Characterizing alcohol-related and metabolic dysfunction-associated steatotic liver disease cirrhosis via fibrotic pattern analysis.

This study aimed to address the diagnostic challenges in distinguishing between alcohol-related liver disease (ALD) and metabolic dysfunction-associated steatotic liver disease (MASLD). We utilized whole-slide imaging technology to conduct a comprehensive digital analysis of liver specimens collected from patients undergoing transplantation. This study included 36 and 17 patients with ALD and MASLD cirrhosis, respectively, who underwent transplantation at our institution. Digital slides were analyzed for fibrosis patterns using FibroNest™. Patient background characteristics were comparable between ALD (n = 36) and MASLD (n = 17) groups, except for sex. The ALD group exhibited thicker collagen per strand, longer and more flexural fibrosis, and a more heterogeneous distribution than the MASLD group. In patients with ALD and concomitant metabolic dysfunction, fiber distribution became relatively uniform, resembling MASLD. Application of the phenotypic fibrosis composite score achieved 100% sensitivity and specificity for ALD/MASLD diagnosis. Digital pathological analysis of the fibrosis patterns showed morphological differences between ALD and MASLD. This approach holds promise for histological differentiation, providing valuable insights beyond the current definitions based solely on alcohol intake. This study emphasizes the potential of digital pathology in refining the diagnostic criteria for hepatic disorders.

Elevated intestinal fatty acid-binding protein levels as a marker of portal hypertension and gastroesophageal varices in cirrhosis.

We measured intestinal fatty acid-binding protein (I-FABP) levels, a useful marker of small intestinal mucosal injury, in patients with cirrhosis to determine their relationship with liver function and complications. This cross-sectional study included 71 patients with cirrhosis admitted for treatment of cirrhotic complications or hepatocellular carcinoma (cohort A) and 104 patients with cirrhosis who received direct-acting antiviral therapy for HCV (cohort B). I-FABP levels, measured by ELISA, were evaluated relative to hepatic reserve and compared with non-invasive scoring systems for diagnostic performance in cirrhotic complications. The median I-FABP level in both cohorts were significantly elevated in patients with reduced hepatic reserve (CTP grade A/BC cohort A, 2.33/3.17 ng/mL, p = 0.032; cohort B, 2.46/3.64 ng/mL, p = 0.008) and complications with gastroesophageal varices (GEV; GEV (-)/(+) cohort A, 1.66/3.67 ng/mL, p < 0.001; cohort B, 2.32/3.36 ng/mL; p = 0.003). Further, multiple logistic regression analysis identified I-FABP as the only factor contributing to GEV presence in both cohorts, which outperformed non-invasive scoring systems for GEV diagnosis (sensitivity 84.6%; specificity 84.2%; sensitivity 69.6%; specificity 63.8%, respectively). In conclusion, elevated small-intestinal mucosal injury in patients with cirrhosis was related to reduced hepatic reserve and GEV presence. I-FABP levels reflect portal hypertension and may be useful in cirrhosis management.

Liver fibrosis analysis using digital pathology.

Digital pathology has enabled the noninvasive quantification of pathological parameters. In addition, the combination of digital pathology and artificial intelligence has enabled the analysis of a vast amount of information, leading to the sharing of much information and the elimination of knowledge gaps. Fibrosis, which reflects chronic inflammation, is the most important pathological parameter in chronic liver diseases, such as viral hepatitis and metabolic dysfunction-associated steatotic liver disease. It has been reported that the quantitative evaluation of various fibrotic parameters by digital pathology can predict the prognosis of liver disease and hepatocarcinogenesis. Liver fibrosis evaluation methods include 1 fiber quantification, 2 elastin and collagen quantification, 3 s harmonic generation/two photon excitation fluorescence (SHG/TPE) microscopy, and 4 Fibronest™..　In this review, we provide an overview of role of digital pathology on the evaluation of fibrosis in liver disease and the characteristics of recent methods to assess liver fibrosis.

Are transmembrane 6 superfamily member 2 gene polymorphisms associated with steatohepatitis after pancreaticoduodenectomy?

