Search

BACKGROUND: There is a need for novel noninvasive markers for metabolic dysfunction-associated steatotic liver disease (MASLD) to stratify patients at high risk for liver-related events including liver cancer and decompensation. In the present study, we used proteomic analysis of proteins in extracellular vesicles (EVs) to identify new biomarkers that change with fibrosis progression and can predict the development of liver-related events. METHODS: We analyzed serum EVs from 50 patients with MASLD assessed for liver fibrosis by biopsy and identified proteins that altered with advanced fibrosis. A further evaluation was conducted on another cohort of 463 patients with MASLD with biopsy. RESULTS: Eight candidate proteins were identified by proteomic analysis of serum EVs. Among them, serum levels of Fibulin-3, Fibulin-1, and Ficolin 1 correlated with their EV levels. In addition, serum Fibulin-3 and serum Fibulin-1 levels changed significantly with advanced fibrosis. Using another cohort with biopsy, we found that the serum Fibulin-3 concentration was significantly greater in those with advanced fibrosis but that the serum Fibulin-1 concentration was not significantly different. Multivariate Cox proportional hazards analysis revealed that a higher Fibrosis-4 (FIB-4) index and higher serum Fibulin-3 concentration were independent risk factors for liver-related events. When the cutoff value for the serum Fibulin-3 concentration was 6.0 µg/mL according to the Youden index of AUROCs, patients with high serum Fibulin-3 significantly more frequently developed liver-related events than did other patients. Validation using another cohort of 226 patients with clinically diagnosed MASLD confirmed that high serum Fibulin-3 levels are associated with a greater frequency of liver-related events. CONCLUSIONS: Serum Fibulin-3 was identified as a biomarker for predicting liver-related events in patients with MASLD.

Currently, there is no effective treatment for obesity and alcohol-associated liver diseases, partially due to the lack of translational human models. Here, we present a protocol to generate 3D human liver spheroids that contain all the liver cell types and mimic "livers in a dish." We describe strategies to induce metabolic and alcohol-associated hepatic steatosis, inflammation, and fibrosis. We outline potential applications, including using human liver spheroids for experimental and translational research and drug screening to identify potential anti-fibrotic therapies.

The mechanisms of hepatic stellate cell (HSC) activation and the development of liver fibrosis are not fully understood. Here, we show that deletion of a nuclear seven transmembrane protein, TM7SF3, accelerates HSC activation in liver organoids, primary human HSCs, and in vivo in metabolic-dysfunction-associated steatohepatitis (MASH) mice, leading to activation of the fibrogenic program and HSC proliferation. Thus, TM7SF3 knockdown promotes alternative splicing of the Hippo pathway transcription factor, TEAD1, by inhibiting the splicing factor heterogeneous nuclear ribonucleoprotein U (hnRNPU). This results in the exclusion of the inhibitory exon 5, generating a more active form of TEAD1 and triggering HSC activation. Furthermore, inhibiting TEAD1 alternative splicing with a specific antisense oligomer (ASO) deactivates HSCs in vitro and reduces MASH diet-induced liver fibrosis. In conclusion, by inhibiting TEAD1 alternative splicing, TM7SF3 plays a pivotal role in mitigating HSC activation and the progression of MASH-related fibrosis.

Alcohol-associated liver disease (ALD) is a substantial cause of morbidity and mortality worldwide and represents a spectrum of liver injury beginning with hepatic steatosis (fatty liver) progressing to inflammation and culminating in cirrhosis. Multiple factors contribute to ALD progression and disease severity. Here, we overview several crucial mechanisms related to ALD end-stage outcome development, such as epigenetic changes, cell death, hemolysis, hepatic stellate cells activation, and hepatic fatty acid binding protein 4. Additionally, in this review, we also present two clinically relevant models using human precision-cut liver slices and hepatic organoids to examine ALD pathogenesis and progression.

