oxygen species (ROS) is key for the progression of fatty liver to steatohepatitis. Cytochrome P450 2E1 (CYP2E1) plays a critical role in ROS generation and CYP2E1 is also induced by alcohol itself. This review summarizes the role of CYP2E1 in ALD and NAFLD.

OSTEOPONTIN, AN OXIDANT STRESS-SENSITIVE CYTOKINE, UP-REGULATES COLLAGEN-I VIA INTEGRIN αVβ3 ENGAGEMENT AND PI3K-pAkt-NFκB SIGNALING

A key feature in the pathogenesis of liver fibrosis is fibrillar Collagen‐I deposition; yet, mediators that could be key therapeutic targets remain elusive. We hypothesized that osteopontin (OPN), an extracellular matrix (ECM) cytokine expressed in hepatic stellate cells (HSCs), could drive fibrogenesis by modulating the HSC pro‐fibrogenic phenotype and Collagen‐I expression. Recombinant OPN (rOPN) up‐regulated Collagen‐I protein in primary HSCs in a transforming growth factor beta (TGFβ)–independent fashion, whereas it down‐regulated matrix metalloprotease‐13 (MMP13), thus favoring scarring. rOPN activated primary HSCs, confirmed by increased α‐smooth muscle actin (αSMA) expression and enhanced their invasive and wound‐healing potential. HSCs isolated from wild‐type (WT) mice were more profibrogenic than those from OPN knockout (Opn−/−) mice and infection of primary HSCs with an Ad‐OPN increased Collagen‐I, indicating correlation between both proteins. OPN induction of Collagen‐I occurred via integrin αvβ3 engagement and activation of the phosphoinositide 3‐kinase/phosphorylated Akt/nuclear factor kappa B (PI3K/pAkt/NFκB)–signaling pathway, whereas cluster of differentiation 44 (CD44) binding and mammalian target of rapamycin/70‐kDa ribosomal protein S6 kinase (mTOR/p70S6K) were not involved. Neutralization of integrin αvβ3 prevented the OPN‐mediated activation of the PI3K/pAkt/NFκB–signaling cascade and Collagen‐I up‐regulation. Likewise, inhibition of PI3K and NFκB blocked the OPN‐mediated Collagen‐I increase. Hepatitis C Virus (HCV) cirrhotic patients showed coinduction of Collagen‐I and cleaved OPN compared to healthy individuals. Acute and chronic liver injury by CCl4 injection or thioacetamide (TAA) treatment elevated OPN expression. Reactive oxygen species up‐regulated OPN in vitro and in vivo and antioxidants prevented this effect. Transgenic mice overexpressing OPN in hepatocytes (OpnHEP Tg) mice developed spontaneous liver fibrosis compared to WT mice. Last, chronic CCl4 injection and TAA treatment caused more liver fibrosis to WT than to Opn−/− mice and the reverse occurred in OpnHEP Tg mice. Conclusion: OPN emerges as a key cytokine within the ECM protein network driving the increase in Collagen‐I protein contributing to scarring and liver fibrosis. (HEPATOLOGY 2012)

High Mobility Group Box-1 (HMGB1) Participates in the Pathogenesis of Alcoholic Liver Disease (ALD)

Background: HMGB1 is a proinflammatory cytokine produced in response to tissue injury, but its role in ALD is unknown. Results: HMGB1 increases; translocates; and undergoes acetylation, phosphorylation, and oxidation in ALD. HMGB1 ablation in hepatocytes protects against steatosis and injury in ALD. Conclusion: HMGB1 plays a key role in ALD. Significance: Dissecting how the increase in HMGB1 causes hepatotoxicity is key for understanding the pathogenesis of ALD. Growing clinical and experimental evidence suggests that sterile inflammation contributes to alcoholic liver disease (ALD). High mobility group box-1 (HMGB1) is highly induced during liver injury; however, a link between this alarmin and ALD has not been established. Thus, the aim of this work was to determine whether HMGB1 contributes to the pathogenesis of ALD. Liver biopsies from patients with ALD showed a robust increase in HMGB1 expression and translocation, which correlated with disease stage, compared with healthy explants. Similar findings were observed in chronic ethanol-fed wild-type (WT) mice. Using primary cell culture, we validated the ability of hepatocytes from ethanol-fed mice to secrete a large amount of HMGB1. Secretion was time- and dose-dependent and responsive to prooxidants and antioxidants. Selective ablation of Hmgb1 in hepatocytes protected mice from alcohol-induced liver injury due to increased carnitine palmitoyltransferase-1, phosphorylated 5′AMP-activated protein kinase-α, and phosphorylated peroxisome proliferator-activated receptor-α expression along with elevated LDL plus VLDL export. Native and post-translationally modified HMGB1 were detected in humans and mice with ALD. In liver and serum from control mice and in serum from healthy volunteers, the lysine residues within the peptides containing nuclear localization signals (NLSs) 1 and 2 were non-acetylated, and all cysteine residues were reduced. However, in livers from ethanol-fed mice, in addition to all thiol/non-acetylated isoforms of HMGB1, we observed acetylated NLS1 and NLS2, a unique phosphorylation site in serine 35, and an increase in oxidation of HMGB1 to the disulfide isoform. In serum from ethanol-fed mice and from patients with ALD, there was disulfide-bonded hyperacetylated HMGB1, disulfide-bonded non-acetylated HMGB1, and HMGB1 phosphorylated in serine 35. Hepatocytes appeared to be a major source of these HMGB1 isoforms. Thus, hepatocyte HMGB1 participates in the pathogenesis of ALD and undergoes post-translational modifications (PTMs) that could condition its toxic effects.