Aim: After pancreaticoduodenectomy, 20-40% of patients develop steatotic liver disease (SLD), and steatohepatitis can be a problem. Although patatin-like phospholipase domain-containing 3 protein (PNPLA3) and transmembrane 6 superfamily member 2 (TM6SF2) polymorphisms are involved in SLD and steatohepatitis development, whether this is the case after pancreaticoduodenectomy is unclear. Methods and Results: Forty-three patients with pancreatic cancer who underwent pancreaticoduodenectomy at our hospital between April 1, 2018, and March 31, 2021, were included. We extracted DNA from noncancerous areas of residual specimens after pancreaticoduodenectomy and determined PNPLA3 and TM6SF2 gene polymorphisms using real-time polymerase chain reaction. SLD was defined as a liver with an attenuation value of ≤40 HU or a liver-to-spleen ratio of ≤0.9 on computed tomography. We defined high hepatic fibrosis indexes (HFI) instead of steatohepatitis as a Fibrosis-4 index of ≥2.67 or nonalcoholic fatty liver disease fibrosis score of ≥0.675 in patients with SLD. The cumulative incidence of SLD (P = 0.299) and high HFI (P = 0.987) after pancreaticoduodenectomy were not significantly different between the PNPLA3 homozygous and minor allele groups. The incidences of high HFI at 1 year after pancreaticoduodenectomy were 16.8% and 27.0% in the TM6SF2 major homozygous and minor allele groups, respectively, with a significant difference in the cumulative incidence (P = 0.046). Conclusion: The TM6SF2 minor allele may contribute to steatohepatitis development after pancreaticoduodenectomy.

Total bile acids levels as a stratification tool for screening portopulmonary hypertension in patients with decompensated cirrhosis.

AIM: Echocardiography is necessary for portopulmonary hypertension diagnosis, and identifying patients with cirrhosis who require it is challenging. In this study, we aimed to investigate the utility of the total bile acid (TBA) levels as a screening tool for identifying patients with decompensated cirrhosis who should undergo echocardiography for portopulmonary hypertension diagnosis. METHODS: We evaluated 135 patients with decompensated cirrhosis who underwent liver transplantation. Subsequently, factors contributing to tricuspid regurgitation pressure gradient (TRPG) elevation (≥30 mmHg) were analyzed using preoperative data, including the TBA levels. RESULTS: The median age of patients was 58 years (61 women), and 45 and 90 patients had Child-Turcotte-Pugh grades of B and C, respectively. The median TRPG level was 21 mmHg, and 17 patients (12.6%) showed TRPG elevation. Multiple logistic regression analysis revealed that elevated TBA (odds ratio 4.322; p = 0.013) and main pulmonary artery diameter ≥33 mm (odds ratio 4.333; p = 0.016) were significantly associated with TRPG elevation. The TBA cut-off value (167.7 µmol/L) showed a high diagnostic performance, with 70.6% sensitivity and 64.4% specificity. Ursodeoxycholic acid (UDCA) administration increased the TBA levels dose-dependently. Analysis stratified by UDCA use revealed that in patients not taking UDCA (n = 59), elevated TBA levels and younger age significantly contributed to TRPG elevation. However, in those taking UDCA (n = 76), this contribution disappeared, suggesting that UDCA consumption reduced TBA levels' efficiency in diagnosing TRPG elevation. CONCLUSIONS: The TBA levels may be a potential screening tool for TRPG elevation; however, caution is warranted when interpreting cases treated with UDCA.

Investigation of deep learning model for predicting immune checkpoint inhibitor treatment efficacy on contrast-enhanced computed tomography images of hepatocellular carcinoma.

Although the use of immune checkpoint inhibitors (ICIs)-targeted agents for unresectable hepatocellular carcinoma (HCC) is promising, individual response variability exists. Therefore, we developed an artificial intelligence (AI)-based model to predict treatment efficacy using pre-ICIs contrast-enhanced computed tomography (CT) imaging characteristics. We evaluated the efficacy of atezolizumab and bevacizumab in 43 patients at the Nagasaki University Hospital from 2020 to 2022 using the modified Response Evaluation Criteria in Solid Tumors. A total of 197 Progressive Disease (PD), 271 Partial Response (PR), and 342 Stable Disease (SD) contrast CT images of HCC were used for training. We used ResNet-18 as the Convolutional Neural Network (CNN) model and YOLOv5, YOLOv7, YOLOv8 as the You Only Look Once (YOLO) model with precision-recall curves and class activation maps (CAMs) for diagnostic performance evaluation and model interpretation, respectively. The 3D t-distributed Stochastic Neighbor Embedding was used for image feature analysis. The YOLOv7 model demonstrated Precision 53.7%, Recall 100%, F1 score 69.8%, mAP@0.5 99.5% for PD, providing accurate and clinically versatile predictions by identifying decisive points. The ResNet-18 model had Precision 100% and Recall 100% for PD. However, the CAMs sites did not align with the tumors, suggesting the CNN model is not predicting that a given CT slice is PD, PR, or SD, but that it accurately predicts Individual Patient's CT slices. Preparing substantial training data for tumor drug effect prediction models is challenging compared to general tumor diagnosis models; hence, large-scale validation using an efficient YOLO model is warranted.

