Effectiveness of 1-year pemafibrate treatment on steatotic liver disease: the influence of alcohol consumption.

BACKGROUND/AIMS: Pemafibrate is a selective peroxisome proliferator-activated receptor α modulator that improves serum alanine aminotransferase (ALT) in dyslipidemia patients. We previously reported that pemafibrate significantly improves liver function, serum triglyceride (TG) levels and liver stiffness in non-alcoholic fatty liver disease patients, however the influence of alcohol consumption was not considered. Therefore, we explored pemafibrate efficacy in patients with steatotic liver disease (SLD) and alcohol-associated liver disease (ALD). METHODS: We retrospectively evaluated pemafibrate efficacy on liver enzymes and lipids in metabolic dysfunction-associated SLD (MASLD) (n = 93), MASLD plus increased alcohol intake (MetALD; n = 23) and ALD (n = 22) patients who had taken pemafibrate for at least 48 weeks. Liver shear wave velocity (SWV, n = 75) was also evaluated. RESULTS: In MASLD group, ALT, aspartate aminotransferase (AST), γ-glutamyl transpeptidase (γ-GTP) and TG values were significantly decreased from baseline to week 24 and week 48 (P < 0.0001). ALT and TG values in MetALD group and ALT and AST values in ALD group were also significantly decreased from baseline to week 24 and week 48. Study participant SWV values decreased from baseline to week 48. We observed no significant difference in changes to ALT, AST, γ-GTP and TG (value at week 24 or week 48 minus value at baseline) among the three groups. CONCLUSION: Pemafibrate improves liver function and liver stiffness thus making it a promising therapeutic agent for SLD, even in patients with excess alcohol consumption (MetALD and ALD groups).

Circulating extracellular vesicles are associated with pathophysiological condition including metabolic syndrome-related dysmetabolism in children and adolescents with obesity.

Obesity of children and adolescents (OCA) is often accompanied by metabolic syndrome (MetS), which often leads to adult obesity and subsequent complications, yet the entire pathophysiological response is not fully understood. The number and composition of circulating extracellular vesicles (EV) reflect overall patient condition; therefore, we investigated the pathophysiological condition of OCA, including MetS-associated dysmetabolism, using circulating EVs. In total, 107 children and adolescents with or without obesity (boys, n = 69; girls, n = 38; median age, 10 years) were enrolled. Circulating EV number and EV protein composition were assessed via flow cytometry and liquid chromatography tandem-mass spectrometry, respectively. In a multivariate analysis, relative body weight (standardized partial regression coefficient (SPRC) 0.469, P = 0.012) and serum triglyceride level (SPRC 0.548, P < 0.001) were detected as independent parameters correlating with circulating EV number. Proteomic analysis identified 31 upregulated and 45 downregulated EV proteins in OCA. Gene ontology analysis revealed upregulated proteins to be involved in various biological processes, including intracellular protein transport, protein folding, stress response, leukocyte activation, innate immune response, and platelet degranulation, which can modulate lipid and glucose metabolism, skeletal and cardiac muscle development, inflammation, immune response, carcinogenesis, and cancer progression. Notably, several identified EV proteins are involved in neuro-development, neurotransmitter release, and neuro-protective agents in OCA. Circulating EVs were derived from adipocytes, hepatocytes, B cell lymphocytes, and neurons. Circulating EV number is significantly associated with MetS-related dysmetabolism and the EV protein cargo carries a special "signature" that reflects the alteration of various biological processes under the pathophysiological condition of OCA. KEY MESSAGES: Circulating EV number correlates with physical and laboratory parameters for obesity in children and adolescents. Relative body weight and triglyceride are independent factors for increased circulating EVs. EV composition is significantly changed in obesity of children and adolescents. Identified EV composition changes associated with obesity and involves in metabolism, immune response, and cancer progression. Circulating EVs are partially derived from adipocyte, hepatocytes, B cells, and neurons.

Serum Zinc-α2-glycoprotein Levels Are Associated with the Hepatorenal Function and Predict the Survival in Cases of Chronic Liver Disease.

