Differences in branched-chain amino acid to tyrosine ratio (BTR) among etiologies of chronic liver disease progression compared to healthy adults.

BACKGROUND: The branched-chain amino acids (BCAAs) to tyrosine (Tyr) ratio (BTR) test is used to evaluate the progression of chronic liver disease (CLD). However, the differences across sex, age, body mass index (BMI) and etiologies are still unclear. METHODS: We retrospectively reviewed data from 2,529 CLD cases with free amino acids (FAAs) in peripheral blood from four hospitals and 16,421 general adults with FAAs data from a biobank database. In total, 1,326 patients with CLD (covering seven etiologies) and 8,086 healthy controls (HCs) were analyzed after exclusion criteria. We investigated the change of BTR in HCs by sex, age and BMI and then compared these to patients divided by modified ALBI (mALBI) grade after propensity score matching. RESULTS: BTR is significantly higher in males than females regardless of age or BMI and decreases with aging in HCs. In 20 types of FAAs, 7 FAAs including BCAAs were significantly decreased, and 11 FAAs including Tyr were significantly increased by mALBI grade in total CLD. The decreasing timings of BTR were at mALBI grade 2b in all CLD etiologies compared to HCs, however in chronic hepatitis C (CHC), chronic hepatitis B (CHB) and alcoholic liver disease (ALD), BTR started to decrease at 2a. There was a positive correlation between BCAAs and albumin among parameters in BTR and mALBI. The correlation coefficients in PBC, ALD and MASLD were higher than those of other etiologies. CONCLUSIONS: BTR varies by sex and age even among healthy adults, and decreasing process and timing of BTR during disease progression is different among CLD etiologies.

Sumoylation of methionine adenosyltransferase alpha 1 promotes mitochondrial dysfunction in alcohol-associated liver disease.

BACKGROUND AND AIMS: Methionine adenosyltransferase alpha1 (MATα1) is responsible for the biosynthesis of S-adenosylmethionine in normal liver. Alcohol consumption enhances MATα1 interaction with peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1), which blocks MATα1 mitochondrial targeting, resulting in lower mitochondrial MATα1 content and mitochondrial dysfunction in alcohol-associated liver disease (ALD) in part through upregulation of cytochrome P450 2E1. Conversely, alcohol intake enhances SUMOylation, which enhances cytochrome P450 2E1 expression. MATα1 has potential SUMOylation sites, but whether MATα1 is regulated by SUMOylation in ALD is unknown. Here, we investigated if MATα1 is regulated by SUMOylation and, if so, how it impacts mitochondrial function in ALD. APPROACH AND RESULTS: Proteomics profiling revealed hyper-SUMOylation of MATα1, and prediction software identified lysine 48 (K48) as the potential SUMOylation site in mice (K47 in humans). Experiments with primary hepatocytes, mouse, and human livers revealed that SUMOylation of MAT1α by SUMO2 depleted mitochondrial MATα1. Furthermore, mutation of MATα1 K48 prevented ethanol-induced mitochondrial membrane depolarization, MATα1 depletion, and triglyceride accumulation. Additionally, CRISPR/CRISPR associated protein 9 gene editing of MATα1 at K48 hindered ethanol-induced MATα1-PIN1 interaction, degradation, and phosphorylation of MATα1 in vitro. In vivo, CRISPR/CRISPR associated protein 9 MATα1 K48 gene-edited mice were protected from ethanol-induced fat accumulation, liver injury, MATα1-PIN1 interaction, mitochondrial MATα1 depletion, mitochondrial dysfunction, and low S-adenosylmethionine levels. CONCLUSIONS: Taken together, our findings demonstrate an essential role for SUMOylation of MATα1 K48 for interaction with PIN1 in ALD. Preventing MATα1 K48 SUMOylation may represent a potential treatment strategy for ALD.

Exercise changes the intrahepatic immune cell profile and inhibits the progression of nonalcoholic steatohepatitis in a mouse model.