Osteopontin induces ductular reaction contributing to liver fibrosis

Objective In human chronic liver disease, there is association between ductular reaction (DR) and fibrosis; yet, the mechanism triggering its onset and its role in scar formation remains unknown. Since we previously showed that osteopontin (OPN) is highly induced during drug-induced liver fibrosis, we hypothesised that OPN could drive oval cells (OC) expansion and DR and signal to hepatic stellate cells (HSC) to promote scarring. Results In vivo studies demonstrated increased OPN expression in biliary epithelial cells (BEC) and in OC in thioacetamide (TAA)-treated mice. OPN ablation protected mice from TAA and bile duct ligation-induced liver injury, DR and scarring. This was associated with greater hepatocyte proliferation, lower OC expansion and DR along with less fibrosis, suggesting that OPN could activate the OC compartment to differentiate into BEC, which could then signal to HSC to enhance scarring. Since TAA-treated wild-type mice and cirrhotic patients showed TGF-β+ BEC, which were lacking in TAA-treated Opn−/− mice and in healthy human explants, this suggested that OPN could regulate TGF-β, a profibrogenic factor. In vitro experiments confirmed that recombinant OPN (rOPN) decreases hepatocyte proliferation and increases OC and BEC proliferation. To evaluate how BEC regulate collagen-I production in HSC, co-cultures were established. Co-cultured BEC upregulated OPN and TGF-β expression and enhanced collagen-I synthesis by HSC. Lastly, recombinant TGF-β (rTGFβ) and rOPN promoted BEC proliferation and neutralisation of OPN and TGF-β reduced collagen-I expression in co-cultured HSC. Conclusions OPN emerges as a key matricellular protein driving DR and contributing to scarring and liver fibrosis via TGF-β.

ARGININOSUCCINATE SYNTHASE CONDITIONS THE RESPONSE TO ACUTE AND CHRONIC ETHANOL-INDUCED LIVER INJURY IN MICE

Argininosuccinate synthase (ASS) is the rate‐limiting enzyme in both the urea and the L‐citrulline/nitric oxide (NO·) cycles regulating protein catabolism, ammonia levels, and NO· generation. Because a proteomics analysis identified ASS and nitric oxide synthase‐2 (NOS2) as coinduced in rat hepatocytes by chronic ethanol consumption, which also occurred in alcoholic liver disease (ALD) and in cirrhosis patients, we hypothesized that ASS could play a role in ethanol binge and chronic ethanol‐induced liver damage. To investigate the contribution of ASS to the pathophysiology of ALD, wildtype (WT) and Ass+/− mice (Ass−/− are lethal due to hyperammonemia) were exposed to an ethanol binge or to chronic ethanol drinking. Compared with WT, Ass+/− mice given an ethanol binge exhibited decreased steatosis, lower NOS2 induction, and less 3‐nitrotyrosine (3‐NT) protein residues, indicating that reducing nitrosative stress by way of the L‐citrulline/NO· pathway plays a significant role in preventing liver damage. However, chronic ethanol‐treated Ass+/− mice displayed enhanced liver injury compared with WT mice. This was due to hyperammonemia, lower phosphorylated AMP‐activated protein kinase alpha (pAMPKα) to total AMPKα ratio, decreased sirtuin‐1 (Sirt‐1) and peroxisomal proliferator‐activated receptor coactivator‐1α (Pgc1α) messenger RNAs (mRNAs), lower fatty acid β‐oxidation due to down‐regulation of carnitine palmitoyl transferase‐II (CPT‐II), decreased antioxidant defense, and elevated lipid peroxidation end‐products in spite of comparable nitrosative stress but likely reduced NOS3. Conclusion: Partial Ass ablation protects only in acute ethanol‐induced liver injury by decreasing nitrosative stress but not in a more chronic scenario where oxidative stress and impaired fatty acid β‐oxidation are key events. (HEPATOLOGY 2012)

Osteopontin binding to lipopolysaccharide lowers tumor necrosis factor-α and prevents early alcohol-induced liver injury in mice.

