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XLS file - 42K, KEGG Pathway Categories of Differentially Up-Regulated Genes Following EHCC miRNA Expression in hPHs

PDF file - 1.3MB, S1. Pearson correlation analysis of hESCs, HCCs and hPHS. S2. EHCC miRNAs are not capable of rescuing hepatic progenitor cell induction in Dgcr8 null mESCs during the in vitro differentiation process of mESCs along a hepatic lineage. S3. EHCC miRNAs cannot rescue the deficiency in hepatocyte differentiation in Dgcr8 null mESCs. S4. Experimental timeline of hPHs. S5. Global gene expression analysis between hPHs and hPHs expressing EHCC miRNAs. S6. Expression patterns of genes reported to be associated with HCCs, ESCs and hepatic progenitor stem cells following EHCC miRNA overexpression in hPHs

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XLS file - 34K, KEGG Pathway Categories of Genes Highly Enriched in hESCs, HCCs and hPHs

Background and aims Older patients with obesity/type II diabetes mellitus frequently present with advanced 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. Approach and results Shc expression increased in livers of older patients with NASH, as assessed by real time quantitative PCR (RT-qPCR) or western blots. Fibrosis, Shc expression, markers of senescence, and nicotinamide adenine dinucleotide phosphate, reduced form oxidases (NOXs) were studied in young/old mice on fast food diet (FFD). To inhibit Shc in old mice, lentiviral (LV)-short hairpin Shc versus control-LV were used during FFD. For hepatocyte-specific effects, floxed (fl/fl) Shc mice on FFD were injected with adeno-associated virus 8-thyroxine-binding globulin-Cre-recombinase versus 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 coimmunoprecipitations. Palmitate-induced p52Shc binding to p47phox , activating the NOX2 complex, more so at an older age. Kinetics of binding were assessed in Src homology 2 domain (SH2) or phosphotyrosine-binding (PTB) domain 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. Conclusions Accelerated fibrosis in the aged is modulated by p52Shc/NOX2. We show a 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.

Background & aimsSrc homology and collagen (Shc) proteins are major adapters to extracellular signals, however, the regulatory role of Shc isoforms in sterile inflammatory responses in alcoholic hepatitis (AH) has not been fully investigated. We hypothesized that in an isoform-specific manner Shc modulates pre-apoptotic signals, calreticulin (CRT) membrane exposure, and recruitment of inflammatory cells.MethodsLiver biopsy samples from patients with AH vs healthy subjects were studied for Shc expression using DNA microarray data and immunohistochemistry. Shc knockdown (hypomorph) and age-matched wild-type mice were pair-fed according to the chronic-plus-binge alcohol diet. To analyze hepatocyte-specific effects, adeno-associated virus 8-thyroxine binding globulin-Cre (hepatocyte-specific Shc knockout)-mediated deletion was performed in flox/flox Shc mice. Lipid peroxidation, proinflammatory signals, redox radicals, reduced nicotinamide adenine dinucleotide/oxidized nicotinamide adenine dinucleotide ratio, as well as cleaved caspase 8, B-cell-receptor-associated protein 31 (BAP31), Bcl-2-associated X protein (Bax), and Bcl-2 homologous antagonist killer (Bak), were assessed invivo. CRT translocation was studied in ethanol-exposed p46ShcẟSH2-transfected hepatocytes by membrane biotinylation in conjunction with phosphorylated-eukaryotic initiation factor 2 alpha, BAP31, caspase 8, and Bax/Bak. The effects of idebenone, a novel Shc inhibitor, was studied in alcohol/pair-fed mice.ResultsShc was significantly induced in patients with AH (P< .01). Alanine aminotransferase, reduced nicotinamide adenine dinucleotide/oxidized nicotinamide adenine dinucleotide ratios, production of redox radicals, and lipid peroxidation improved (P < .05), and interleukin 1β, monocyte chemoattractant protein 1, and C-X-C chemokine ligand 10 were reduced in Shc knockdown and hepatocyte-specific Shc knockout mice. Invivo, Shc-dependent induction, and, in hepatocytes, a p46Shc-dependent increase in pre-apoptotic proteins Bax/Bak, caspase 8, BAP31 cleavage, and membrane translocation of CRT/endoplasmic reticulum-resident protein 57 were seen. Idebenone protected against alcohol-mediated liver injury.ConclusionsAlcohol induces p46Shc-dependent activation of pre-apoptotic pathways and translocation of CRT to the membrane, where it acts as a damage-associated molecular pattern, instigating immunogenicity. Shc inhibition could be a novel treatment strategy in AH.