BACKGROUND: NASH is an increasingly common cause of chronic liver disease and can progress to cirrhosis and HCC. Although exercise suppresses inflammation during acute hepatitis, its impact on the progression of chronic liver disease remains unclear. Here, we investigated the effects of exercise on disease progression and intrahepatic immune cell composition in a mouse model of NASH. METHOD: Mice were assigned to 4 groups: 2 control groups (normal diet) and 2 NASH groups (western diet and low-dose carbon tetrachloride injection). One of each group remained sedentary and one was exercised on a treadmill for 12 weeks (60 min/d, 5 times/wk). All mice were then analyzed for liver histomorphology, steatosis, inflammation, and fibrosis; liver, adipose tissue, and skeletal muscle expression of genes related to metabolism and inflammation; and intrahepatic immune cell composition. RESULT: Compared with the normal diet mice, NASH mice exhibited enhanced liver steatosis, inflammation, and fibrosis; upregulated expression of liver lipogenesis-related and inflammation-related genes; and increased frequencies of intrahepatic F4/80 int CD11b hi bone marrow-derived macrophages and programmed death receptor-1 (PD-1) + CD8 + T cells. Expression of inflammatory cytokines and the frequencies of bone marrow-derived macrophages and PD-1 + CD8 + T cells correlated positively with liver steatosis, inflammation, and fibrosis. Exercise was shown to reduce NASH-induced hepatic steatosis, liver inflammation, and fibrosis; induce alterations in metabolism-related genes and inflammatory cytokines in the liver; and suppress accumulation of liver bone marrow-derived macrophages and PD-1 + CD8 + T cells. In addition, we showed that exercise induced increased expression of IL-15 in muscle and its deficiency exacerbated the pathology of NASH. CONCLUSIONS: Exercise alters the intrahepatic immune cell profile and protects against disease progression in a mouse model of NASH.

Experimental Workflow for Preclinical Studies of Human Antifibrotic Therapies.

Chronic liver diseases accompanied by liver fibrosis have caused significant morbidity and mortality in the world with increasing prevalence. Nonetheless, there are no approved antifibrotic therapies. Although numerous preclinical studies showed satisfactory results in targeting fibrotic pathways, these animal studies have not led to success in humans. In this chapter, we summarize the experimental approaches currently available, including in vitro cell culture models, in vivo animal models, and new experimental tools relevant to humans, and discuss how we translate laboratory results to clinical trials. We will also address the obstacles in transitioning promising therapies from preclinical studies to human antifibrotic treatments.

Extracellular vesicles in fatty liver promote a metastatic tumor microenvironment.

Liver metastasis is a major cause of death in patients with colorectal cancer (CRC). Fatty liver promotes liver metastasis, but the underlying mechanism remains unclear. We demonstrated that hepatocyte-derived extracellular vesicles (EVs) in fatty liver enhanced the progression of CRC liver metastasis by promoting oncogenic Yes-associated protein (YAP) signaling and an immunosuppressive microenvironment. Fatty liver upregulated Rab27a expression, which facilitated EV production from hepatocytes. In the liver, these EVs transferred YAP signaling-regulating microRNAs to cancer cells to augment YAP activity by suppressing LATS2. Increased YAP activity in CRC liver metastasis with fatty liver promoted cancer cell growth and an immunosuppressive microenvironment by M2 macrophage infiltration through CYR61 production. Patients with CRC liver metastasis and fatty liver had elevated nuclear YAP expression, CYR61 expression, and M2 macrophage infiltration. Our data indicate that fatty liver-induced EV-microRNAs, YAP signaling, and an immunosuppressive microenvironment promote the growth of CRC liver metastasis.

Peripheral-dominant liver fibrosis and tumor distribution in a mouse model of congestive hepatopathy.

