Natalie J. Török

Phagocytosis of apoptotic bodies by hepatic stellate cells induces NADPH oxidase and is associated with liver fibrosis in vivo.

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 causes downregulation of miRNA-150 and miRNA-194 in hepatic stellate cells, and their overexpression causes decreased stellate cell activation

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.

Liver fibrosis and hepatocyte apoptosis are attenuated by GKT137831 a novel NOX4/NOX1 inhibitor in vivo

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 TNFα/actinomycin D-induced apoptosis was studied in wild-type and NOX4(-/-) hepatocytes. 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 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.

De novo nonalcoholic fatty liver disease after liver transplantation

Hepatic steatosis is a recognized problem in patients after orthotopic liver transplant (OLT). However, de novo development of nonalcoholic fatty liver disease (NAFLD) has not been well described. The aim of this study was to determine the prevalence and predictors of de novo NAFLD after OLT. A retrospective analysis was performed on 68 OLT patients with donor liver biopsies and posttransplantation liver biopsies. Individual medical charts were reviewed for demographics, indication for OLT, serial histology reports, genotypes for hepatitis C, comorbid conditions, and medications. Liver biopsies were reviewed blindly and graded according to the Brunt Scoring System. Multivariate logistic regression analysis was used to study the risk factors for developing NAFLD. The interval time from OLT to subsequent follow‐up liver biopsy was 28 ± 18 months. A total of 12 patients (18%) developed de novo NAFLD, and 6 (9%) developed de novo NASH. The regression model indicated that the use of angiotensin‐converting enzyme inhibitors (ACE‐I) was associated with a reduced risk of developing NAFLD after OLT (odds ratio, 0.09; 95% confidence interval, 0.010‐0.92; P = 0.042). Increase in body mass index (BMI) of greater than 10% after OLT was associated with a higher risk of developing NAFLD (odds ratio, 19.38; 95% confidence interval, 3.50‐107.40; P = 0.001). In conclusion, de novo NAFLD is common in the post‐OLT setting, with a significant association with weight gain after transplant. The use of an ACE‐I may reduce the risk of developing post‐OLT NAFLD. Liver Transpl, 2006.

Apoptotic body engulfment by hepatic stellate cells promotes their survival by the JAK/STAT and Akt/NF-κB-dependent pathways ☆

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-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. 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-kappaB 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.

Adiponectin Decreases C-Reactive Protein Synthesis and Secretion From Endothelial Cells Evidence for an Adipose Tissue-Vascular Loop

Background and Objective—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). Methods and Results—Adiponectin dose-dependently reduced CRP mRNA and protein from HAECs. Adiponectin treatment of HAECs significantly decreased I&kgr;B phosphorylation and NF&kgr;B binding activity. There was no effect of adiponectin on STAT or C/EBP transcriptional activity. Adiponectin also activated AMP kinase resulting in decreased NF&kgr;B 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 NF&kgr;B. Similar findings were observed in rat primary hepatocytes. Conclusions—Thus, in obesity and diabetes, the hypoadiponectinemia could exacerbate the proinflammatory state by inducing CRP production.

Reduced Nicotinamide Adenine Dinucleotide Phosphate Oxidase 2 Plays a Key Role in Stellate Cell Activation and Liver Fibrogenesis In Vivo

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.

Minocycline in the treatment of patients with primary sclerosing cholangitis: results of a pilot study.

OBJECTIVES: 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. METHODS: 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. RESULTS: A statistically significant improvement in serum alkaline phosphatase activity (330 U/I vs. 265 U/I, 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. CONCLUSIONS: 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.

NOX1/nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase promotes proliferation of stellate cells and aggravates liver fibrosis induced by bile duct ligation.

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‐1α mRNA and hydroxyproline were significantly suppressed in Nox1KO with a reduced number of activated HSCs and less severe fibrotic lesions. The expression levels of α‐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 p27kip1, 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 p27kip1 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.

Adenosine induces loss of actin stress fibers and inhibits contraction in hepatic stellate cells via Rho inhibition

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 μM, 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. (HEPATOLOGY 2009;49:185‐194)