Shc expression rises in human nonalcoholic steatohepatitis (NASH) livers, and Shc-deficient mice are protected from NASH–thus Shc inhibition could be a novel therapeutic strategy for NASH. Idebenone was recently identified as the first small-molecule Shc inhibitor drug. We tested idebenone in the fibrotic methionine-choline deficient (MCD) diet and the metabolic fast food diet (FFD) mouse models of NASH. In the fibrotic MCD NASH model, idebenone reduced Shc expression and phosphorylation in peripheral blood mononuclear cells and Shc expression in the liver; decreased serum alanine aminotransferase and aspartate aminotransferase; and attenuated liver fibrosis as observed by quantitative polymerase chain reaction (qPCR) and hydroxyproline quantification. In the metabolic FFD model, idebenone administration improved insulin resistance, and reduced inflammation and fibrosis shown with qPCR, hydroxyproline measurement, and histology. Thus, idebenone ameliorates NASH in two mouse models. As an approved drug with a benign safety profile, Idebenone could be a reasonable human NASH therapy.

The cardiac glycoside digoxin was identified as a potent suppressor of pyruvate kinase isoform 2-hypoxia-inducible factor-α (PKM2-HIF-1α) pathway activation in liver injury mouse models via intraperitoneal injection. We have assessed the therapeutic effects of digoxin to reduce nonalcoholic steatohepatitis (NASH) by the clinically relevant oral route 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 wk and started concurrently with the gavage of digoxin (2.5, 0.5, 0.125 mg/kg twice a week) for 5 wk. 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-1α sustained NLRP3 inflammasome activation in macrophages. We have reported for the first time that PKM2 is upregulated 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-1α transactivation without affecting PKM2 enzyme activation. Thus, oral digoxin showed potential to therapeutically inhibit liver injury in NASH through the regulation of PKM2-HIF-1α pathway activation with involvement of multiple cell types. Because of the large clinical experience with oral digoxin, this may have significant clinical applicability in human NASH. NEW & NOTEWORTHY This study is the first to assess the therapeutic efficacy of oral digoxin on nonalcoholic steatohepatitis (NASH) in a high-fat diet (HFD) mouse model and to determine the divergent of cell type-specific effects. Oral digoxin reduced liver damage, steatosis, and inflammation in HFD mice. Digoxin attenuated hypoxia-inducible factor (HIF)-1α axis-sustained inflammasome activity in macrophages and hepatic oxidative stress response in hepatocytes via the regulation of PKM2-HIF-1α axis pathway activation. Oral digoxin may have significant clinical applicability in human NASH.

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.

Saturated fatty acids (SFAs) (the "bad" fat), especially palmitate (PA), in the human diet are blamed for potential health risks such as obesity and cancer because of SFA-induced lipotoxicity. However, epidemiological results demonstrate a latent benefit of SFAs, and it remains elusive whether a certain low level of SFAs is physiologically essential for maintaining cell metabolic hemostasis. Here, we demonstrate that although high-level PA (HPA) indeed induces lipotoxic effects in liver cells, low-level PA (LPA) increases mitochondrial functions and alleviates the injuries induced by HPA or hepatoxic agent carbon tetrachloride (CCl4). LPA treatment in mice enhanced liver mitochondrial activity and reduced CCl4 hepatotoxicity with improved blood levels of aspartate aminotransferase (AST), alanine transaminase (ALT), and mitochondrial aspartate transaminase (m-AST). LPA-mediated mitochondrial homeostasis is regulated by CDK1-mediated SIRT3 phosphorylation, which in turn deacetylates and dimerizes CPT2 to enhance fatty acid oxidation. Thus, an advantageous effect is suggested by the consumption of LPA that augments mitochondrial metabolic homeostasis via CDK1-SIRT3-CPT2 cascade.

