Background and Aims
Nonalcoholic fatty liver disease encompasses a spectrum of diseases ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), cirrhosis, and liver cancer. At present, ...how simple steatosis progresses to NASH remains obscure and effective pharmacological therapies are lacking. Hepatic expression of C‐X‐C motif chemokine ligand 1 (CXCL1), a key chemokine for neutrophil infiltration (a hallmark of NASH), is highly elevated in NASH patients but not in fatty livers in obese individuals or in high‐fat diet (HFD)‐fed mice. The aim of this study was to test whether overexpression of CXCL1 itself in the liver can induce NASH in HFD‐fed mice and to test the therapeutic potential of IL‐22 in this new NASH model.
Approach and Results
Overexpression of Cxcl1 in the liver alone promotes steatosis‐to‐NASH progression in HFD‐fed mice by inducing neutrophil infiltration, oxidative stress, and stress kinase (such as apoptosis signal‐regulating kinase 1 and p38 mitogen‐activated protein kinase) activation. Myeloid cell‐specific deletion of the neutrophil cytosolic factor 1 (Ncf1)/p47phox gene, which encodes a component of the NADPH oxidase 2 complex that mediates neutrophil oxidative burst, markedly reduced CXCL1‐induced NASH and stress kinase activation in HFD‐fed mice. Treatment with interleukin (IL)‐22, a cytokine with multiple targets, ameliorated CXCL1/HFD‐induced NASH or methionine‐choline deficient diet‐induced NASH in mice. Mechanistically, IL‐22 blocked hepatic oxidative stress and its associated stress kinases via the induction of metallothionein, one of the most potent antioxidant proteins. Moreover, although it does not target immune cells, IL‐22 treatment attenuated the inflammatory functions of hepatocyte‐derived, mitochondrial DNA‐enriched extracellular vesicles, thereby suppressing liver inflammation in NASH.
Conclusions
Hepatic overexpression of CXCL1 is sufficient to drive steatosis‐to‐NASH progression in HFD‐fed mice through neutrophil‐derived reactive oxygen species and activation of stress kinases, which can be reversed by IL‐22 treatment via the induction of metallothionein.
Gene Expression database of Normal and Tumor tissues 2 (GENT2) is an updated version of GENT, which has provided a user-friendly search platform for gene expression patterns across different normal ...and tumor tissues compiled from public gene expression data sets.
We refactored GENT2 with recent technologies such as Apache Lucene indexing for fast search and Google Web Toolkit (GWT) framework for a user-friendly web interface. Now, GENT2 contains more than 68,000 samples and has several new useful functions. First, GENT2 now provides gene expression across 72 different tissues compared to 57 in GENT. Second, with increasing importance of tumor subtypes, GENT2 provides an option to study the differential expression and its prognostic significance based on tumor subtypes. Third, whenever available, GENT2 provides prognostic information of a gene of interest. Fourth, GENT2 provides a meta-analysis of survival information to provide users more reliable prognostic value of a gene of interest.
In conclusion, with these significant improvements, GENT2 will continue to be a useful tool to a wide range of researchers. GENT2 is freely available at http://gent2.appex.kr .
ARPC2 is a subunit of the Arp2/3 complex, which is essential for lamellipodia, invadopodia and filopodia, and ARPC2 has been identified as a migrastatic target molecule. To identify ARPC2 inhibitors, ...we generated an ARPC2 knockout DLD‐1 human colon cancer cell line using the clustered regularly interspaced short palindromic repeats/CRISPR‐associated protein 9 (CRISPR/Cas9) system and explored gene signature‐based strategies, such as a connectivity map (CMap) using the gene expression profiling data of ARPC2 knockout and knockdown cells. From the CMap‐based drug discovery strategy, we identified pimozide (a clinically used antipsychotic drug) as a migrastatic drug and ARPC2 inhibitor. Pimozide inhibited the migration and invasion of various cancer cells. Through drug affinity responsive target stability (DARTS) analysis and cellular thermal shift assay (CETSA), it was confirmed that pimozide directly binds to ARPC2. Pimozide increased the lag phase of Arp2/3 complex‐dependent actin polymerization and inhibited the vinculin‐mediated recruitment of ARPC2 to focal adhesions in cancer cells. To validate the likely binding of pimozide to ARPC2, mutant cells, including ARPC2F225A, ARPC2F247A and ARPC2Y250F cells, were prepared using ARPC2 knockout cells prepared by gene‐editing technology. Pimozide strongly inhibited the migration of mutant cells because the mutated ARPC2 likely has a larger binding pocket than the wild‐type ARPC2. Therefore, pimozide is a potential ARPC2 inhibitor, and ARPC2 is a new molecular target. Taken together, the results of the present study provide new insights into the molecular mechanism and target that are responsible for the antitumor and antimetastatic activity of pimozide.
