Natalie J. Torok

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. (Hepatology 2015;62:627–634

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.

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 the main producers of the extracellular matrix in the liver. On liver injury, the quiescent stellate cells transdifferentiate into myofibroblasts, a process highlighted by the loss of vitamin A stores, upregulation of interstitial type collagens, α-smooth muscle actin, matrix metalloproteinases, and 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 the extrahepatic milieu such as the gut microbiome. In this review we focus on the pathomechanism of stellate cell activation following chronic liver injury with the aim of identifying possible treatment targets for antifibrogenic 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. 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)

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 -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. Conclusion: 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.

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 α_vβ₃ heterodimer-dependent binding, indicating that galectin-3 modulates HSC phagocytosis via cross-linking this integrin and enhancing the tethering of apoptotic cells. Blocking integrin α_vβ₃ 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.

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.