Liver fibrosis of different etiologies is a serious health problem worldwide. There is no effective therapy available for liver fibrosis except the removal of the underlying cause of injury or liver transplantation. Development of liver fibrosis is caused by fibrogenic myofibroblasts that are not present in the normal liver, but rather activate from liver resident mesenchymal cells in response to chronic toxic or cholestatic injury. Many studies indicate that liver fibrosis is reversible when the causative agent is removed. Regression of liver fibrosis is associated with the disappearance of activated myofibroblasts and resorption of the fibrous scar. In this review, we discuss the results of genetic tracing and cell fate mapping of hepatic stellate cells and portal fibroblasts, their specific characteristics, and potential phenotypes. We summarize research progress in the understanding of the molecular mechanisms underlying the development and reversibility of liver fibrosis, including activation, apoptosis, and inactivation of myofibroblasts.

Here, we present a protocol for isolating human hepatocytes and neural progenitor cells from normal and nonalcoholic steatohepatitis livers. We describe steps for perfusion for scaled-up liver cell isolation and optimization of chemical digestion to achieve maximal yield and cell viability. We then detail a liver cell cryopreservation and potential applications, such as the use of human liver cells as a tool to link experimental and translational research.

Summary: Background and Aims: Although metabolic dysfunction‐associated steatotic liver disease (MASLD) patients with a Fib‐4 index >1.3 are recommended for fibrosis evaluation via elastography or biopsy, a more convenient method identifying high‐risk populations requiring follow‐up is needed. We explored the utility of serum levels of growth differentiation factor‐15 (GDF15), a cell stress‐responsive cytokine related to metabolic syndrome, for stratifying the risk of clinical events in MASLD patients. Methods: Serum GDF15 levels were measured in 518 biopsy‐performed MASLD patients, 216 MASLD patients for validation, and 361 health checkup recipients with MASLD. Results: In the biopsy‐MASLD cohort, multivariate analysis indicated that the serum GDF15 level was a risk factor for liver cancer, independent of the fibrosis stage or Fib‐4 index. Using a GDF15 cutoff of 1.75 ng/mL based on the Youden index, high‐GDF15 patients, regardless of fibrosis status, had a higher liver cancer incidence rate. While patients with a Fib‐4 index <1.3 or low‐GDF15 rarely developed liver cancer, high‐GDF15 patients with a Fib‐4 index >1.3 developed liver cancer and decompensated liver events at significantly higher rates and had poorer prognoses. In the validation cohort, high‐GDF15 patients had significantly higher incidences of liver cancer and decompensated liver events and poorer prognoses than low‐GDF15 patients, whether limited to high‐Fib‐4 patients. Among health checkup recipients with MASLD, 23.0% had a Fib‐4 index >1.3, 2.7% had a Fib‐4 index >1.3 and >1.75 ng/mL GDF15. Conclusions: Serum GDF15 is a biomarker for liver cancer with high predictive capability and is useful for identifying MASLD patients requiring regular surveillance. [ABSTRACT FROM AUTHOR]

This article, titled "Featured Cover," discusses a study conducted by Shusuke Kumazaki et al. on the use of serum growth differentiation factor 15 (GDF15) as a biomarker for liver cancer occurrence in patients with metabolic-associated fatty liver disease (MASLD), regardless of liver fibrosis. The study found that GDF15 showed high predictive capability for liver cancer in these patients. The authors of the study include a diverse group of researchers from various institutions. The article provides a valuable resource for library patrons researching liver cancer and its biomarkers. [Extracted from the article]