Objective Zinc-α2-glycoprotein (ZAG) is secreted by various organs, such as liver, kidney and adipose tissue, is involved in lipolysis, and may contribute to the pathogenesis of chronic liver disease (CLD). We therefore assessed whether or not ZAG is a surrogate marker for the hepatorenal function, body composition and all causes of mortality, as well as complications, including ascites, hepatic encephalopathy (HE) and portosystemic shunts (PSS) in CLD. Methods Serum ZAG levels were measured in 180 CLD patients upon hospital admission. The associations of ZAG levels with the liver functional reserve and clinical parameters were investigated using a multiple regression analysis. Kaplan-Meier analyses were performed to evaluate the associations of the ZAG/creatinine ratio (ZAG/Cr) and prognostic factors with mortality. Results High serum ZAG levels were associated with preserving the liver function and renal insufficiency. A multiple regression analysis showed that the estimated glomerular filtration rate (p<0.0001), albumin-bilirubin (ALBI) score (p=0.0018) and subcutaneous fat area (p=0.0023) had a significant independent correlation with serum ZAG levels. Serum ZAG levels were elevated in the absence of HE (p=0.0023) and PSS (p=0.0003). In all patients and those without hepatocellular carcinoma (HCC), the cumulative mortality rate was significantly decreased in patients with a high ZAG/Cr compared with those with a low ZAG/Cr (p=0.0018 and p=0.0002, respectively). The ZAG/Cr, presence of HCC, ALBI score and psoas muscle index were independent predictors of the prognosis in CLD patients. Conclusion Serum ZAG levels are associated with the hepatorenal function and can be used to predict the survival in CLD patients.

Extracellular Vesicles as Therapeutic and Diagnostic Tools for Chronic Liver Diseases.

Chronic liver diseases can lead to fibrotic changes that may progress to the development of cirrhosis, which poses a significant risk for morbidity and increased mortality rates. Metabolic dysfunction-associated steatotic liver disease (MASLD), alcohol-associated liver disease (ALD), and viral hepatitis are prevalent liver diseases that may lead to cirrhosis. The advanced stages of cirrhosis can be further complicated by cancer development or end-stage liver disease and liver failure. Hence, early detection and diagnosis of liver fibrosis is crucial for preventing the progression to cirrhosis and improving patient outcomes. Traditionally, invasive liver biopsy has been considered the gold standard for diagnosing and staging liver fibrosis. In the last decade, research has focused on non-invasive methods, known as liquid biopsies, which involve the identification of disease-specific biomarkers in human fluids, such as blood. Among these alternative approaches, extracellular vesicles (EVs) have emerged as promising diagnostic and therapeutic tools for various diseases, including chronic liver diseases. EVs are released from stressed or damaged cells and can be isolated and quantified. Moreover, EVs facilitate cell-to-cell communication by transporting various cargo, and they have shown the potential to reduce the expression of profibrogenic markers, making them appealing tools for novel anti-fibrotic treatments. This review focuses on the impact of EVs in chronic liver diseases and exploring their potential applications in innovative therapeutic and diagnostic approaches.

Effect of pemafibrate on liver enzymes and shear wave velocity in non-alcoholic fatty liver disease patients.

Background/Aims: Pemafibrate is a selective peroxisome proliferator-activated receptor α modulator that improves serum alanine aminotransferase (ALT) in dyslipidemia patients. Pemafibrate was reported to reduce ALT in non-alcoholic fatty liver disease (NAFLD) patients, but efficacy was not clearly elucidated due to the small size of previous study populations. Therefore, we explored pemafibrate efficacy in NAFLD patients. Methods: We retrospectively evaluated pemafibrate efficacy on liver enzymes (n = 132) and liver shear wave velocity (SWV, n = 51) in NAFLD patients who had taken pemafibrate for at least 24 weeks. Results: Patient ALT levels were decreased from 81.0 IU/L at baseline to 48.0 IU/L at week 24 (P < 0.0001). Serum levels of aspartate aminotransferase (AST), γ-glutamyl transpeptidase (γ-GTP) and triglyceride (TG) were significantly decreased, and high-density lipoprotein cholesterol and platelet count were significantly increased, with no change in body weight being observed. Study participant SWV values decreased from 1.45 m/s at baseline to 1.32 m/s at week 48 (P < 0.001). Older age (P = 0.035) and serum TG levels (P = 0.048) were significantly associated with normalized ALT. Changes in AST, ALT, γ-GTP and body weight were significantly correlated with change in SWV. Conclusion: Pemafibrate significantly improves liver function, serum TG and liver stiffness in NAFLD patients. Pemafibrate is a promising therapeutic agent for NAFLD and may be a candidate for NAFLD patients with elevated TG.