Liver is a unique organ in displaying a reparative and regenerative response after acute/chronic damage or partial hepatectomy, when all the cell types must proliferate to re-establish the liver mass. The NADPH oxidase NOX4 mediates Transforming Growth Factor-beta (TGF-β) actions, including apoptosis in hepatocytes and activation of stellate cells to myofibroblasts. Aim of this work was to analyze the impact of NOX4 in liver regeneration by using two mouse models where Nox4 was deleted: 1) general deletion of Nox4 (NOX4−/−) and 2) hepatocyte-specific deletion of Nox4 (NOX4hepKO). Liver regeneration was analyzed after 2/3 partial hepatectomy (PH). Results indicated an earlier recovery of the liver-to-body weight ratio in both NOX4−/− and NOX4hepKO mice and an increased survival, when compared to corresponding WT mice. The regenerative hepatocellular fat accumulation and the parenchyma organization recovered faster in NOX4 deleted livers. Hepatocyte proliferation, analyzed by Ki67 and phospho-Histone3 immunohistochemistry, was accelerated and increased in NOX4 deleted mice, coincident with an earlier and increased Myc expression. Primary hepatocytes isolated from NOX4 deleted mice showed higher proliferative capacity and increased expression of Myc and different cyclins in response to serum. Transcriptomic analysis through RNA-seq revealed significant changes after PH in NOX4−/− mice and support a relevant role for Myc in a node of regulation of proliferation-related genes. Interestingly, RNA-seq also revealed changes in the expression of genes related to activation of the TGF-β pathway. In fact, levels of active TGF-β1, phosphorylation of Smads and levels of its target p21 were lower at 24 h in NOX4 deleted mice. Nox4 did not appear to be essential for the termination of liver regeneration in vivo, neither for the in vitro hepatocyte response to TGF-β1 in terms of growth inhibition, which suggest its potential as therapeutic target to improve liver regeneration, without adverse effects., Graphical abstract Image 1, Highlights • General or hepatocyte-specific Nox4 deletion accelerates mice liver regeneration. • Increased hepatocyte proliferation is observed, coincident with higher Myc expression. • RNA-seq analysis reveals a role for Myc in a node of regulation of gene expression. • Transcriptional and functional attenuation of the TGF-β1 pathway is observed in vivo. • In vitro, Nox4 deleted hepatocytes maintain the growth inhibitory response to TGF-β1.

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.

Background & Aims Hepatic infiltration of neutrophils is a hallmark of steatohepatitis; however, the role of neutrophils in the progression of steatohepatitis remains unknown. Methods 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. Results 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 p47phox 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. Conclusions 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)., Graphical abstract

Recruitment of inflammatory cells is a major feature of alcoholic liver injury however; the signals and cellular sources regulating this are not well defined. C-C chemokine receptor type 2 (CCR2) is expressed by active hepatic stellate cells (HSC) and is a key monocyte recruitment signal. Activated HSC are also important sources of hydrogen peroxide resulting from the activation of NADPH oxidase 4 (NOX4). As the role of this NOX in early alcoholic liver injury has not been addressed, we studied NOX4-mediated regulation of CCR2/CCL2 mRNA stability. NOX4 mRNA was significantly induced in patients with alcoholic liver injury, and was co-localized with αSMA-expressing activated HSC. We generated HSC-specific NOX4 KO mice and these were pair-fed on alcohol diet. Lipid peroxidation have not changed significantly however, the expression of CCR2, CCL2, Ly6C, TNFα, and IL-6 was significantly reduced in NOX4HSCKO compared to fl/fl mice. NOX4 promoter was induced in HSC by acetaldehyde treatment, and NOX4 has significantly increased mRNA half-life of CCR2 and CCL2 in conjunction with Ser221 phosphorylation and cytoplasmic shuttling of HuR. In conclusion, NOX4 is induced in early alcoholic liver injury and regulates CCR2/CCL2 mRNA stability thereby promoting recruitment of inflammatory cells and production of proinflammatory cytokines.