Summary: Background: After hepatitis C virus (HCV) elimination, patients should be followed up due to risk of hepatocellular carcinoma (HCC). Growth differentiation factor 15 (GDF15) is a cytokine induced by mitochondrial dysfunction or oxidative stress. Aim To evaluate the prognostic value of GDF15 for HCC occurrence after HCV elimination. Methods: We measured GDF15 levels in stored serum from patients with chronic HCV infection without a history of HCC who had achieved sustained virological response with direct‐acting antiviral agents (DAAs). The patients were randomly divided into derivation (n = 964) and validation (n = 642) cohorts. Results: In the derivation cohort, serum GDF15 levels were higher in those with HCC occurrence after DAA treatment than in those without. Multivariate Cox proportional hazards analysis revealed baseline GDF15 (>1350 pg/mL, HR 2.54), AFP (>5 ng/mL, HR 2.00), and the FIB‐4 index (>3.25, HR 2.69) to be independent risk factors for HCC. Scoring based on GDF15, AFP and the FIB‐4 index stratified HCC occurrence risk. In the validation cohort, the cumulative HCC occurrence rate at 3 years was 0.64%, 3.27% and 15.3% in low‐score (N = 171), medium‐score (N = 300) and high‐score (N = 166) groups, respectively. In the total cohort, scoring divided patients with a FIB‐4 index ≤3.25, whose HCC occurrence rate was 2.0% at 3 years, into medium‐score and low‐score groups with HCC occurrence rates at 3 years of 3.76% and 0.24%, respectively. Conclusions: Serum GDF15 predicts de novo HCC occurrence. Scoring using GDF15, AFP, and the FIB‐4 index can predict de novo HCC occurrence risk after HCV elimination. [ABSTRACT FROM AUTHOR]

Radiation therapy is one of the treatment methods for hepatocellular carcinoma. However, radiation tolerance of the liver is low, and the detailed effect of radiation on liver regeneration has not been clarified. C57BL/6J mice or hepatocyte‐specific p53 knockout (KO) mice (albumin [Alb]‐Cre Trp53flox/flox) were irradiated with a single fraction of 10 Gy localized to the upper abdomen. We performed 70% partial hepatectomy (PHx) 24 hours after irradiation. Liver regeneration was assessed by proliferation cell nuclear antigen (PCNA)‐ and Ki‐67‐positive hepatocyte ratios and liver‐to‐body weight ratio after PHx. To establish a fibrosis model, CCl4 was orally administered for 8 weeks. The murine hepatocyte cell line BNL CL.2 (CL2) was irradiated with 10 Gy. Irradiation activated p53, induced downstream p21 in the liver, and delayed liver regeneration after PHx. While PHx increased hepatocyte growth factor (HGF) levels and activated Met with or without irradiation in the regenerative liver, it activated Akt and extracellular kinase 1 and 2 (Erk 1/2) less in irradiated mice than in nonirradiated mice. In CL2 cells cultured with HGF, irradiation suppressed cell growth by decreasing phosphorylated Akt and Erk 1/2 levels, which was abolished by small interfering RNA‐mediated p53 knockdown but not by p21 knockdown. Hepatocyte‐specific knockout of p53 in mice abolished the irradiation‐induced suppression of both liver regeneration and Akt and Erk 1/2 activation after PHx. In the fibrotic mouse model, the survival rate after PHx of irradiated p53 KO mice was higher than that of wild‐type mice. Conclusion: p53 but not p21 is involved in the impaired regenerative ability of the irradiated liver. [ABSTRACT FROM AUTHOR]