Non-liver-related mortality in the DAA era: Insights from post-SVR patients with and without previous HCC history.

BACKGROUND AND AIMS: Mortality after sustained virological response (SVR) with interferon-free direct-acting antiviral (IFN-free DAA) therapy is crucial for optimizing post-SVR patient care, but it remains unclear, especially regarding non-liver-related mortality. METHODS: Consecutive post-SVR patients from 14 institutions were stratified into three cohorts: A (without advanced fibrosis and without prior HCC), B (with advanced fibrosis and without prior HCC), and C (curative HCC treatment). We assessed mortality (per 1000 person-years [/1000PY]) post-SVR. Mortality rates were compared between cohorts A and B and the general population using age- and sex-adjusted standardized mortality ratio (SMR). Comparison of survival between each cohort was performed using propensity-score (PS) matching with sex, age, and comorbidity. RESULTS: In cohort A (n = 762; median age, 65 years), 22 patients died (median follow-up, 36 months); all-cause mortality was 10.0/1000PY, with 86.4% non-liver-related deaths. In cohort B (n = 519; median age, 73 years), 27 patients died (median follow-up, 39 months); all-cause mortality was 16.7/1000PY, with 88.9% non-liver-related deaths. In both cohorts, malignant neoplasm was the most common cause of death; all-cause mortality was comparable to that of the general population (SMR: 0.96 and 0.92). In cohort C (n = 108; median age, 75 years), 15 patients died (median follow-up, 51 months); all-cause mortality was 36.0/1000PY, with 53.3% liver-related deaths. PS matching showed no significant survival differences between cohorts A and B, both of which had better survival than cohort C. CONCLUSIONS: Mortality varies based on HCC history in the DAA era; nevertheless, attention should be paid to non-liver-related deaths in all post-SVR patients.

Comparison of the novel Franseen needle versus the fine-needle aspiration needle in endoscopic ultrasound-guided tissue acquisition for cancer gene panel testing: A propensity score-matching analysis.

Background and Aim: Reports have indicated that a surface area of 4 mm2 or more of collected tissue sections could provide the recommended total DNA for the OncoGuide NCC Oncopanel system, which is a cancer gene panel test developed in Japan. We wished to compare the percentage of tissue sections collected by endoscopic ultrasound-assisted tissue acquisition (EUS-TA) with surface areas of ≥4 mm2 between a conventional needle, namely the EZ Shot 3 Plus (Olympus Medical Japan, Tokyo, Japan) (EZ3), and the recent SonoTip TopGain (MediGlobe, Rohrdorf, Germany) (TopGain). Method: From April 2010 to December 2021, among 693 EUS-TA cases, EZ3 was used in 390 cases and TopGain in 45. The EZ3 and TopGain groups were matched in a 1:1 ratio with a tolerance of 0.2, with 35 patients each matched using propensity score analysis. Results: The TopGain group had a significantly higher percentage of cases with a tissue area of ≥4 mm2 than the EZ3 group (42.9% vs 68.6%, P = 0.030). Multivariate analysis revealed an association between TopGain and tissue areas of ≥4 mm2 (odds ratio 2.996, 95% confidence interval 1.068-8.403, P = 0.037). Conclusions: EUS-TA using TopGain significantly collected more ≥4 mm2 tissue area compared with EZ3, suggesting its usefulness for cancer gene panel testing.

Hypoglycemia measured by flash glucose monitoring system predicts liver-related events in chronic liver disease patients.