Pimozide is identified as a migrastatic drug and ARPC2 inhibitor from connectivity map‐based drug discovery strategy. Pimozide inhibits migration and invasion in various cancer cell lines, and suppresses metastasis in an in vivo antimetastatic assay. Through drug affinity responsive target stability (DARTS) analysis and cellular thermal shift assay (CETSA), it was confirmed that pimozide directly binds to ARPC2.
Adipose tissue represents a large volume of biologically active tissue that exerts substantial systemic effects in health and disease. Alcohol consumption can profoundly disturb the normal functions ...of adipose tissue by inducing adipocyte death and altering secretion of adipokines, pro-inflammatory mediators and free fatty acids from adipose tissue, which have important direct and indirect effects on the pathogenesis of alcoholic liver disease (ALD). Cessation of alcohol intake quickly reverses inflammatory changes in adipose tissue, and pharmacological treatment that normalizes adipose tissue function improves experimental ALD. Obesity exacerbates liver injury induced by chronic or binge alcohol consumption, and obesity and alcohol can synergize to increase risk of ALD and progression. Physicians who care for individuals with ALD should be aware of the effects of adipose tissue dysfunction on liver function, and consider strategies to manage obesity and insulin resistance. This Review examines the effect of alcohol on adiposity and adipose tissue and the relationship between alcohol, adipose tissue and the liver.
Background and Aims
Neutrophil infiltration is a hallmark of nonalcoholic steatohepatitis (NASH), but how this occurs during the progression from steatosis to NASH remains obscure. Human NASH ...features hepatic neutrophil infiltration and up‐regulation of major neutrophil‐recruiting chemokines (e.g., chemokine C‐X‐C motif ligand 1 CXCL1 and interleukin IL‐8). However, mice fed a high‐fat diet (HFD) only develop fatty liver without significant neutrophil infiltration or elevation of chemokines. The aim of this study was to determine why mice are resistant to NASH development and the involvement of p38 mitogen‐activated protein kinase (p38) activated by neutrophil‐derived oxidative stress in the pathogenesis of NASH.
Approach and Results
Inflamed human hepatocytes attracted neutrophils more effectively than inflamed mouse hepatocytes because of the greater induction of CXCL1 and IL‐8 in human hepatocytes. Hepatic overexpression of Cxcl1 and/or IL‐8 promoted steatosis‐to‐NASH progression in HFD‐fed mice by inducing liver inflammation, injury, and p38 activation. Pharmacological inhibition of p38α/β or hepatocyte‐specific deletion of p38a (a predominant form in the liver) attenuated liver injury and fibrosis in the HFD+Cxcl1‐induced NASH model that is associated with strong hepatic p38α activation. In contrast, hepatocyte‐specific deletion of p38a in HFD‐induced fatty liver where p38α activation is relatively weak exacerbated steatosis and liver injury. Mechanistically, weak p38α activation in fatty liver up‐regulated the genes involved in fatty acid β‐oxidation through peroxisome proliferator‐activated receptor alpha phosphorylation, thereby reducing steatosis. Conversely, strong p38α activation in NASH promoted caspase‐3 cleavage, CCAAT‐enhancer‐binding proteins homologous protein expression, and B cell lymphoma 2 phosphorylation, thereby exacerbating hepatocyte death.
Conclusions
Genetic ablation of hepatic p38a increases simple steatosis but ameliorates oxidative stress‐driven NASH, indicating that p38α plays distinct roles depending on the disease stages, which may set the stage for investigating p38α as a therapeutic target for the treatment of NASH.
System x
contributes to glutathione (GSH) synthesis and protects cells against ferroptosis by importing cystine and exchanging it with glutamate. Transforming growth factor β1 (TGF-β1) induces redox ...imbalance; however, its role in system x
regulation remains poorly understood. The present study was the first to show that TGF-β1 repressed the protein and mRNA levels of xCT, a catalytic subunit of system x
, in PLC/PRF/5, Huh7, Huh6, and HepG2 cells with an early TGF-β1 gene signature but not in SNU387, SNU449, SNU475, and SK-Hep1 cells with a late TGF-β1 gene signature. TGF-β1 treatment for 24 h reduced xCT expression in a dose-dependent manner but this TGF-β1-induced repression was blunted by pretreatment with a TGF-β1 receptor inhibitor. TGF-β1-mediated xCT repression was prevented by Smad3, but not Smad2 or Smad4, knockdown, whereas it was enhanced by Smad3 overexpression. TGF-β1 decreased GSH levels in control cells but not xCT-overexpressed cells. Furthermore, TGF-β1 increased reactive oxygen species (ROS) levels in PLC/PRF/5 cells and enhanced tert-butyl hydroperoxide-induced ROS levels in Huh7 cells; these changes were reversed by xCT overexpression. TGF-β1 treatment ultimately induced the ferrostatin-1- and deferoxamine-dependent lipid peroxidation after 2 days and 8 days in PLC/PRF/5 and Huh7 cells but not in SNU475 and SK-Hep1 cells. Pre-treatment of TGF-β1 for 2 days enhanced the reduction of cell viability induced by RSL3, a GSH peroxidase 4 (GPX4) inhibitor, in PLC/PRF/5 and Huh7 cells. In conclusion, TGF-β1 represses xCT expression via Smad3 activation and enhances lipid peroxidation in hepatocellular carcinoma cells with an early TGF-β1 signature, which would benefit from the targeting of GPX4.