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-α)-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-α production as well as induction of fibrogenic transcripts. Ad-Timp3 injection resulted in a significant decline in TACE activity, procollagen α1 (I), alpha smooth muscle actin (α-SMA) and transforming growth factor beta (TGF-β) 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. Conclusion: TACE activation is central to the pathogenesis of NASH and is mediated by AGEs through NOX2 induction and down-regulation of Sirt1/Timp3 pathways. (Hepatology 2013;58:1339–1348)

UnlabelledHepatic 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-α) levels. Dnmt3b was down-regulated in tx-j mice together with global DNA hypomethylation. PCA treatment of tx-j mice reduced Tnf-α and ALT levels, betaine treatment increased S-adenosylmethionine and up-regulated Dnmt3b levels, and both treatments restored global DNA methylation levels.ConclusionReduced 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.

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-βRII 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-1β. 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-1β. 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-βRII/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., Torok, N. J., Jiang, J. X. Galectin-3 regulates inflammasome activation in cholestatic liver injury.

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 TGFβ 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. Mol Cancer Res; 10(7); 979–91. ©2012 AACR.

Hepatic stellate cells (HSC), the key fibrogenic cells of the liver, transdifferentiate into myofibroblasts upon phagocytosis of apoptotic hepatocytes. Galectin-3, a β-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-β1, and procollagen α1(I). Recombinant galectin-3 reversed this defect, suggesting that extracellular galectin-3 is required for HSC activation. Galectin-3 facilitated the αvβ3 heterodimer-dependent binding, indicating that galectin-3 modulates HSC phagocytosis via cross-linking this integrin and enhancing the tethering of apoptotic cells. Blocking integrin αvβ3 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-κB 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 α-smooth muscle actin and procollagen α1(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.

Lipotoxicity-associated diseases such as obesity and cancer are linked to mitochondrial dysfunction due to over digestion of saturated fatty acids (SFAs), especially palmitate (PA) in diet. However, the physiological level of SFAs consumption required for maintaining normal mitochondrial homeostasis and liver functions remains to be elucidated. Here, we demonstrate that, in contrast to the lipotoxic effects induced by high-level PA (HPA), low-level PA (LPA) improves mitochondrial functions which alleviate cellular injuries induced by HPA and CCl4. Mice treated with LPA boost the mitochondrial functions in liver and lessen CCl4-induced hepatotoxicity with improved blood levels of AST, ALT, and m-AST. Restitution of SIRT3 and CPT2 in CRISPR-edited cells demonstrates that LPA-activated mitochondrial function is initiated by SIRT3 that is activated by CDK1 to boost CPT2 activity and fatty acid oxidation. These results provide the evidence for an overlooked advantageous effect of SFAs in mitochondrial homeostasis mediated by a unique CDK1-SIRT3-CPT2 cascade.

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 α-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.

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. Conclusion: 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. (HEPATOLOGY 2008.)

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.

Oxidative stress is a common feature observed in a wide spectrum of chronic liver diseases including viral hepatitis, alcoholic, and nonalcoholic steatohepatitis. The nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs) are emerging as major sources of reactive oxygen species (ROS). Several major isoforms are expressed in the liver, including NOX1, NOX2, and NOX4. While the phagocytic NOX2 has been known to play an important role in Kupffer cell and neutrophil phagocytic activity and inflammation, the nonphagocytic NOX homologues are increasingly recognized as key enzymes in oxidative injury and wound healing. In this review, we will summarize the current advances in knowledge on the regulatory pathways of NOX activation, their cellular distribution, and their role in the modulation of redox signaling in liver diseases.

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 β-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-κB 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.

The aryl hydrocarbon receptor (AhR) is a highly evolutionary conserved, ligand-activated transcription factor that is best known to mediate the toxicities of dioxins and dioxin-like compounds. Phenotype of AhR-null mice, together with the recent discovery of a variety of endogenous and plant-derived ligands, point to the integral roles of AhR in normal cell physiology, in addition to its roles in sensing the environmental chemicals. Here, we summarize the current knowledge about AhR signaling pathways, its ligands and AhR-mediated effects on cell specialization, host defense and detoxification. AhR-mediated health effects particularly in liver, immune, and nervous systems, as well as in tumorgenesis are discussed. Dioxin-initiated embryotoxicity and immunosuppressive effects in fish and birds are reviewed. Recent data demonstrate that AhR is a convergence point of multiple signaling pathways that inform the cell of its external and internal environments. As such, AhR pathway is a promising potential target for therapeutics targeting nervous, liver, and autoimmune diseases through AhR ligand-mediated interventions and other perturbations of AhR signaling. Additionally, using available laboratory data obtained on animal models, AhR-centered adverse outcome pathway analysis is useful in reexamining known and potential adverse outcomes of specific or mixed compounds on wildlife.