AIM: Congestive hepatopathy often leads to liver fibrosis and hepatocellular carcinoma. Imaging modalities provided clinical evidence that elevation of liver stiffness and tumor occurrence are mainly induced in the periphery of the liver in patients with congestive hepatopathy. However, clinical relevance of liver stiffness and liver fibrosis is unclear because liver congestion itself increases liver stiffness in congestive hepatopathy. It also unclear which factors configure such regional disparity of tumor development in patients with congestive hepatopathy. To answer these questions, we evaluated the macroscopic spatial distribution of liver fibrosis and tumors in the murine model of congestive hepatopathy. METHODS: Chronic liver congestion was induced by partial ligation of the suprahepatic inferior vena cava. Distribution of liver congestion, fibrosis, and tumors in partial ligation of the suprahepatic inferior vena cava mice were assessed by histological findings, laser microdissection (LMD)-based qPCR and enhanced computed tomography. LMD-based RNA-sequencing was performed to identify causal factors that promote tumor development in congestive hepatopathy. RESULTS: Liver fibrosis was mainly induced in the periphery of the liver and co-localized with distribution of liver congestion. Liver tumors were also induced in the periphery of the liver where liver congestion and fibrosis occurred. LMD-based RNA-sequencing revealed the upregulation of extracellular matrix/collagen fibril-, wound healing-, angiogenesis-, morphogenesis-, and cell motility-related signaling pathways in periphery of liver compared with liver center. CONCLUSIONS: Our findings showed the experimental relevance of liver congestion, fibrosis, and tumor development in congestive hepatopathy, and may provide important locational information. Macroscopic regional disparity observed in this murine model should be considered to manage patients with congestive hepatopathy.

Targeting A-kinase anchoring protein 12 phosphorylation in hepatic stellate cells regulates liver injury and fibrosis in mouse models.

Trans-differentiation of hepatic stellate cells (HSCs) to activated state potentiates liver fibrosis through release of extracellular matrix (ECM) components, distorting the liver architecture. Since limited antifibrotics are available, pharmacological intervention targeting activated HSCs may be considered for therapy. A-kinase anchoring protein 12 (AKAP12) is a scaffolding protein that directs protein kinases A/C (PKA/PKC) and cyclins to specific locations spatiotemporally controlling their biological effects. It has been shown that AKAP12's scaffolding functions are altered by phosphorylation. In previously published work, observed an association between AKAP12 phosphorylation and HSC activation. In this work, we demonstrate that AKAP12's scaffolding activity toward the endoplasmic reticulum (ER)-resident collagen chaperone, heat-shock protein 47 (HSP47) is strongly inhibited by AKAP12's site-specific phosphorylation in activated HSCs. CRISPR-directed gene editing of AKAP12's phospho-sites restores its scaffolding toward HSP47, inhibiting HSP47's collagen maturation functions, and HSC activation. AKAP12 phospho-editing dramatically inhibits fibrosis, ER stress response, HSC inflammatory signaling, and liver injury in mice. Our overall findings suggest a pro-fibrogenic role of AKAP12 phosphorylation that may be targeted for therapeutic intervention in liver fibrosis.

Depletion of mitochondrial methionine adenosyltransferase α1 triggers mitochondrial dysfunction in alcohol-associated liver disease.

MATα1 catalyzes the synthesis of S-adenosylmethionine, the principal biological methyl donor. Lower MATα1 activity and mitochondrial dysfunction occur in alcohol-associated liver disease. Besides cytosol and nucleus, MATα1 also targets the mitochondria of hepatocytes to regulate their function. Here, we show that mitochondrial MATα1 is selectively depleted in alcohol-associated liver disease through a mechanism that involves the isomerase PIN1 and the kinase CK2. Alcohol activates CK2, which phosphorylates MATα1 at Ser114 facilitating interaction with PIN1, thereby inhibiting its mitochondrial localization. Blocking PIN1-MATα1 interaction increased mitochondrial MATα1 levels and protected against alcohol-induced mitochondrial dysfunction and fat accumulation. Normally, MATα1 interacts with mitochondrial proteins involved in TCA cycle, oxidative phosphorylation, and fatty acid ß-oxidation. Preserving mitochondrial MATα1 content correlates with higher methylation and expression of mitochondrial proteins. Our study demonstrates a role of CK2 and PIN1 in reducing mitochondrial MATα1 content leading to mitochondrial dysfunction in alcohol-associated liver disease.