Nonalcoholic fatty liver disease (NAFLD) represents a spectrum of diseases ranging from simple steatosis to more severe forms of liver injury including nonalcoholic steatohepatitis (NASH), fibrosis, ...and hepatocellular carcinoma (HCC). In humans, only 20%‐40% of patients with fatty liver progress to NASH, and mice fed a high‐fat diet (HFD) develop fatty liver but are resistant to NASH development. To understand how simple steatosis progresses to NASH, we examined hepatic expression of anti‐inflammatory microRNA‐223 (miR‐223) and found that this miRNA was highly elevated in hepatocytes in HFD‐fed mice and in human NASH samples. Genetic deletion of miR‐223 induced a full spectrum of NAFLD in long‐term HFD‐fed mice including steatosis, inflammation, fibrosis, and HCC. Furthermore, microarray analyses revealed that, compared to wild‐type mice, HFD‐fed miR‐223 knockout (miR‐223KO) mice had greater hepatic expression of many inflammatory genes and cancer‐related genes, including (C‐X‐C motif) chemokine 10 (Cxcl10) and transcriptional coactivator with PDZ‐binding motif (Taz), two well‐known factors that promote NASH development. In vitro experiments demonstrated that Cxcl10 and Taz are two downstream targets of miR‐223 and that overexpression of miR‐223 reduced their expression in cultured hepatocytes. Hepatic levels of miR‐223, CXCL10, and TAZ mRNA were elevated in human NASH samples, which positively correlated with hepatic levels of several miR‐223 targeted genes as well as several proinflammatory, cancer‐related, and fibrogenic genes. Conclusion: HFD‐fed miR‐223KO mice develop a full spectrum of NAFLD, representing a clinically relevant mouse NAFLD model; miR‐223 plays a key role in controlling steatosis‐to‐NASH progression by inhibiting hepatic Cxcl10 and Taz expression and may be a therapeutic target for the treatment of NASH.
Acetaminophen (APAP) overdose is a leading cause of acute liver failure worldwide, in which mitochondrial DNA (mtDNA) released by damaged hepatocytes activates neutrophils through binding of ...Toll‐like receptor 9 (TLR9), further aggravating liver injury. Here, we demonstrated that mtDNA/TLR9 also activates a negative feedback pathway through induction of microRNA‐223 (miR‐223) to limit neutrophil overactivation and liver injury. After injection of APAP in mice, levels of miR‐223, the most abundant miRNAs in neutrophils, were highly elevated in neutrophils. Disruption of the miR‐223 gene exacerbated APAP‐induced hepatic neutrophil infiltration, oxidative stress, and injury and enhanced TLR9 ligand‐mediated activation of proinflammatory mediators in neutrophils. An additional deletion of the intercellular adhesion molecule 1 (ICAM‐1) gene ameliorated APAP‐induced neutrophil infiltration and liver injury in miR‐223 knockout mice. In vitro experiments revealed that miR‐223‐deficient neutrophils were more susceptible to TLR9 agonist‐mediated induction of proinflammatory mediators and nuclear factor kappa B (NF‐κB) signaling, whereas overexpression of miR‐223 attenuated these effects in neutrophils. Moreover, inhibition of TLR9 signaling by either treatment with a TLR9 inhibitor or by disruption of TLR9 gene partially, but significantly, suppressed miR‐223 expression in neutrophils post‐APAP injection. In contrast, activation of TLR9 up‐regulated miR‐223 expression in neutrophils in vivo and in vitro. Mechanistically, activation of TLR9 up‐regulated miR‐223 by enhancing NF‐κB binding on miR‐223 promoter, whereas miR‐223 attenuated TLR9/NF‐κB‐mediated inflammation by targeting IκB kinase α expression. Collectively, up‐regulation of miR‐223 plays a key role in terminating the acute neutrophilic response and is a therapeutic target for treatment of APAP‐induced liver failure. (Hepatology 2017;66:220–234).