Background & Aims 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. Methods 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-2α kinase−mediated stress signaling pathways. We performed hyperinsulinemic-euglycemic clamp studies and immunoprecipitation analyses to determine the oxidation and phosphatase activity of PP1C. Results 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. Conclusions 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.

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 α1 (I) and transforming growth factor beta (TGF-β) expression in vitro. Here we have further investigated the downstream effects of phagocytosis by studying NADPH oxidase activation and its link to procollagen α1 (I) and TGF-β1 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 α1 (I); in contrast, TGF-β1 expression was independent of NADPH oxidase activation. This was also confirmed by using siRNA to inhibit TGF-β1 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. (HEPATOLOGY 2006;43:435–443.)

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 α 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.

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-α)-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-α production as well as induction of fibrogenic transcripts. Ad-Timp3 injection resulted in a significant decline in TACE activity, procollagen α1 (I), alpha smooth muscle actin (α-SMA) and transforming growth factor beta (TGF-β) 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.

Reactive oxygen species (ROS) play a key role in chronic liver injury and fibrosis. Homologues of NADPH oxidases (NOXs) are major sources of ROS, but the exact role of the individual homologues 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. GKT136731 was either applied for the full term of BDL (preventive arm), or starting at 10 days post-operatively (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 TNFα/actinomycin D induced apoptosis was studied in wild type and NOX4−/− hepatocytes. Results: NOX4 was upregulated by a TGF-β/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 TNFα/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 both 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.

The self-renewal capacity ascribed to hESCs is paralleled in cancer cell proliferation, suggesting that a common network of genes may facilitate the promotion of these traits. However, the molecular mechanisms that are involved in regulating the silencing of these genes as stem cells differentiate into quiescent cellular lineages remain poorly understood. Here, we show that a differentiated cell specific miR-122 exemplifies this regulatory attribute by suppressing the translation of a gene, Pkm2, which is commonly enriched in hESCs and liver cancer cells (HCCs), and facilitates self-renewal and proliferation. Through a series of gene expression analysis, we show that miR-122 expression is highly elevated in quiescent human primary hepatocytes (hPHs) but lost or attenuated in hESCs and HCCs, while an opposing expression pattern is observed for Pkm2. Depleting hESCs and HCCs of Pkm2, or overexpressing miR-122, leads to a common deficiency in self-renewal and proliferation. Likewise, during the differentiation process of hESCs into hepatocytes, a reciprocal expression pattern is observed between miR-122 and Pkm2. An examination of the genomic region upstream of miR-122 uncovered hyper-methylation in hESCs and HCCs, while the same region is de-methylated and occupied by a transcription initiating protein, RNA polymerase II (RNAPII), in hPHs. These findings indicate that one possible mechanism by which hESC self-renewal is modulated in quiescent hepatic derivatives of hESCs is through the regulatory activity of a differentiated cell-specific miR-122, and that a failure to properly turn “on” this miRNA is observed in uncontrollably proliferating HCCs.