Sphingosine-1-phosphate promotes tumor development and liver fibrosis in mouse model of congestive hepatopathy.

BACKGROUND AND AIMS: Chronic liver congestion reflecting right-sided heart failure (RHF), Budd-Chiari syndrome, or Fontan-associated liver disease (FALD) is involved in liver fibrosis and HCC. However, molecular mechanisms of fibrosis and HCC in chronic liver congestion remain poorly understood. APPROACH AND RESULTS: Here, we first demonstrated that chronic liver congestion promoted HCC and metastatic liver tumor growth using murine model of chronic liver congestion by partial inferior vena cava ligation (pIVCL). As the initial step triggering HCC promotion and fibrosis, gut-derived lipopolysaccharide (LPS) appeared to induce LSECs capillarization in mice and in vitro. LSEC capillarization was also confirmed in patients with FALD. Mitogenic factor, sphingosine-1-phosphate (S1P), was increased in congestive liver and expression of sphingosine kinase 1, a major synthetase of S1P, was increased in capillarized LSECs after pIVCL. Inhibition of S1P receptor (S1PR) 1 (Ex26) and S1PR2 (JTE013) mitigated HCC development and liver fibrosis, respectively. Antimicrobial treatment lowered portal blood LPS concentration, LSEC capillarization, and liver S1P concentration accompanied by reduction of HCC development and fibrosis in the congestive liver. CONCLUSIONS: In conclusion, chronic liver congestion promotes HCC development and liver fibrosis by S1P production from LPS-induced capillarized LSECs. Careful treatment of both RHF and liver cancer might be necessary for patients with RHF with primary or metastatic liver cancer.

Deregulated 14-3-3ζ and methionine adenosyltransferase α1 interplay promotes liver cancer tumorigenesis in mice and humans.

Methionine adenosyltransferase 1A (MAT1A) is a tumor suppressor downregulated in hepatocellular carcinoma and cholangiocarcinoma, two of the fastest rising cancers worldwide. We compared MATα1 (protein encoded by MAT1A) interactome in normal versus cancerous livers by mass spectrometry to reveal interactions with 14-3-3ζ. The MATα1/14-3-3ζ complex was critical for the expression of 14-3-3ζ. Similarly, the knockdown and small molecule inhibitor for 14-3-3ζ (BV02), and ChIP analysis demonstrated the role of 14-3-3ζ in suppressing MAT1A expression. Interaction between MATα1 and 14-3-3ζ occurs directly and is enhanced by AKT2 phosphorylation of MATα1. Blocking their interaction enabled nuclear MATα1 translocation and inhibited tumorigenesis. In contrast, overexpressing 14-3-3ζ lowered nuclear MATα1 levels and promoted tumor progression. However, tumor-promoting effects of 14-3-3ζ were eliminated when liver cancer cells expressed mutant MATα1 unable to interact with 14-3-3ζ. Taken together, the reciprocal negative regulation that MATα1 and 14-3-3ζ exert is a key mechanism in liver tumorigenesis.

Promotion of cholangiocarcinoma growth by diverse cancer-associated fibroblast subpopulations.

Cancer-associated fibroblasts (CAF) are a poorly characterized cell population in the context of liver cancer. Our study investigates CAF functions in intrahepatic cholangiocarcinoma (ICC), a highly desmoplastic liver tumor. Genetic tracing, single-cell RNA sequencing, and ligand-receptor analyses uncovered hepatic stellate cells (HSC) as the main source of CAF and HSC-derived CAF as the dominant population interacting with tumor cells. In mice, CAF promotes ICC progression, as revealed by HSC-selective CAF depletion. In patients, a high panCAF signature is associated with decreased survival and increased recurrence. Single-cell RNA sequencing segregates CAF into inflammatory and growth factor-enriched (iCAF) and myofibroblastic (myCAF) subpopulations, displaying distinct ligand-receptor interactions. myCAF-expressed hyaluronan synthase 2, but not type I collagen, promotes ICC. iCAF-expressed hepatocyte growth factor enhances ICC growth via tumor-expressed MET, thus directly linking CAF to tumor cells. In summary, our data demonstrate promotion of desmoplastic ICC growth by therapeutically targetable CAF subtype-specific mediators, but not by type I collagen.