Hepatocellular Carcinoma Pseudoprogression Involving the Main Portal Vein, Right Ventricular Invasion, and Exacerbation of Lung Metastases in a Patient on Atezolizumab Plus Bevacizumab.

A 70-year-old man was diagnosed with hepatocellular carcinoma (HCC) with portal vein invasion and lung metastases, for which atezolizumab plus bevacizumab (ATZ/BEV) was initiated. After two months, computed tomography revealed tumor growth accompanied by ascites, right ventricular invasion, exacerbation of the lung metastases, and main portal vein invasion. However, continuation of ATZ/BEV caused remarkable size reductions in all lesions, finally resulting in the disappearance of the vascular invasion and lung metastases after nine cycles of treatment. The tumor growth was considered to reflect pseudoprogression, which is difficult to distinguish from hyperprogression. We herein report a remarkable HCC case of pseudoprogression on ATZ/BEV.

Extracellular vesicles in fatty liver disease and steatohepatitis: Role as biomarkers and therapeutic targets.

Non-alcoholic fatty liver disease (NAFLD) and alcohol-associated liver disease (ALD) are characterized by lipid deposition in hepatocytes in the absence or presence of excessive alcohol consumption, respectively, ranging from simple steatosis to non-alcoholic steatohepatitis (NASH) or alcoholic hepatitis (AH) and from mild fibrosis to cirrhosis. Fatty liver disease and steatohepatitis similarly occur in individuals who have both metabolic syndrome and excessive alcohol intake; therefore, the single overarching term metabolic associated fatty liver disease (MAFLD) has been proposed to better reflect these risk factors and the continuity of disease progression. Extracellular vesicles (EVs) are membrane-bound endogenous nanoparticles released into the extracellular space by a majority of cell types. Liver disease-related EVs contain a variety of cellular cargo and are internalized into target cells resulting in the transfer of bioinformation reflecting the state of the donor cell to the recipient. Furthermore, EV composition can be used to identify the degree and type of liver disease, suggesting that EV composition may be a useful biomarker. With regard to MAFLD, the presence of metabolic risk factors, such as insulin resistance, will be indicated by adipose tissue-derived EVs and with that comes the potential to use as a clinical monitor of overall metabolic status. However, the inhibition of specific EV composition may be difficult to implement as a real-world therapeutic approach. Current global evidence shows that mesenchymal stem cell (MSCs)-derived EVs (MSC-EVs) play an important role in regulating the immune response, which has spawned a clinical trial to treat liver disease.

The prognostic role of controlling nutritional status and skeletal muscle mass in patients with hepatocellular carcinoma after curative treatment.

BACKGROUND: Nutritional status and skeletal muscle mass affect the prognosis of hepatocellular carcinoma (HCC). Nutritional status is closely associated with skeletal muscle mass. Here, we investigate the effect of controlling preoperative nutritional status and skeletal muscle mass on the prognosis of HCC after curative treatment. METHODS: This retrospective analysis contained 181 patients who received curative treatment of HCC including liver resection or radiofrequency ablation (RFA) therapy. Nutritional status and skeletal muscle mass were evaluated prior to therapy using the controlling nutritional status (CONUT) score and psoas muscle mass index (PMI), respectively. Associations of predictor variables with overall survival (OS) and progression-free survival (PFS) were determined using Cox proportional hazards regression and CHAID decision tree algorithm analysis. RESULTS: A total of 111 patients (61.3%) were determined to be of poor nutritional status and 100 patients (55.2%) had muscle mass depletion. Patients with PS 0, Barcelona clinic liver cancer (BCLC) stage 0, low CONUT score, and high PMI showed significantly better OS than those with PS 1, BCLC stage A, high CONUT score, and low PMI. Multivariate analysis indicated that a high CONUT score [hazard ratio (HR) 4.130; 95% confidence interval (CI), 1.713-9.958; P < 0.01) and low PMI (HR 4.625; 95% CI, 1.704-12.549; P < 0.01) found to be useful for predicting OS in patients after curative treatment of HCC. Regarding PFS, a significant predictor was only tumor numbers in univariate analysis (HR 2.147; 95% CI, 1.350-3.414; P = 0.001). In decision tree analysis, the mortality rate was 28.8%, 12.5%, and 1.9% in patients with a high CONUT score, with a low CONUT score-low PMI, or with a low CONUT score-high PMI, respectively. CONCLUSION: The combined CONUT score and PMI were found to be independent predictors of OS in HCC patients after liver resection or RFA.