Impaired glucose tolerance, glucose fluctuations, and hypoglycemia have been observed in patients with chronic liver disease (CLD). The flash glucose monitoring (FGM) system, which recognises continuous and dynamic glucose changes in real time, is used in daily clinical practice. This study aimed to examine the association between glucose fluctuations and hypoglycemia, as measured by the FGM system, and liver-related events. Seventy-two patients with CLD and type 2 DM who had their blood glucose measured using Freestyle Libre Pro between April 2017 and July 2018 at our institution were enrolled in this retrospective study. We assessed the results of the FGM system measurements and liver-related events, as defined by gastrointestinal bleeding, infection, ascites, encephalopathy, and liver-related death. The standard deviation (SD) of mean glucose as measured by the FGM system was 41.55 mg/dl, and hypoglycemia was observed in 48.6% (35/72) of the patients. Liver-related event-free survival was not significant when stratified based on SD; however, the event-free survival was significantly lower when stratified by hypoglycemia (p = 0.007). In a multivariate analysis using the Cox proportional hazards model, Child-Pugh class B [Hazards ratio (HR) 2.347 (95% confidence interval (CI): 1.042-5.283), p = 0.039] and hypoglycemia [HR 2.279 (95% CI: 1.064-4.881), p = 0.034] were identified as factors contributing to event-free survival. Hypoglycemia, as determined by the FGM system, was identified as a significant factor that was closely associated with liver-related events. In addition to measuring glucose levels, the FGM system is useful in predicting the occurrence of liver-related events.

Modulating sphingosine 1-phosphate receptor signaling skews intrahepatic leukocytes and attenuates murine nonalcoholic steatohepatitis.

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid associated with nonalcoholic steatohepatitis (NASH). Immune cell-driven inflammation is a key determinant of NASH progression. Macrophages, monocytes, NK cells, T cells, NKT cells, and B cells variably express S1P receptors from a repertoire of 5 receptors termed S1P1 - S1P5. We have previously demonstrated that non-specific S1P receptor antagonism ameliorates NASH and attenuates hepatic macrophage accumulation. However, the effect of S1P receptor antagonism on additional immune cell populations in NASH remains unknown. We hypothesized that S1P receptor specific modulation may ameliorate NASH by altering leukocyte recruitment. A murine NASH model was established by dietary feeding of C57BL/6 male mice with a diet high in fructose, saturated fat, and cholesterol (FFC) for 24 weeks. In the last 4 weeks of dietary feeding, the mice received the S1P1,4,5 modulator Etrasimod or the S1P1 modulator Amiselimod, daily by oral gavage. Liver injury and inflammation were determined by histological and gene expression analyses. Intrahepatic leukocyte populations were analyzed by flow cytometry, immunohistochemistry, and mRNA expression. Alanine aminotransferase, a sensitive circulating marker for liver injury, was reduced in response to Etrasimod and Amiselimod treatment. Liver histology showed a reduction in inflammatory foci in Etrasimod-treated mice. Etrasimod treatment substantially altered the intrahepatic leukocyte populations through a reduction in the frequency of T cells, B cells, and NKT cells and a proportional increase in CD11b+ myeloid cells, polymorphonuclear cells, and double negative T cells in FFC-fed and control standard chow diet (CD)-fed mice. In contrast, FFC-fed Amiselimod-treated mice showed no changes in the frequencies of intrahepatic leukocytes. Consistent with the improvement in liver injury and inflammation, hepatic macrophage accumulation and the gene expression of proinflammatory markers such as Lgals3 and Mcp-1 were decreased in Etrasimod-treated FFC-fed mice. Etrasimod treated mouse livers demonstrated an increase in non-inflammatory (Marco) and lipid associated (Trem2) macrophage markers. Thus, S1P1,4,5 modulation by Etrasimod is more effective than S1P1 antagonism by Amiselimod, at the dose tested, in ameliorating NASH, likely due to the alteration of leukocyte trafficking and recruitment. Etrasimod treatment results in a substantial attenuation of liver injury and inflammation in murine NASH.

Peripheral lymphocyte fluctuation as an indicator of severe immune-related adverse events in patients treated with immune checkpoint inhibitors.