Increased Frequency of Dysfunctional Siglec-7-CD57+PD-1+ Natural Killer Cells in Patients With Non-alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) is a progressive disorder that can develop into liver fibrosis and hepatocellular carcinoma. Natural killer (NK) cells have been shown to protect against liver fibrosis and tumorigenesis, suggesting that they may also play a role in the pathogenesis of NAFLD. Sialic acid-binding immunoglobulin-like lectins (Siglecs) are a family of inhibitory and activating receptors expressed by many cell types, including NK cells. Here, we investigated the phenotypic profiles of peripheral blood and intrahepatic NK cells, including expression of Siglecs and immune checkpoint molecules, and their association with NK cell function in patients with NAFLD. Immune cells in the peripheral blood of 42 patients with biopsy-proven NAFLD and 13 healthy volunteers (HVs) were identified by mass cytometry. The function of various NK cell subpopulations was assessed by flow cytometric detection of intracellular IFN-γ and CD107a/LAMP-1, a degranulation marker, after in vitro stimulation. We found that peripheral blood from NAFLD patients, regardless of fibrosis stage, contained significantly fewer total CD56+ NK cell and CD56dim NK cell populations compared with HVs, and the CD56dim cells from NAFLD patients were functionally impaired. Among the Siglecs examined, NK cells predominantly expressed Siglec-7 and Siglec-9, and both the expression levels of Siglec-7 and Siglec-9 on NK cells and the frequencies of Siglec-7+CD56dim NK cells were reduced in NAFLD patients. Notably, Siglec-7 levels on CD56dim NK cells were inversely correlated with PD-1, CD57, and ILT2 levels and positively correlated with NKp30 and NKp46 levels. Further subtyping of NK cells identified a highly dysfunctional Siglec-7-CD57+PD-1+CD56dim NK cell subset that was increased in patients with NAFLD, even those with mild liver fibrosis. Intrahepatic NK cells from NAFLD patients expressed elevated levels of NKG2D and CD69, suggesting a more activated phenotype than normal liver NK cells. These data identify a close association between NK cell function and expression of Siglec-7, CD57, and PD-1 that could potentially be therapeutically targeted in NAFLD.

Inhibition of hyaluronan synthesis by 4-methylumbelliferone ameliorates non-alcoholic steatohepatitis in choline-deficient L-amino acid-defined diet-induced murine model.

Hyaluronan (HA) as a glycosaminoglycan can bind to cell-surface receptors, such as TLR4, to regulate inflammation, tissue injury, repair, and fibrosis. 4-methylumbelliferone (4-MU), an inhibitor of HA synthesis, is a drug used for the treatment of biliary spasms. Currently, therapeutic interventions are not available for non-alcoholic steatohepatitis (NASH). In this study, we investigated the effects of 4-MU on NASH using a choline-deficient amino acid (CDAA) diet model. CDAA diet-fed mice showed NASH characteristics, including hepatocyte injury, hepatic steatosis, inflammation, and fibrogenesis. 4-MU treatment significantly reduced hepatic lipid contents in CDAA diet-fed mice. 4-MU reversed CDAA diet-mediated inhibition of Ppara and induction of Srebf1 and Slc27a2. Analysis of serum ALT and AST levels revealed that 4-MU treatment protected against hepatocellular damage induced by CDAA diet feeding. TLR4 regulates low molecular weight-HA-induced chemokine expression in hepatocytes. In CDAA diet-fed, 4-MU-treated mice, the upregulated chemokine/cytokine expression, such as Cxcl1, Cxcl2, and Tnf was attenuated with the decrease of macrophage infiltration into the liver. Moreover, HA inhibition repressed CDAA diet-induced mRNA expression of fibrogenic genes, Notch1, and Hes1 in the liver. In conclusion, 4-MU treatment inhibited liver steatosis and steatohepatitis in a mouse model of NASH, implicating that 4-MU may have therapeutic potential for NASH.