Complement complex 1 subunit q-mediated hepatic stellate cell activation with connective tissue growth factor elevation is a prognostic factor for survival in rat and human chronic liver diseases.

Complement complex 1 subunit q (C1q) has multiple functions, including cell migration, in addition to its traditional complement-activating effect. Research shows C1q is a ligand for frizzled receptors (FZDs). FZD-induced yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ) alternate Wnt signaling activation induces connective tissue growth factor (CTGF) production and hepatic stellate cell (HSC) activation. However, no study exists in which C1q directly induces CTGF in HSCs. Here, we investigated the role of C1q in HSC activation. Human HSCs (LX2) were incubated with C1q to assess HSC activation. C1q and fibrotic markers were assessed using immunohistochemistry, immunoblotting, and quantitative reverse-transcription polymerase chain reaction in cirrhotic rats administered CCl4 for 21 weeks. Serum C1q, liver function, and fibrosis score were measured in 91 patients with chronic liver disease. The correlations between serum C1q and liver function, fibrosis score, and survival prognosis were examined. C1q-activated LX2s showed morphologic changes, up-regulation of CTGF, tissue inhibitors of metalloproteinases (TIMP-1), and alternate Wnt signal genes FZD2, TAZ, and cysteine-rich angiogenic inducer 61 (Cyr61). Cirrhotic rat liver C1q expression correlated with the Azan-positive area and expression of CTGF, TIMP-1, hyaluronan synthase (HAS)1, HAS3, and CD44. Expression of C1q protein and C1q, CTGF, and TIMP-1 genes were higher in deceased cirrhotic rat livers compared to surviving rats. Human serum C1q levels increased in liver cirrhosis compared to chronic hepatitis and correlated with liver fibrosis and functional markers. Ten patients suffered liver-related death over a 66-month observation period. The C1q cut-off value (11 mg/dl) showed patients with serum values < 11 mg/dl had longer rates of survival compared to C1q ≥ 11 mg/dl. Conclusion: C1q-mediated HSC activation in liver fibrosis is associated with CTGF elevation. Additionally, serum C1q may be diagnostic for survival in human chronic liver diseases.

Association of lithocholic acid with skeletal muscle hypertrophy through TGR5-IGF-1 and skeletal muscle mass in cultured mouse myotubes, chronic liver disease rats and humans.

Background: Hepatic sarcopenia is one of many complications associated with chronic liver disease (CLD) and has a high mortality rate; however, the liver-muscle axis is not fully understood. Therefore, few effective treatments exist for hepatic sarcopenia, the best of which being branched-chain amino acid (BCAA) supplementation to help increase muscle mass. Our aim was to investigate the molecular mechanism(s) of hepatic sarcopenia focused on bile acid (BA) composition. Methods: The correlation between serum BA levels and psoas muscle mass index (PMI) was examined in 73 CLD patients. Gastrocnemius muscle phenotype and serum BA levels were assessed in CLD rats treated with BCAA. Mouse skeletal muscle cells (C2C12) were incubated with lithocholic acid (LCA), G-protein-coupled receptor 5 (TGR5) agonist or TGR5 antagonist to assess skeletal muscle hypertrophy. Results: In human CLD, serum LCA levels were the sole factor positively correlated with PMI and were significantly decreased in both the low muscle mass group and the deceased group. Serum LCA levels were also shown to predict patient survival. Gastrocnemius muscle weight significantly increased in CLD rats treated with BCAA via suppression of protein degradation pathways, coupled with a significant increase in serum LCA levels. LCA treated C2C12 hypertrophy occurred in a concentration-dependent manner linked with TGR5-Akt pathways based upon inhibition results via a TGR5 antagonist. Conclusions: Our results indicate LCA-mediated skeletal muscle hypertrophy via activation of TGR5-IGF1-Akt signaling pathways. In addition, serum LCA levels were associated with skeletal muscle mass in cirrhotic rats, as well as CLD patients, and predicted overall patient survival. Funding: This research was supported by JSPS KAKENHI Grant Number 22K08011 and 21H02892, and AMED under Grant Number JP21fk0210090 and JP22fk0210115. Maintaining cirrhotic rats were partially supported by Otsuka Pharmaceutical Company.