AIM: Immune checkpoint inhibitors (ICIs) have proven to be effective treatments for various cancers, but can also elicit immune-related adverse events (irAEs). Given that severe irAEs can be life-threatening, biomarkers that can predict the occurrence of irAEs are of paramount importance. ICIs affect the dynamics of lymphocytes, and alterations in these dynamics may play a role in the development and severity of irAEs. The aim of this study was to investigate the correlation between irAEs and changes in lymphocyte counts. METHODS: Information on irAEs was collected from 226 ICI cases from 2014 to 2020. We compared lymphocyte counts before treatment and at the onset of irAE and investigated the association between lymphocyte count fluctuations and the presence and severity of irAE, the course after steroid treatment, and overall survival. RESULTS: Of the 226 cases, 27 patients developed grade 3 or higher irAE. Compared to the other groups, the lymphocyte count in this group was significantly decreased at the time of irAE (p < 0.01). There was a trend toward a rapid increase in lymphocyte count in the steroid responder group compared to the non-responder group. Regarding overall survival, patients with irAE had significantly longer survival than those without irAE (p = 0.0025). However, there was no association between changes in lymphocyte count and survival in patients with irAE. CONCLUSION: The percentage change in lymphocyte count was found to correlate with the incidence of severe irAEs. Close monitoring of the patient's condition is crucial when the lymphocyte count decreases during ICI treatment.

Evans' syndrome induced by atezolizumab plus bevacizumab combination therapy in advanced hepatocellular carcinoma.

An 86-year-old man presented with recurrence of hepatocellular carcinoma (HCC) after surgery. Atezolizumab plus bevacizumab was initiated. After the third course of atezolizumab plus bevacizumab therapy, petechial purpura appeared on the extremities and trunk. Laboratory tests revealed isolated severe thrombocytopenia without evidence of combined coagulopathy. He was diagnosed with immune thrombocytopenic purpura (ITP), and high-dose immunoglobulin and Helicobacter pylori eradication therapies were administered. Improvement in thrombocytopenia was observed; however, 20 days after the onset of ITP, laboratory data revealed hemolytic anemia. Both direct and indirect Coombs tests were positive, and he was diagnosed with Evan's syndrome complicated by ITP and autoimmune hemolytic anemia (AIHA) induced by immune-related adverse events (irAEs). After treatment with prednisolone, the hemoglobin level increased, and hemolytic findings improved on blood tests. We encountered a rare case of Evans' syndrome due to atezolizumab plus bevacizumab therapy for HCC. In atezolizumab plus bevacizumab therapy, hematologic toxicities are not rare adverse events and attention is required.

Evaluation of a PEGylated Fibroblast Growth Factor 21 Variant Using Novel Preclinical Magnetic Resonance Imaging and Magnetic Resonance Elastography in a Mouse Model of Nonalcoholic Steatohepatitis.

BACKGROUND: Treatments for nonalcoholic steatohepatitis (NASH) are urgently needed. Hepatic fat fraction and shear stiffness quantified by magnetic resonance imaging (MRI-HFF) and magnetic resonance elastography (MRE-SS), respectively, are biomarkers for hepatic steatosis and fibrosis. PURPOSE: This study assessed the longitudinal effects of fibroblast growth factor 21 variant (polyethylene glycol [PEG]-FGF21v) on MRI-HFF and MRE-SS in a NASH mouse model. STUDY TYPE: Preclinical. ANIMAL MODEL: This study included a choline-deficient, amino acid-defined, high-fat diet (CDAHFD) model and 6-week-old, male C57BL/6J mice (N = 78). FIELD STRENGTH/SEQUENCE: This study was performed using: 3T: gradient-echo two-point Dixon and spin-echo (SE) echo-planar imaging elastography (200 Hz) and 7T: SE two-point Dixon and SE elastography (200 Hz). ASSESSMENT: MRI and MRE were performed before control diet (CD) or CDAHFD (BD), before PEG-FGF21v dosing (baseline), and after PEG-FGF21v treatment (WK4/8). Regions of interest for MRI-HFF and MRE-SS were delineated by J.L. and H.T. (>5 years of experience each). Fibrosis and steatosis were measured histologically after picrosirius red and H&E staining. Alkaline phosphatase, alanine transaminase, bile acids, and triglycerides (TGs) were measured. STATISTICAL TESTS: Two-tailed Dunnett's tests were used for statistical analysis; untreated CDAHFD or baseline was used for comparisons. Imaging and histology/biochemistry data were determined using Spearman correlations. Bayesian posterior distributions for MRE-SS at WK8, posterior means, and 95% credible intervals were presented. RESULTS: CDAHFD significantly increased baseline MRI-HFF (3T: 21.97% ± 0.29%; 7T: 40.12% ± 0.35%) and MRE-SS (3T: 1.25 ± 0.02; 7T: 1.78 ± 0.06 kPa) vs. CD (3T: 3.45% ± 0.7%; 7T: 12.06% ± 1.4% and 3T: 1.01 ± 0.02; 7T: 0.89 ± 0.06 kPa). At 7T, PEG-FGF21v significantly decreased MRI-HFF (WK4: 28.97% ± 1.22%; WK8: 20.93% ± 1.15%) and MRE-SS (WK4: 1.57 ± 0.04; WK8: 1.36 ± 0.05 kPa) vs. untreated (WK4: 36.36% ± 0.62%; WK8: 30.58% ± 0.81% and WK4: 2.03 ± 0.06; WK8: 2.01 ± 0.04 kPa); 3T trends were similar. WK8 SS posterior mean percent attenuation ratios (RDI ) were -68% (-90%, -44%; 3T) and -64% (-78%, -52%; 7T). MRI-HFF was significantly correlated with H&E (3T, r = 0.93; 7T, r = 0.94) and TGs (both, r = 0.92). DATA CONCLUSIONS: MRI-HFF and MRE-SS showed PEG-FGF21v effects on hepatic steatosis and fibrosis across 3 and 7T, consistent with histological and biochemical data. LEVEL OF EVIDENCE: 1 TECHNICAL EFFICACY STAGE: 2.