Oral administration of PEGylated TLR7 ligand ameliorates alcohol-associated liver disease via the induction of IL-22.

Effective therapies for alcohol-associated liver disease (ALD) are limited; therefore, the discovery of new therapeutic agents is greatly warranted. Toll-like receptor 7 (TLR7) is a pattern recognition receptor for single-stranded RNA, and its activation prevents liver fibrosis. We examined liver and intestinal damage in Tlr7-/- mice to determine the role of TLR7 in ALD pathogenesis. In an alcoholic hepatitis (AH) mouse model, hepatic steatosis, injury, and inflammation were induced by chronic binge ethanol feeding in mice, and Tlr7 deficiency exacerbated these effects. Because these results demonstrated that endogenous TLR7 signaling activation is protective in the AH mouse model, we hypothesized that TLR7 activation may be an effective therapeutic strategy for ALD. Therefore, we investigated the therapeutic effect of TLR7 agonistic agent, 1Z1, in the AH mouse model. Oral administration of 1Z1 was well tolerated and prevented intestinal barrier disruption and bacterial translocation, which thus suppressed ethanol-induced hepatic injury, steatosis, and inflammation. Furthermore, 1Z1 treatment up-regulated the expression of antimicrobial peptides, Reg3b and Reg3g, in the intestinal epithelium, which modulated the microbiome by decreasing and increasing the amount of Bacteroides and Lactobacillus, respectively. Additionally, 1Z1 up-regulated intestinal interleukin (IL)-22 expression. IL-22 deficiency abolished the protective effects of 1Z1 in ethanol-induced liver and intestinal damage, suggesting intestinal IL-22 as a crucial mediator for 1Z1-mediated protection in the AH mouse model. Collectively, our results indicate that TLR7 signaling exerts protective effects in the AH mouse model and that a TLR7 ligand, 1Z1, holds therapeutic potential for the treatment of AH.

The liver fibrosis niche: Novel insights into the interplay between fibrosis-composing mesenchymal cells, immune cells, endothelial cells, and extracellular matrix.

Liver fibrosis is a hepatic wound-healing response caused by chronic liver diseases that include viral hepatitis, alcoholic liver disease, non-alcoholic steatohepatitis, and cholestatic liver disease. Liver fibrosis eventually progresses to cirrhosis that is histologically characterized by an abnormal liver architecture that includes distortion of liver parenchyma, formation of regenerative nodules, and a massive accumulation of extracellular matrix (ECM). Despite intensive investigations into the underlying mechanisms of liver fibrosis, developments of anti-fibrotic therapies for liver fibrosis are still unsatisfactory. Recent novel experimental approaches, such as single-cell RNA sequencing and proteomics, have revealed the heterogeneity of ECM-producing cells (mesenchymal cells) and ECM-regulating cells (immune cells and endothelial cells). These approaches have accelerated the identification of fibrosis-specific subpopulations among these cell types. The ECM also consists of heterogenous components. Their production, degradation, deposition, and remodeling are dynamically regulated in liver fibrosis, further affecting the functions of cells responsible for fibrosis. These cellular and ECM elements cooperatively form a unique microenvironment: a fibrotic niche. Understanding the complex interplay between these elements could lead to a better understanding of underlying fibrosis mechanisms and to the development of effective therapies.

Hepatic Stellate Cell-Macrophage Crosstalk in Liver Fibrosis and Carcinogenesis.