Although osteopontin (OPN) is induced in alcoholic patients, its role in the pathophysiology of alcoholic liver disease (ALD) remains unclear. Increased translocation of lipopolysaccharide (LPS) from the gut is key for the onset of ALD because it promotes macrophage infiltration and activation, tumor necrosis factor‐α (TNFα) production, and liver injury. Since OPN is protective for the intestinal mucosa, we postulated that enhancing OPN expression in the liver and consequently in the blood and/or in the gut could protect from early alcohol‐induced liver injury. Wild‐type (WT), OPN knockout (Opn−/−), and transgenic mice overexpressing OPN in hepatocytes (OpnHEPTg) were fed either the control or the ethanol Lieber‐DeCarli diet. Ethanol increased hepatic, plasma, biliary, and fecal OPN more in OpnHEPTg than in WT mice. Steatosis was less in ethanol‐treated OpnHEPTg mice as shown by decreased liver‐to‐body weight ratio, hepatic triglycerides, the steatosis score, oil red‐O staining, and lipid peroxidation. There was also less inflammation and liver injury as demonstrated by lower alanine aminotransferase (ALT) activity, hepatocyte ballooning degeneration, LPS levels, the inflammation score, and the number of macrophages and TNFα+ cells. To establish if OPN could limit LPS availability and its noxious effects in the liver, binding studies were performed. OPN showed binding affinity for LPS which prevented macrophage activation, reactive oxygen, and nitrogen species generation and TNFα production. Treatment with milk OPN (m‐OPN) blocked LPS translocation in vivo and protected from early alcohol‐induced liver injury. Conclusion: Natural induction plus forced overexpression of OPN in the liver or treatment with m‐OPN protect from early alcohol‐induced liver injury by blocking the gut‐derived LPS and TNFα effects in the liver. (Hepatology 2014;59:1600‐1616)

Osteopontin delays resolution of liver fibrosis.

To date, considerable progress has been made both in the mechanisms driving liver fibrosis and in the prevention of disease progression. Resolution of liver fibrosis is an emerging field in hepatology; yet, the mediators involved remain elusive. Earlier work from our laboratory demonstrated that the matricellular cytokine osteopontin (OPN) is pro-fibrogenic by promoting hepatic stellate cell (HSC) activation and extracellular matrix (ECM) deposition in vitro and in vivo and specifically by governing fibrillar collagen-I expression, the key pro-fibrogenic protein. Here we hypothesized that OPN could also delay the resolution of liver fibrosis by sustaining collagen-I synthesis or by preventing its degradation. To demonstrate this, wild-type (WT) and OPN-knockout (Opn−/−) mice were administered thioacetamide (TAA) in the drinking water for 4 months. Half of the mice were killed at 4 months to assess the extent of fibrosis at the peak of injury, and the rest of the mice were killed 2 months after TAA withdrawal to determine the rate of fibrosis resolution. Following TAA cessation, livers from Opn−/− mice showed no centrilobular and parenchymal necrosis along with faster ECM remodeling than WT mice. The latter was quantified by less fibrillar collagen-I immunostaining. Western blot analysis demonstrated a significant decrease in fibrillar collagen-I and in tissue inhibitor of metalloproteinase-1 (TIMP-1) in Opn−/− mice undergoing fibrosis resolution compared with WT mice. In conclusion, these results suggest that OPN delays liver fibrosis resolution due to sustained fibrillar collagen-I deposition; hence, inhibiting OPN could be an effective therapeutic strategy for resolving liver fibrosis.