A Comparative Proteomic Analysis of Extracellular Vesicles Associated With Lipotoxicity.

Extracellular vesicles (EVs) are emerging mediators of intercellular communication in nonalcoholic steatohepatitis (NASH). Palmitate, a lipotoxic saturated fatty acid, activates hepatocellular endoplasmic reticulum stress, which has been demonstrated to be important in NASH pathogenesis, including in the release of EVs. We have previously demonstrated that the release of palmitate-stimulated EVs is dependent on the de novo synthesis of ceramide, which is trafficked by the ceramide transport protein, STARD11. The trafficking of ceramide is a critical step in the release of lipotoxic EVs, as cells deficient in STARD11 do not release palmitate-stimulated EVs. Here, we examined the hypothesis that protein cargoes are trafficked to lipotoxic EVs in a ceramide-dependent manner. We performed quantitative proteomic analysis of palmitate-stimulated EVs in control and STARD11 knockout hepatocyte cell lines. Proteomics was performed on EVs isolated by size exclusion chromatography, ultracentrifugation, and density gradient separation, and EV proteins were measured by mass spectrometry. We also performed human EV proteomics from a control and a NASH plasma sample, for comparative analyses with hepatocyte-derived lipotoxic EVs. Size exclusion chromatography yielded most unique EV proteins. Ceramide-dependent lipotoxic EVs contain damage-associated molecular patterns and adhesion molecules. Haptoglobin, vascular non-inflammatory molecule-1, and insulin-like growth factor-binding protein complex acid labile subunit were commonly detected in NASH and hepatocyte-derived ceramide-dependent EVs. Lipotoxic EV proteomics provides novel candidate proteins to investigate in NASH pathogenesis and as diagnostic biomarkers for hepatocyte-derived EVs in NASH patients.

Circulating extracellular vesicles are a biomarker for NAFLD resolution and response to weight loss surgery.

There is increasing interest in the development of minimally invasive biomarkers for the diagnosis and prognosis of NAFLD via extracellular vesicles (EV). Plasma EVs were isolated by differential ultracentrifugation and quantified by nanoparticle tracking analysis from pre (n = 28) and post (n = 28) weight loss patients. In the pre weight loss group 22 had NAFLD. Nanoplasmon enhanced scattering (nPES) of gold nanoparticles conjugated to hepatocyte-specific antibodies was employed to identify hepatocyte-specific EVs. Complex lipid panel and targeted sphingolipids were performed. Logistic regression analysis was used to identify predictors of NAFLD. Plasma levels of EVs and hepatocyte-derived EVs are dynamic and decrease following NAFLD resolution due to weight loss surgery. Hepatocyte-derived EVs correlate with steatosis in NAFLD patients and steatosis and inflammation in NASH patients. Plasma levels of small EVs correlate with EV sphingolipids in patients with NASH. Hepatocyte-derived EVs measured by the nPES assay could serve as a point-of-care test for NAFLD.

Geranylgeranylacetone decreases the production of hepatitis B virus-related antigen by comprehensive downregulation of mRNA transcription activity.