Chronic liver injury due to viral hepatitis, alcohol abuse, and metabolic disorders is a worldwide health concern. Insufficient treatment of chronic liver injury leads to fibrosis, causing liver dysfunction and carcinogenesis. Most cases of hepatocellular carcinoma (HCC) develop in the fibrotic liver. Pathological features of liver fibrosis include extracellular matrix (ECM) accumulation, mesenchymal cell activation, immune deregulation, and angiogenesis, all of which contribute to the precancerous environment, supporting tumor development. Among liver cells, hepatic stellate cells (HSCs) and macrophages play critical roles in fibrosis and HCC. These two cell types interplay and remodel the ECM and immune microenvironment in the fibrotic liver. Once HCC develops, HCC-derived factors influence HSCs and macrophages to switch to protumorigenic cell populations, cancer-associated fibroblasts and tumor-associated macrophages, respectively. This review aims to summarize currently available data on the roles of HSCs and macrophages in liver fibrosis and HCC, with a focus on their interaction.

Serum soluble sialic acid-binding immunoglobulin-like lectin-7 concentration as an indicator of liver macrophage activation and advanced fibrosis in patients with non-alcoholic fatty liver disease.

AIM: Non-alcoholic fatty liver disease (NAFLD) is a leading cause of liver disease worldwide. Because liver fibrosis is associated with the long-term prognosis of patients with NAFLD, there is an urgent need for non-invasive markers of liver fibrosis. Sialic acid-binding immunoglobulin-like lectin-7 (Siglec-7) is an immunomodulatory molecule expressed on various immune cells, including macrophages, which plays a key role in liver inflammation and fibrosis in NAFLD. We aimed to determine whether serum levels of soluble Siglec-7 (sSiglec-7) could have utility at a marker of fibrosis in this patient population. METHODS: We examined serum samples from 93 NAFLD patients and 19 healthy donors for macrophage-associated protein, including sSiglec-7, soluble CD163, and YKL-40, and examined their correlation with liver fibrosis scores, tissue elastography, and histological findings. Independent factors associated with advanced fibrosis were analyzed using a logistic regression model and a decision tree. To clarify the source of sSiglec-7, we examined its expression in liver tissue-derived macrophages and cultured monocyte-derived macrophages. RESULTS: Serum sSiglec-7 levels were significantly higher in NAFLD patients compared with healthy donors, and correlated positively with sCD163 and YKL-40 levels. Serum sSiglec-7 was an independent diagnostic marker with high specificity (96.3%) for advanced fibrosis (F3 and F4) in NAFLD patients. Siglec-7 was mainly expressed on CCR2+ macrophages in the liver, and sSiglec-7 production by monocyte-derived macrophages in vitro was increased after stimulation by pro-inflammatory factors. CONCLUSIONS: Elevated serum sSiglec-7 could serve as an independent marker with high specificity for advanced liver fibrosis in patients with NAFLD.

Serum milk fat globule-EGF factor 8 (MFG-E8) as a diagnostic and prognostic biomarker in patients with hepatocellular carcinoma.

Current serum hepatocellular carcinoma (HCC) biomarkers are insufficient for early diagnosis. We aimed to clarify whether serum MFG-E8 can serve as a diagnostic or prognostic biomarker of HCC. Serum MFG-E8 levels of 282 HCC patients, who underwent primary hepatectomy, were examined by ELISA. We also quantified serum MFG-E8 levels in patients with chronic hepatitis (CH), liver cirrhosis (LC), as well as in healthy volunteers (HVs). Serum MFG-E8 levels were significantly lower in HCC patients than in HVs regardless of the etiology of liver disease (3.6 ± 0.1 vs 5.8 ± 0.2 ng/mL, p < 0.0001), and recovered after treatment of HCC. Serum MFG-E8 levels in CH and LC patients were comparable to those in HVs. Serum MFG-E8 could detect HCCs, even α-fetoprotein (AFP)-negative or des-γ-carboxy prothrombin (DCP)-negative HCCs, in CH and LC patients. Our new HCC prediction model using MFG-E8 and DCP (Logit(p) = 2.619 - 0.809 × serum MFG-E8 + 0.0226 × serum DCP) distinguished HCC patients from CH and LC patients with an area under the curve of 0.923, a sensitivity of 81.1%, and a specificity of 89.8%. Futhermore, low preoperative serum MFG-E8 was an independent predictor of poor overall survival. Thus, serum MFG-E8 could serve as a feasible diagnostic and prognostic biomarker for HCC.