Nonalcoholic steatohepatitis (NASH) is an advanced stage of fatty liver disease characterized by liver damage, inflammation, and fibrosis. Although neutrophil infiltration is consistently observed in ...the livers of patients with NASH, the precise role of neutrophil-recruiting chemokines and infiltrating neutrophils in NASH pathogenesis remains poorly understood. Here, we aimed to elucidate the role of neutrophil infiltration in the transition from fatty liver to NASH by examining hepatic overexpression of interleukin-8 (IL8), a major chemokine responsible for neutrophil recruitment in humans. Mice fed a high-fat diet (HFD) for 3 months developed fatty liver without concurrent liver damage, inflammation, and fibrosis. Subsequent infection with an adenovirus overexpressing human IL8 for an additional 2 weeks increased IL8 levels, neutrophil infiltration, and liver injury in mice. Mechanistically, IL8-induced liver injury was associated with the upregulation of components of the NADPH oxidase 2 complex, which participate in neutrophil oxidative burst. IL8-driven neutrophil infiltration promoted macrophage aggregate formation and upregulated the expression of chemokines and inflammatory cytokines. Notably, IL8 overexpression amplified factors associated with fibrosis, including collagen deposition and hepatic stellate cell activation, in HFD-fed mice. Collectively, hepatic overexpression of human IL8 promotes neutrophil infiltration and fatty liver progression to NASH in HFD-fed mice.
•IRB/AML fixed-dose regimens show superior antihypertensive efficacy over IRB monotherapy.•A potential benefit of IRB/AML regimens was noted in the elderly and T2DM patients.•IRB/AML combinations are ...well-tolerated and have comparable safety to IRB monotherapy.
This study aimed to evaluate the efficacy and tolerability of irbesartan (IRB) and amlodipine (AML) combination therapy in patients with essential hypertension whose blood pressure (BP) was not controlled by IRB monotherapy.
Two multicenter, randomized, double-blind, placebo-controlled, phase III studies were conducted in Korea (the I-DUO 301 study and the I-DUO 302 study). After a 4-week run-in period with either 150 mg IRB (I-DUO 301 study) or 300 mg IRB (I-DUO 302 study), patients with uncontrolled BP (ie, mean sitting systolic BP MSSBP ≥140 mmHg to <180 mmHg and mean sitting diastolic BP <110 mmHg) were randomized to the placebo, AML 5 mg, or AML 10 mg group. A total of 428 participants were enrolled in the 2 I-DUO studies. In the I-DUO 301 study, 271 participants were randomized in a 1:1:1 ratio to receive either IRB/AML 150/5 mg, IRB/AML 150/10 mg, or IRB 150 mg/placebo. In the I-DUO 302 study, 157 participants were randomized in a 1:1 ratio to receive IRB/AML 300/5 mg or IRB 300 mg/placebo. The primary endpoint was the change in MSSBP from baseline to week 8. Tolerability was assessed according to the development of treatment-emergent adverse events (TEAEs) and clinically significant changes in physical examination, laboratory tests, pulse, and 12-lead electrocardiography.
In I-DUO 301, the mean (SD) changes of MSSBP at week 8 from baseline were −14.78 (12.35) mmHg, −21.47 (12.78) mmHg, and −8.61 (12.19) mmHg in the IRB/AML 150/5 mg, IRB/AML 150/10 mg, and IRB 150 mg/placebo groups, respectively. In I-DUO 302, the mean (SD) changes of MSSBP at week 8 from baseline were −13.30 (12.47) mmHg and −7.19 (15.37) mmHg in the IRB/AML 300/5 mg and IRB 300 mg/placebo groups, respectively. In both studies, all combination groups showed a significantly higher reduction in MSSBP than the IRB monotherapy groups (P < 0.001 for both). TEAEs occurred in 10.00%, 10.99%, and 12.22% of participants in the IRB/AML 150/5 mg, IRB/AML 150/10 mg, and IRB 150 mg/placebo groups, respectively, in I-DUO 301 and in 6.33% and 10.67% of participants in the IRB/AML 300/5 mg and IRB 300 mg/placebo groups, respectively, in I-DUO 302, with no significant between-group differences. Overall, there was one serious adverse event throughout I-DUO study.
The combination of IRB and AML has superior antihypertensive effects compared with IRB alone over an 8-week treatment period, with placebo-like tolerability.
ClinicalTrials.gov identifier: NCT05476354 (I-DUO 301), NCT05475665 (I-DUO 302).
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