Dysregulated Expression of the Nuclear Exosome Targeting Complex Component Rbm7 in Nonhematopoietic Cells Licenses the Development of Fibrosis.

Fibrosis is an incurable disorder of unknown etiology. Segregated-nucleus-containing atypical monocytes (SatMs) are critical for the development of fibrosis. Here we examined the mechanisms that recruit SatMs to pre-fibrotic areas. A screen based on cytokine expression in the fibrotic lung revealed that the chemokine Cxcl12, which is produced by apoptotic nonhematopoietic cells, was essential for SatM recruitment. Analyses of lung tissues at fibrosis onset showed increased expression of Rbm7, a component of the nuclear exosome targeting complex. Rbm7 deletion suppressed bleomycin-induced fibrosis and at a cellular level, suppressed apoptosis of nonhematopoietic cells. Mechanistically, Rbm7 bound to noncoding (nc)RNAs that form subnuclear bodies, including Neat1 speckles. Dysregulated expression of Rbm7 resulted in the nuclear degradation of Neat1 speckles, the dispersion of the DNA repair protein BRCA1, and the triggering of apoptosis. Thus, Rbm7 in epithelial cells plays a critical role in the development of fibrosis by regulating ncRNA decay and thereby the production of chemokines that recruit SatMs.

Exploring the genetic diversity of the Japanese population: Insights from a large-scale whole genome sequencing analysis.

The Japanese archipelago is a terminal location for human migration, and the contemporary Japanese people represent a unique population whose genomic diversity has been shaped by multiple migrations from Eurasia. We analyzed the genomic characteristics that define the genetic makeup of the modern Japanese population from a population genetics perspective from the genomic data of 9,287 samples obtained by high-coverage whole-genome sequencing (WGS) by the National Center Biobank Network. The dataset comprised populations from the Ryukyu Islands and other parts of the Japanese archipelago (Hondo). The Hondo population underwent two episodes of population decline during the Jomon period, corresponding to the Late Neolithic, and the Edo period, corresponding to the Early Modern era, while the Ryukyu population experienced a population decline during the shell midden period of the Late Neolithic in this region. Haplotype analysis suggested increased allele frequencies for genes related to alcohol and fatty acid metabolism, which were reported as loci that had experienced positive natural selection. Two genes related to alcohol metabolism were found to be 12,500 years out of phase with the time when they began to increase in the allele frequency; this finding indicates that the genomic diversity of Japanese people has been shaped by events closely related to agriculture and food production.

Epidemiology of Fontan-associated liver disease in Japan: Results from a nationwide survey in 2021.

AIM: Although the Fontan procedure has improved the survival of patients with single-ventricle heart disease, the long-term consequences of the procedure have been a concern. This study aimed to explore the patients' postoperative clinical characteristics, including a diagnosis of Fontan-associated liver disease (FALD). METHODS: A nationwide Japanese epidemiological survey of post-Fontan patients was undertaken in 2021. The survey targets were selected from all departments of pediatrics, pediatric surgery, cardiology, cardiovascular surgery, and gastroenterology using stratified random sampling by the number of beds. Each department was asked to complete a mail-back questionnaire on the numbers of patients and their clinical characteristics. The diagnosis of FALD was made by each attending physician. RESULTS: The estimated number of post-Fontan patients was 7810 (95% confidence interval, 5430-10 200) in 2020, with a period prevalence of 61.9 per million. During the follow-up of 13.8 years after the Fontan procedure, 40% of patients were diagnosed with FALD. An elevated γ-glutamyl transpeptidase level was the most common finding leading to the FALD diagnosis (41%), and 45% of the patients also showed liver fibrosis. Compared with non-FALD patients, FALD patients were older, had longer duration since the Fontan procedure, and had more severe cardiac or liver conditions. However, more than half of the non-FALD patients had elevated liver enzyme levels, suggesting underestimation of the number of FALD patients. CONCLUSIONS: In 2020, approximately 40% of post-Fontan patients underwent follow-up with a diagnosis of FALD, although the lack of established diagnostic criteria for FALD could affect the reported prevalence of FALD.

Integrated policy of medical expense subsidies and clinical registry for patients with liver cancer and decompensated cirrhosis in Japan.