BACKGROUND AND AIM: Elimination of hepatitis B virus (HBV) is infrequently achieved with current therapies. Therefore, more effective anti-HBV therapy is needed. We previously reported that geranylgeranylacetone (GGA) showed anti-hepatitis C virus activity in human hepatoma cells. In this study, we examined the anti-HBV activity of GGA. METHODS: We used HepG2.2.15.7 cells, PXB cells infected with HBV, Huh7 cells transfected with linear HBV, and PLC/PRF/5 cells as HBV-infected hepatocyte models. After GGA treatment, HBV-related antigen was measured by chemiluminescent immunoassay. HBV-related mRNA was examined by Northern blot. cccDNA and endoplasmic reticulum stress markers were measured by real-time polymerase chain reaction. The activities of HBV promoters and enhancer regions were examined using luciferase vectors. RESULTS: After GGA treatment, hepatitis B surface antigen and hepatitis B e antigen secretion was decreased in all HBV-infected hepatocyte models. HBV-related mRNA was also decreased by GGA treatment, although cccDNA levels were not affected. Additionally, the activity of HBV S1 and S2 promoter region and Enhancer 1/Enhancer 2/core promoter region was reduced by GGA treatment. The mRNA expression of the main transcription factors, hepatocyte nuclear factor 3 and 4 and CCAAT/enhancer binding protein, was also decreased. Further, the expression levels of endoplasmic reticulum stress markers were increased by GGA treatment, which reflected the change in HBV-related antigen secretion. CONCLUSIONS: Geranylgeranylacetone treatment reduces HBV-related protein levels by suppressing comprehensive downregulation of HBV promoter and enhancer activity, which might be caused by decreased hepatic transcription factor expression. GGA treatment may enhance anti-HBV effects in combination with other therapies.

Lipid-induced endothelial vascular cell adhesion molecule 1 promotes nonalcoholic steatohepatitis pathogenesis.

Monocyte homing to the liver and adhesion to the liver sinusoidal endothelial cells (LSECs) are key elements in nonalcoholic steatohepatitis (NASH) pathogenesis. We reported previously that VCAM-1 mediates monocyte adhesion to LSECs. However, the pathogenic role of VCAM-1 in NASH is unclear. Herein, we report that VCAM-1 was a top upregulated adhesion molecule in the NASH mouse liver transcriptome. Open chromatin landscape profiling combined with genome-wide transcriptome analysis showed robust transcriptional upregulation of LSEC VCAM-1 in murine NASH. Moreover, LSEC VCAM-1 expression was significantly increased in human NASH. LSEC VCAM-1 expression was upregulated by palmitate treatment in vitro and reduced with inhibition of the mitogen-activated protein 3 kinase (MAP3K) mixed lineage kinase 3 (MLK3). Likewise, LSEC VCAM-1 expression was reduced in the Mlk3-/- mice with diet-induced NASH. Furthermore, VCAM-1 neutralizing Ab or pharmacological inhibition attenuated diet-induced NASH in mice, mainly via reducing the proinflammatory monocyte hepatic population as examined by mass cytometry by time of flight (CyTOF). Moreover, endothelium-specific Vcam1 knockout mice were also protected against NASH. In summary, lipotoxic stress enhances the expression of LSEC VCAM-1, in part, through MLK3 signaling. Inhibition of VCAM-1 was salutary in murine NASH and might serve as a potential therapeutic strategy for human NASH.

Circulating Extracellular Vesicles Carrying Sphingolipid Cargo for the Diagnosis and Dynamic Risk Profiling of Alcoholic Hepatitis.