The prognostic potential of fragmented CK18 serum levels in HCC patients reflecting disease progression and overall hepatocyte damage.

Background: Fragmented cytokeratin 18 (fCK18) is released from damaged hepatocytes undergoing apoptosis and is recognized as a liver condition biomarker. We have developed a highly sensitive serum fCK18 CLEIA and reported that serum levels of this caspase-derived protein were significantly associated with hepatocyte ballooning, thus assisting in the accurate diagnosis of nonalcoholic steatohepatitis (NASH). We aim to investigate serum fCK18 levels in a variety of chronic liver diseases and to explore its potential as a prognostic marker of survival in hepatocellular carcinoma (HCC) patients. Methods: Serum fCK18 levels were measured using a highly sensitive CLEIA in 497 chronic liver disease patients (297 outpatients and 200 hospitalized with HCC). Results: In 497 chronic liver disease patients, serum fCK18 levels were significantly correlated with overall liver condition, including ALT, FIB-4 index and albumin-bilirubin (ALBI) score and were significantly increased in patients with HCC. In 200 HCC patients, serum fCK18 levels were significantly correlated with alpha-fetoprotein (AFP) and des-gamma-carboxy prothrombin (DCP), and were significantly associated with HCC stage, whereas FIB-4 index and ALBI score were not changed based on HCC stage. The Survival group had significantly lower levels of serum fCK18, AFP, DCP, FIB-4 index and ALBI score. A ROC analysis yield area under the curve (AUC) value of 0.728 for serum fCK18 is a significantly high value when compared to AUC measurements for other factors. Notably, AUROC values for serum fCK18 levels were constant in the short- and long-term by time-dependent ROC analysis for the prediction of HCC patient survival. HCC patients with serum fCK18 measured at < 1.15 ng/mL, AFP < 7.7 ng/mL, DCP < 133 mAU/mL, ALBI score < -2.97 or FIB-4 index < 6.4 had significantly longer rates of survival when compared to patients with values exceeding these thresholds. Serum fCK18 (HR, 3.5; P < 0.0001), DCP (HR, 3.2; P < 0.0001) and Barcelona Clinic Liver Cancer (BCLC) (HR, 2.4; P = 0.001) values were independent predictors of patient survival. [Conclusion] Serum fCK18 levels reflect overall liver function, the level of liver fibrosis and the progression of HCC, and are a potential predictor of survival in HCC patients.

Elevation of enterococcus-specific antibodies associated with bacterial translocation is predictive of survival rate in chronic liver disease.

Introduction/purpose: The gut-liver axis contributes to disease progression, a rise in infection rate, organ failure and a poor overall outcome in chronic liver diseases (CLD). Monitoring of the gut-liver axis is critical in understanding disease status, but biomarkers have not been elucidated. The aim of this study is to determine the level of serum antibodies against Enterococcus (E.) faecalis in evaluating patients with CLD, including those treated with rifaximin (a minimally absorbed antibiotic), and in patients with alcohol-associated liver disease (ALD). Materials and methods: We enrolled 109 CLD patients (cohort 1), 30 hepatic encephalopathy patients treated with rifaximin (cohort 2), 53 inpatients with ALD undergoing alcohol cessation (cohort 3) and 33 healthy subjects. To assess the consequences of E. faecalis translocation, we developed an assay for the detection of a serum antibody against E. faecalis capsular polysaccharide (E.CPS). Results: Serum E.CPS antibody titer was elevated only in those patients with advanced CLD and ALD. The E.CPS antibody titer was an independent prognostic factor (p < 0.05), while Mac-2 binding protein and albumin-bilirubin score were not independent predictors of survival. The improvement of predictive model in integrated factors was significant [continuous net reclassification index (value 0.699, p < 0.05) and integrated discrimination improvement (value 0.164, p = 0.051)]. Furthermore, rifaximin treatment led to a decrease of serum E.CPS antibody titer resulting in a significantly longer overall rate of survival. Conclusion: The E.CPS antibody titer appears to be a strong predictor of survival in CLD patients. Serum E.CPS levels decrease in CLD patients receiving rifaximin, and may be associated with an overall improvement in rate of survival.