Non-alcoholic steatohepatitis (NASH) is one of the most common chronic liver diseases worldwide (1). Despite the large number of studies published in the field, the molecular signals triggering the progression of NASH from simple steatosis to necroinflammation are still poorly understood. One of the most important and early features of progressive NASH is lipoapoptosis of hepatocytes that creates a proinflammatory and fibrogenic environment (2, 3). The activation of c-jun N-terminal kinase 1(JNK1) in both hepatocytes and macrophages has been described as a key event in NASH (4, 5), thus the signals governing its induction need to be better elucidated. The article published by Ibrahim et al. (6) has focused on the role of the mixed-lineage kinase 3 (MLK3) as an important proximal JNK activator that has a major role in progressive liver injury in diet-induced NASH. Several studies demonstrated that MLK3/JNK activation plays an essential role in saturated fatty acid-induced insulin resistance and hepatocyte lipotoxicity (7–9). Exposure to free fatty acid induced MLK3/JNK in mouse embryonic fibroblasts, and the MLK3-/- cells displayed increased insulin sensitivity (8); the same trend was found in the MLK3-/- mice on high-fat diet (8, 10). In hepatocytes, palmitate induced the recruitment of cdc42/Rac1 and the activation of MLK3/JNK, leading to downstream ER stress signalling (7). In a different study, the palmitate-induced M1 macrophage polarization was diminished in the MLK3-/- cells (10). All of these suggest that MLK3/JNK signalling plays an important role in the progression of NASH. MLKs are MAPK-kinase kinases (MKKKs) that activate JNK and p38 signalling cascades via the MAPK kinases (MKKs) by either forming a homodimer or associating with Rho GTPases (11–13). The selective activation of MKKs and the downstream induction of JNK1/2 or p38 by MLKs are mediated by the MAPK scaffold proteins such as JNK-interacting proteins (JIPs) (13, 14). How these JIPs direct selective activation of their targets is still under investigation. JIP3 was shown to bind to MLK3/MKK7 inducing JNK1 activation in neurons (14–16). It is however, not clear which JIPs are predominant in the liver and whether they selectively mediate JNK1 and 2 activation. c-Jun N-terminal kinase activation plays a key role in saturated fatty acid-induced hepatocyte apoptosis (5), both in humans and in animal models (17–19). In HSC, phosphorylated-JNK1 directly mediates transdifferentiation contributing to fibrosis (20). MLK3/JNK/P38 activation in LX-2 cells has been observed when cells were stimulated with a PPARβ/δ ligand, leading to cell proliferation (21). JNK1 and JNK2 have differential effects in NASH: JNK1 mediates steatohepatitis and lipotoxicity, whereas JNK2 activation is more protective (22, 23). It is not yet known whether MLK3 has differential effects on JNK1 or JNK2 in the liver, and if so, how the differential induction is mediated. This study is in agreement with previous findings demonstrating that the MLK3-/- mice developed less hepatic steatosis, decreased liver injury, inflammation and fibrosis. The diet used in this study mimics the fast food diet consumed by humans inducing insulin resistance, steatohepatitis and fibrosis (24). It has been recently reported that compared to the WT mice, the global MLK3-deficient mice on high-fat diet displayed significantly less weight gain, and reduced macrophage infiltration in the adipose tissue and also decreased systemic inflammation (10). Interestingly, these mice had increased energy expenditure that could have accounted for the slower weight gain. The improved inflammation in the liver could be explained by the decreased macrophage recruitment and JNK inactivation in the MLK3-/- mice (10). Using the MLK2/3 double knockout mice on the high-fat diet model, Davis and colleagues showed that the obesity-resistant phenotype of these animals was related to the upregulation of the sympatho-adrenal system, as using a selective antagonist to the β3-adrenergic receptor prevented the increase in body temperature and decreased the expression of the adrenergic target genes (25). Distinct from the studies described above, the authors in this study chose high-fat diet combined with high fructose consumption. The mice in this study gained similar amount of weight in both treatment arms, and this could be attributed to the high fructose intake. As fibrosis was diminished in the MLK3-/- mice, it is likely that MLK3 in stellate cells was involved in their transdifferentiation, which has been observed in lung fibroblasts by Lin et al. (26). Macrophage polarization could also be affected by MLK3 (10); and there is evidence showing that MLK3 interacts with TLR signalling by directly binding to Myd88 (27). The dominant cell type and mechanisms for the pro-inflammatory and fibrogenic activity of MLK3 in the liver would require further investigation, and could be addressed in the future by the generation of conditional knockout mice.

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.

Background/Aims 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. Methods Apoptosis of phagocytosing HSC was studied in the presence of known apoptotic agents. The JAK/STAT- and PI3K/Akt-dependent pathways, NF-κB activation and expression of the anti-apoptotic proteins Mcl-1 and A1 were evaluated. Apoptosis was assessed after blocking A1 by an siRNA approach. Results 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-κB activation and subsequent upregulation of A1 was observed, and A1 inhibition induced apoptosis of HSC. Conclusion 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.

Background & Aims 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. Methods 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. Results In the phagocytosis model, engulfment of GFP-labeled apoptotic bodies was seen, and the expression of α-smooth muscle actin (α-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. Conclusions 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.