GCN2 kinase is a key regulator of fibrogenesis and acute and chronic liver injury induced by carbon tetrachloride in mice

General control nonderepresible 2 (GCN2) is a highly conserved cytosolic kinase that modulates a complex response for coping with the stress owing to lack of amino acids. GCN2 has been recently shown to be involved in the regulation of metabolic balance and lipid degradation rate in the liver. We hypothesized that GCN2 could have a role in in hepatic fibrogenesis and in the response to acute or chronic liver injury. Activation of GCN2 in primary or immortalized human hepatic stellate cells by incubation with medium lacking the essential amino acid histidine correlated with decreased levels of collagen type I protein and mRNA, suggesting an antifibrogenic effect of GCN2. In vivo studies with Gcn2 knock-out mice (Gcn2−/−) showed increased susceptibility to both acute or chronic liver damage induced by CCl4, as shown by higher alanine aminotransferase and aspartate aminotransferase activities, increased necrosis and higher inflammatory infiltrates compared with wild-type mice (WT). Chronic CCl4 treatment increased deposition of interstitial collagen type I more in Gcn2−/− mice than in WT mice. Col1a1 and col1a2 mRNA levels also increased in CCl4-treated Gcn2−/− mice compared with WT mice. These results suggest that GCN2 is a key regulator of the fibrogenic response to liver injury.

Partial deletion of argininosuccinate synthase protects from pyrazole plus lipopolysaccharide-induced liver injury by decreasing nitrosative stress

Argininosuccinate synthase (ASS) is the rate-limiting enzyme in the urea cycle. Along with nitric oxide synthase (NOS)-2, ASS endows cells with the L-citrulline/nitric oxide (NO·) salvage pathway to continually supply L-arginine from L-citrulline for sustained NO· generation. Because of the relevant role of NOS in liver injury, we hypothesized that downregulation of ASS could decrease the availability of intracellular substrate for NO· synthesis by NOS-2 and, hence, decrease liver damage. Previous work demonstrated that pyrazole plus LPS caused significant liver injury involving NO· generation and formation of 3-nitrotyrosine protein adducts; thus, wild-type (WT) and Ass+/- mice (Ass+/+ mice are lethal) were treated with pyrazole plus LPS, and markers of nitrosative stress, as well as liver injury, were analyzed. Partial ablation of Ass protected from pyrazole plus LPS-induced liver injury by decreasing nitrosative stress and hepatic and circulating TNFα. Moreover, apoptosis was prevented, since pyrazole plus LPS-treated Ass+/- mice showed decreased phosphorylation of JNK; increased MAPK phosphatase-1, which is known to deactivate JNK signaling; and lower cleaved caspase-3 than treated WT mice, and this was accompanied by less TdT-mediated dUTP nick end labeling-positive staining. Lastly, hepatic neutrophil accumulation was almost absent in pyrazole plus LPS-treated Ass+/- compared with WT mice. Partial Ass ablation prevents pyrazole plus LPS-mediated liver injury by reducing nitrosative stress, TNFα, apoptosis, and neutrophil infiltration.

Alcoholic liver disease: from CYP2E1 to CYP2A5.

This article reviews recent studies on CYP2E1-mediated alcoholic liver injury, the induction of CYP2A5 by alcohol and the mechanism for this upregulation, especially the permissive role of CYP2E1 in the induction of CYP2A5 by alcohol and the CYP2E1-ROS-Nrf2 pathway, and protective effects of CYP2A5 against ethanol-induced oxidative liver injury. Ethanol can induce CYP2E1, an active generator of reactive oxygen species (ROS), and CYP2E1 is a contributing factor for alcoholinduced oxidative liver injury. CYP2A5, another isoform of cytochrome P450, can also be induced by ethanol. Chronic feeding of ethanol to wild type mice increased CYP2A5 catalytic activity, protein and mRNA levels as compared to pair-fed controls. This induction was blunted in CYP2E1 knockout (cyp2e1-/-) mice but was restored when human CYP2E1 was reintroduced and expressed in cyp2e1-/- mice. Ethanol-induced CYP2E1 co-localized with CYP2A5 and preceded the elevation of CYP2A5. The antioxidants N-acetyl cysteine and vitamin C lowered the alcohol elevation of ROS and blunted the alcohol induction of CYP2A5, but not CYP2E1, suggesting ROS play a novel role in the crosstalk between CYP2E1 and CYP2A5. The antioxidants blocked the activation of Nrf2, a transcription factor known to upregulate expression of CYP2A5. When alcohol-induced liver injury was enhanced in Nrf2 knockout (Nrf2-/-) mice, alcohol elevation of CYP2A5 but not CYP2E1 was also lower in Nrf2-/- mice. CYP2A5 knockout (cyp2a5-/-) mice exhibited an enhanced alcoholic liver injury compared with WT mice as indicated by serum ALT, steatosis and necroinflammation. Alcohol-induced hyperglycemia were observed in cyp2a5-/- mice but not in WT mice.