Development of a New Index to Distinguish Hepatic Encephalopathy through Automated Quantification of Globus Pallidal Signal Intensity Using MRI.

Hyperintensities within the bilateral globus pallidus on T1-weighted magnetic resonance images were present in some liver cirrhosis patients with hepatic encephalopathy. The symptoms of covert hepatic encephalopathy are similar to those of mild dementia. We aimed to develop a new diagnostic index in which to distinguish hepatic encephalopathy from dementia. The globus pallidus signal hyperintensity was quantified using three-dimensional images. In addition, the new index value distribution was evaluated in a cohort of dementia patients. Signal intensity of globus pallidus significantly increased in liver cirrhosis patients with hepatic encephalopathy compared to those without hepatic encephalopathy (p < 0.05), healthy subjects (p < 0.05) or dementia patients (p < 0.001). Only 12.5% of liver cirrhosis patients without hepatic encephalopathy and 2% of dementia patients exceeded the new index cut-off value of 0.994, which predicts hepatic encephalopathy. One dementia patient in our evaluation had a history of liver cancer treatment and was assumed to have concomitant hepatic encephalopathy. The automatic assessment of signal intensity in globus pallidus is useful for distinguishing liver cirrhosis patients with hepatic encephalopathy from healthy subjects and liver cirrhosis patients without hepatic encephalopathy. Our image analyses exclude possible cases of hepatic encephalopathy from patients with neurocognitive impairment, including dementia.

A new detection system for serum fragmented cytokeratin 18 as a biomarker reflecting histologic activities of human nonalcoholic steatohepatitis.

Caspase-generated fragmented cytokeratin 18 (fCK18) is recognized as a useful noninvasive biomarker in the diagnosis of nonalcoholic fatty liver disease (NAFLD), particularly nonalcoholic steatohepatitis (NASH). However, fCK18 measurement is not applied clinically due to widely variable cut-off values under the current enzyme-linked immunosorbent assay platform. Therefore, we developed a highly sensitive chemiluminescent enzyme immunoassay using newly developed monoclonal antibodies against fCK18 and investigated its relevance in NASH diagnosis. Serum fCK18 levels were measured in the derivation and validation cohort. The correlation between serum fCK18 levels and NAFLD activity score (NAS), fibrosis stage, and liver function was examined. Serum fCK18 levels were significantly correlated with alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transpeptidase. Serum fCK18 levels were significantly associated with NAS, Brunt's grade/stage, Matteoni's classification, portal inflammation, and fat accumulation in the liver. Notably, hepatocyte ballooning was the only independent variable significantly associated with serum fCK18 in the multivariate linear regression analysis. Serum fCK18 levels were significantly elevated in patients with NAFLD and nonalcoholic fatty liver (NAFL) compared to healthy individuals. They were also significantly elevated in patients with NAFL compared to NASH defined by NAS or Matteoni's classification, with area under the curve values being 0.961 (NAFLD vs. healthy), 0.913 (NAFL vs. healthy), 0.763 (NASH vs. NAFL), and 0.796 (NASH type 3-4 vs. NAFL type 1-2). These results were confirmed by a validation cohort. Notably, changes over time in serum fCK18 levels were significantly correlated with changes in ALT, AST, and the fibrosis-4 index in 25 patients who underwent lifestyle modification. Serum fCK18 levels were significantly correlated with liver damage associated with NASH pathology. Serum fCK18 levels are accurate in distinguishing patients with NAFL or NASH from healthy individuals and may be useful to monitor NASH over time.