BACKGROUND AND AIMS: Alcoholic hepatitis (AH) is diagnosed by clinical criteria, although several objective scores facilitate risk stratification. Extracellular vesicles (EVs) have emerged as biomarkers for many diseases and are also implicated in the pathogenesis of AH. Therefore, we investigated whether plasma EV concentration and sphingolipid cargo could serve as diagnostic biomarkers for AH and inform prognosis to permit dynamic risk profiling of AH subjects. APPROACH AND RESULTS: EVs were isolated and quantified from plasma samples from healthy controls, heavy drinkers, and subjects with end-stage liver disease (ESLD) attributed to cholestatic liver diseases and nonalcoholic steatohepatitis, decompensated alcohol-associated cirrhosis (AC), and AH. Sphingolipids were quantified by tandem mass spectroscopy. The median plasma EV concentration was significantly higher in AH subjects (5.38 × 1011 /mL) compared to healthy controls (4.38 × 1010 /mL; P < 0.0001), heavy drinkers (1.28 × 1011 /mL; P < 0.0001), ESLD (5.35 × 1010 /mL; P < 0.0001), and decompensated AC (9.2 × 1010 /mL; P < 0.0001) disease controls. Among AH subjects, EV concentration correlated with Model for End-Stage Liver Disease score. When EV counts were dichotomized at the median, survival probability for AH subjects at 90 days was 63.0% in the high-EV group and 90.0% in the low-EV group (log-rank P value = 0.015). Interestingly, EV sphingolipid cargo was significantly enriched in AH when compared to healthy controls, heavy drinkers, ESLD, and decompensated AC (P = 0.0001). Multiple sphingolipids demonstrated good diagnostic and prognostic performance as biomarkers for AH. CONCLUSIONS: Circulating EV concentration and sphingolipid cargo signature can be used in the diagnosis and differentiation of AH from heavy drinkers, decompensated AC, and other etiologies of ESLD and predict 90-day survival permitting dynamic risk profiling.

IRE1A Stimulates Hepatocyte-Derived Extracellular Vesicles That Promote Inflammation in Mice With Steatohepatitis.

BACKGROUND & AIMS: Endoplasmic reticulum to nucleus signaling 1 (ERN1, also called IRE1A) is a sensor of the unfolded protein response that is activated in the livers of patients with nonalcoholic steatohepatitis (NASH). Hepatocytes release ceramide-enriched inflammatory extracellular vesicles (EVs) after activation of IRE1A. We studied the effects of inhibiting IRE1A on release of inflammatory EVs in mice with diet-induced steatohepatitis. METHODS: C57BL/6J mice and mice with hepatocyte-specific disruption of Ire1a (IRE1αΔhep) were fed a diet high in fat, fructose, and cholesterol to induce development of steatohepatitis or a standard chow diet (controls). Some mice were given intraperitoneal injections of the IRE1A inhibitor 4µ8C. Mouse liver and primary hepatocytes were transduced with adenovirus or adeno-associated virus that expressed IRE1A. Livers were collected from mice and analyzed by quantitative polymerase chain reaction and chromatin immunoprecipitation assays; plasma samples were analyzed by enzyme-linked immunosorbent assay. EVs were derived from hepatocytes and injected intravenously into mice. Plasma EVs were characterized by nanoparticle-tracking analysis, electron microscopy, immunoblots, and nanoscale flow cytometry; we used a membrane-tagged reporter mouse to detect hepatocyte-derived EVs. Plasma and liver tissues from patients with NASH and without NASH (controls) were analyzed for EV concentration and by RNAscope and gene expression analyses. RESULTS: Disruption of Ire1a in hepatocytes or inhibition of IRE1A reduced the release of EVs and liver injury, inflammation, and accumulation of macrophages in mice on the diet high in fat, fructose, and cholesterol. Activation of IRE1A, in the livers of mice, stimulated release of hepatocyte-derived EVs, and also from cultured primary hepatocytes. Mice given intravenous injections of IRE1A-stimulated, hepatocyte-derived EVs accumulated monocyte-derived macrophages in the liver. IRE1A-stimulated EVs were enriched in ceramides. Chromatin immunoprecipitation showed that IRE1A activated X-box binding protein 1 (XBP1) to increase transcription of serine palmitoyltransferase genes, which encode the rate-limiting enzyme for ceramide biosynthesis. Administration of a pharmacologic inhibitor of serine palmitoyltransferase to mice reduced the release of EVs. Levels of XBP1 and serine palmitoyltransferase were increased in liver tissues, and numbers of EVs were increased in plasma, from patients with NASH compared with control samples and correlated with the histologic features of inflammation. CONCLUSIONS: In mouse hepatocytes, activated IRE1A promotes transcription of serine palmitoyltransferase genes via XBP1, resulting in ceramide biosynthesis and release of EVs. The EVs recruit monocyte-derived macrophages to the liver, resulting in inflammation and injury in mice with diet-induced steatohepatitis. Levels of XBP1, serine palmitoyltransferase, and EVs are all increased in liver tissues from patients with NASH. Strategies to block this pathway might be developed to reduce liver inflammation in patients with NASH.