Background and Aims: Metabolic dysfunction-associated steatotic liver disease (MASLD) ranges from Metabolic dysfunction-associated steatotic liver (MASL) to Metabolic dysfunction-associated steatohepatitis (MASH) with fibrosis. Activation of Hepatic Stellate Cells (HSCs) into fibrogenic myofibroblasts plays a critical role in the pathogenesis of MASH liver fibrosis. We compared transcriptome and chromatin accessibility of human HSCs from NORMAL, MASL, and MASH livers at single cell resolution. We aimed to identify genes that are upregulated in activated HSCs and to determine which of these genes are key in the pathogenesis of MASH fibrosis., Methods: 18 human livers were profiled using single-nucleus (sn)RNA-seq and snATAC-seq. High priority targets were identified, then tested in 2D human HSC cultures, 3D human liver spheroids, and HSC-specific gene knockout mice., Results: MASH-enriched activated (A) HSC subclusters are the major source of extracellular matrix proteins. We identified a set of concurrently upregulated and more accessible core genes (GAS7, SPON1, SERPINE1, LTBP2, KLF9, EFEMP1) that drive activation of (A) HSC subclusters. Expression of these genes was regulated via crosstalk between lineage-specific (JUNB/AP1), cluster-specific (RUNX1/2) and signal-specific (FOXA1/2) transcription factors. The pathological relevance of the selected targets, such as SERPINE1 (PAI-1), was demonstrated using dsiRNA-based HSC-specific gene knockdown or pharmacological inhibition of PAI-1 in 3D human MASH liver spheroids, and HSC-specific Serpine1 knockout mice., Conclusion: This study identified novel gene targets and regulatory mechanisms underlying activation of MASH fibrogenic HSCs and demonstrated that genetic or pharmacological inhibition of select genes suppressed liver fibrosis., Impact and Implications: Here we present snRNA-seq and snATAC-seq analysis of human HSCs from NORMAL, MASL, and MASH livers. We identified additional subclusters that were not detected by previous studies and characterized the mechanism by which HSCs activate in the MASH livers, including the transcriptional machinery that activates HSCs into myofibroblasts. For the first time, we described the pathogenic role of activated HSC-derived PAI-1 (a product of SERPINE1 gene) in the development of MASH liver fibrosis. Targeting of RUNX1/2-SERPINE1 axis may provide a novel strategy for treatment of liver fibrosis in patients., Competing Interests: Declaration of Competing Interest Nothing to declare, (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Grb2-associated binder 1 (Gab1) is an adaptor protein that is important for intracellular signal transduction by receptor tyrosine kinases that are receptors for various growth factors and plays an important role in rapid liver regeneration after partial hepatectomy and during acute hepatitis. On the other hand, mild liver regeneration is induced in livers of individuals with chronic hepatitis, where hepatocyte apoptosis is persistent; however, the impact of Gab1 on such livers remains unclear. We examined the role of Gab1 in chronic hepatitis. Gab1 knockdown enhanced the decrease in cell viability and apoptosis induced by ABT-737, a Bcl-2/-xL/-w inhibitor, in BNL.CL2 cells, while cell viability and caspase activity were unchanged in the absence of ABT-737. ABT-737 treatment induced Gab1 cleavage to form p35-Gab1. p35-Gab1 was also detected in the livers of mice with hepatocyte-specific Mcl-1 knockout (KO), which causes persistent hepatocyte apoptosis. Gab1 deficiency exacerbated hepatocyte apoptosis in Mcl-1 KO mice with posttranscriptional downregulation of Bcl-XL. In BNL.CL2 cells treated with ABT-737, Gab1 knockdown posttranscriptionally suppressed Bcl-xL expression, and p35-Gab1 overexpression enhanced Bcl-xL expression. Gab1 deficiency in Mcl-1 KO mice activated STAT3 signaling in hepatocytes, increased hepatocyte proliferation, and increased the incidence of liver cancer with the exacerbation of liver fibrosis. In conclusion, Gab1 is cleaved in the presence of apoptotic stimuli and forms p35-Gab1 in hepatocytes. In chronic liver injury, the role of Gab1 in suppressing apoptosis and reducing liver damage, fibrosis, and tumorigenesis is more important than its role in liver regeneration. NEW & NOTEWORTHY Grb2-associated binder 1 (Gab1) is known to contribute to liver regeneration after acute liver injury. However, in chronic liver diseases, Gab1 plays a greater role in suppressing hepatocyte apoptosis than in liver regeneration, resulting in suppression of hepatocyte proliferation, liver fibrosis, and liver carcinogenesis.