Inhibiting SCAP/SREBP exacerbates liver injury and carcinogenesis in murine nonalcoholic steatohepatitis.

Enhanced de novo lipogenesis mediated by sterol regulatory element-binding proteins (SREBPs) is thought to be involved in nonalcoholic steatohepatitis (NASH) pathogenesis. In this study, we assessed the impact of SREBP inhibition on NASH and liver cancer development in murine models. Unexpectedly, SREBP inhibition via deletion of the SREBP cleavage-activating protein (SCAP) in the liver exacerbated liver injury, fibrosis, and carcinogenesis despite markedly reduced hepatic steatosis. These phenotypes were ameliorated by restoring SREBP function. Transcriptome and lipidome analyses revealed that SCAP/SREBP pathway inhibition altered the fatty acid (FA) composition of phosphatidylcholines due to both impaired FA synthesis and disorganized FA incorporation into phosphatidylcholine via lysophosphatidylcholine acyltransferase 3 (LPCAT3) downregulation, which led to endoplasmic reticulum (ER) stress and hepatocyte injury. Supplementation with phosphatidylcholines significantly improved liver injury and ER stress induced by SCAP deletion. The activity of the SCAP/SREBP/LPCAT3 axis was found to be inversely associated with liver fibrosis severity in human NASH. SREBP inhibition also cooperated with impaired autophagy to trigger liver injury. Thus, excessively strong and broad lipogenesis inhibition was counterproductive for NASH therapy; this will have important clinical implications in NASH treatment.

Cell-to-Cell Communications in Alcohol-Associated Liver Disease.

This review covers some important new aspects of the alcohol-induced communications between liver parenchymal and non-parenchymal cells leading to liver injury development. The information exchange between various cell types may promote end-stage liver disease progression and involves multiple mechanisms, such as direct cell-to-cell interactions, extracellular vesicles (EVs) or chemokines, cytokines, and growth factors contained in extracellular fluids/cell culture supernatants. Here, we highlighted the role of EVs derived from alcohol-exposed hepatocytes (HCs) in activation of non-parenchymal cells, liver macrophages (LM), and hepatic stellate cells (HSC). The review also concentrates on EV-mediated crosstalk between liver parenchymal and non-parenchymal cells in the settings of HIV- and alcohol co-exposure. In addition, we overviewed the literature on the crosstalk between cell death pathways and inflammasome activation in alcohol-activated HCs and macrophages. Furthermore, we covered highly clinically relevant studies on the role of non-inflammatory factors, sinusoidal pressure (SP), and hepatic arterialization in alcohol-induced hepatic fibrogenesis. We strongly believe that the review will disclose major mechanisms of cell-to-cell communications pertained to alcohol-induced liver injury progression and will identify therapeutically important targets, which can be used for alcohol-associated liver disease (ALD) prevention.

Protein and miRNA profile of circulating extracellular vesicles in patients with primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is an idiopathic and heterogenous cholestatic liver disease characterized by chronic inflammation and fibrosis of the biliary tree. Currently, no effective therapies are available for this condition, whose incidence is rising. At present, specificity and sensitivity of current serum markers used to diagnose PSC are limited and often unreliable. In this study, we characterize circulating extracellular vesicles and provide supporting data on their potential use as novel surrogate biomarkers for PSC. EVs are membrane surrounded structures, 100-1000 nm in size, released by cells under various conditions and which carry a variety of bioactive molecules, including small non-coding RNAs, lipids and proteins. In recent years, a large body of evidence has pointed to diagnostic implications of EVs and relative cargo in various human diseases. We isolated EVs from serum of well-characterized patients with PSC or control subjects by differential centrifugation and size-exclusion chromatography. A complete characterization identified elevated levels of circulating EVs in PSC patients compared to healthy control subjects (2000 vs. 500 Calcein-FITC + EVs/µL). Tissue and cell specificity of circulating EVs was assessed by identification of liver-specific markers and cholangiocyte marker CK-19. Further molecular characterization identified 282 proteins that were differentially regulated in PSC-derived compared to healthy control-EVs. Among those, IL-13Ra1 was the most significantly and differentially expressed protein in PSC-derived EVs and correlated with the degree of liver fibrosis. In addition to protein profiling, we performed a miRNA-sequencing analysis which identified 11 among established, liver-specific (e.g., miR-122 and miR-192) and novel miRNAs. One of the newly identified miRNAs, miR-4645-3p, was significantly up-regulated fourfold in PSC-derived EVs compared to circulating EVs isolated from healthy controls. This study provides supporting evidence of the potential role of circulating EVs and associated protein and miRNA cargo as surrogate noninvasive and reliable biomarker for PSC.

Accuracy of carbohydrate-deficient transferrin as a biomarker of chronic alcohol abuse during treatment for alcoholism.

AIM: Clinical evaluations are generally used to verify the effectiveness of detoxification treatments for alcohol dependence, but new objective biomarkers are essential for accurate diagnosis. We aim to assess the accuracy of carbohydrate-deficient transferrin (%CDT) in a cohort of Japanese patients admitted to a psychiatric hospital specializing in alcohol dependence. In addition, we investigated the kinetics of %CDT during alcohol moderation or cessation. METHODS: The study cohort consisted of 126 alcohol-dependent patients. The levels of serum %CDT were assessed by the N Latex CDT direct immunonephelometric assay. RESULTS: Alcohol consumption was significantly correlated with %CDT. The only independent predictive factor of alcohol consumption was %CDT, with glutamyltranspeptidase (GGT) and albumin-bilirubin score proving insufficient. The cut-off value of %CDT was 1.9% with high sensitivity and specificity in detecting alcohol abstinence beyond 30 days (68.6% sensitivity, 91.8% specificity) and excessive alcohol drinking (77.9% sensitivity, 77.1% specificity). The %CDT levels were significantly decreased at 30 days of abstinence when compared with baseline. Notably, %CDT values were significantly changed even in the light alcohol drinking cohort (p = 0.0009), whereas GGT levels were not significantly changed. CONCLUSIONS: Our results indicate that %CDT is an accurate and specific biomarker of alcohol consumption and is useful in detecting alcohol abstinence even in a low alcohol intake patient cohort. These results suggest that %CDT could be a useful objective biomarker of chronic alcohol abuse during clinical treatment for alcoholism.

Extracellular vesicles from pancreatic ductal adenocarcinoma endoscopic ultrasound-fine needle aspiration samples contain a protein barcode.

BACKGROUND: The survival rate of pancreatic ductal adenocarcinoma (PDAC) is very poor because early detection is difficult. Extracellular vesicles (EVs) are released from cells associating with the cellular condition and circulated in the blood. We aimed to identify EV proteins from endoscopic ultrasound-fine needle aspiration (EUS-FNA) biopsy samples in order to develop novel biomarkers for PDAC. METHODS: Extracellular vesicles were isolated from EUS-FNA samples of 40 PDAC patients and six autoimmune pancreatitis (AIP) patients to be used as a control. EV proteins were identified using nanoLC-MS/MS. RESULTS: Intact EVs approximately 200 nm in diameter were detected from EUS-FNA samples. We identified 2059 or 1032 EV proteins in PDAC or AIP, respectively, and 1071 EV proteins were detected only in PDAC. One hundred and fifty-three EV proteins were significantly different between PDAC and AIP: 64 proteins were down-regulated in PDAC whereas 89 EV proteins were up-regulated in PDAC including mucins, keratins, Ras-related proteins, and olfactomedin-4, which proteins have been reported to be elevated in PDAC tissue/blood, or cultured pancreatic cancer cell lines. Notably, in the 89 up-regulated PDAC EV proteins we identified novel proteins including ADP-ribosylation factor 3, CD55, pyruvate kinase, and lipopolysaccharide-induced tumor necrosis factor. Out of 89 proteins, a total of 13 proteins including Ras-related proteins were significantly elevated in PDAC stages II-IV compared to PDAC stage I, including Ras-related proteins, moesin, and CD55. CONCLUSIONS: The EV proteins obtained from EUS-FNA samples contain a PDAC-specific protein barcode. The EV proteins identified from EUS-FNA samples include promising biomarkers for the diagnosis and clinical staging of PDAC.