Chronic hepatitis B and C are among the most significant infectious diseases worldwide, and are major risk factors for liver cirrhosis and liver cancer. In Japan, comprehensive hepatitis measures are implemented for the testing and treatment of viral hepatitis, thus enabling the early diagnosis of liver cancer. Nevertheless, patients with decompensated cirrhosis and liver cancer often have unfavorable prognoses and require repetitive long-term treatment. In fiscal year 2018, an integrated policy of medical expense subsidies and research was established in Japan that aimed to alleviate patients' financial burden and launch the clinical registry of advanced liver disease. Over time, updates to the eligibility for the subsidy increased access to patients and has led to an increased number of beneficiaries. Additionally, the accumulation of clinical data in the registry has revealed the treatment choices for these diseases. However, the disparities in efforts across prefectures have also become evident. Raising public awareness of the policy and tightening the multisector healthcare network are keys to success in supporting qualifying patients with advanced liver disease.

Hepatitis C virus modulates signal peptide peptidase to alter host protein processing.

Immunoevasins are viral proteins that prevent antigen presentation on major histocompatibility complex (MHC) class I, thus evading host immune recognition. Hepatitis C virus (HCV) evades immune surveillance to induce chronic infection; however, how HCV-infected hepatocytes affect immune cells and evade immune recognition remains unclear. Herein, we demonstrate that HCV core protein functions as an immunoevasin. Its expression interfered with the maturation of MHC class I molecules catalyzed by the signal peptide peptidase (SPP) and induced their degradation via HMG-CoA reductase degradation 1 homolog, thereby impairing antigen presentation to CD8+ T cells. The expression of MHC class I in the livers of HCV core transgenic mice and chronic hepatitis C patients was impaired but was restored in patients achieving sustained virological response. Finally, we show that the human cytomegalovirus US2 protein, possessing a transmembrane region structurally similar to the HCV core protein, targets SPP to impair MHC class I molecule expression. Thus, SPP represents a potential target for the impairment of MHC class I molecules by DNA and RNA viruses.

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.

Toll-like receptor 7 agonist, GS-986, is an immune-stimulant inducing follicular helper T cells and expanding HBs antigen-specific B cells in vitro.

BACKGROUNDS AND AIMS: Toll-like receptor (TLR) agonists have been developed as adjuvants to efficiently induce antiviral immune responses. Specificity and potency of these compounds are essential requirements for clinical trial applications. In patients with hepatitis B virus (HBV) infections, sustained loss of hepatitis B surface antigen (HBsAg) is a therapeutic goal, which may be achievable by the sequential activation of follicular helper T cells (Tfh) and antibody-secreting B cells. We aimed to elucidate whether novel TLR7 agonist, GS-986, could activate immune responses involved in HBV elimination. METHODS: To clarify the impact of GS-986 on pDCs, we quantified the expression levels of surface markers and evaluated for Tfh induction in a culture model consisting of human pDCs with allogeneic naïve CD4+ T cells. In addition, we examined whether GS-986 could enhance HBs antibody production capacity using PBMC from CHB patients. RESULTS: pDCs from CHB patients had lower OX40L expression and as well as impaired capacity for Tfh induction compared with those from healthy donors. However, GS-986-stimulated pDCs from CHB patients expressed OX40L and produced IL-6 and IL-12, resulting in the induction of IL-21-producing Tfh cells (CXCR5+ PD-1+ CD4+ ) from naïve CD4+ T cells. The Tfh-inducing capacity of GS-986 was reduced in the presence of an anti-OX40L blocking antibody. Furthermore, GS-986 promoted HBsAg-specific antibody production in PBMCs from CHB patients. CONCLUSIONS: GS-986 is an adjuvant that stimulates pDCs to induce Tfh differentiation and antigen-specific B-cell production. This immune profile may be beneficial for therapeutic application as an immune modulator in CHB patients.

Preoperative serum brain-derived neurotrophic factor as a predictive biomarker for sepsis after living-donor liver transplantation.

AIM: Although the survival rate after living-donor liver transplantation (LDLT) is improving, sepsis still limits the prognosis. Immune dysfunction and sarcopenia are often observed in LDLT patients, and increase susceptibility to infection. Brain-derived neurotrophic factor (BDNF) is a myokine produced by immune cells and skeletal muscle. We aimed to determine whether serum BDNF could be a feasible biomarker for sepsis of LDLT patients. METHODS: We measured serum samples from 124 patients who underwent LDLT and 9 healthy volunteers for BDNF. We examined its correlation with incidence rate of sepsis. To clarify the source of BDNF, we examined its expression in lymphocytes, skeletal muscle cells, and hepatocytes. RESULTS: Patients who experienced sepsis showed worse short-term survival. Preoperative serum BDNF was lower in LDLT patients compared with healthy volunteers, and was also lower in Child-Pugh C compared with Child-Pugh A or B. Serum BDNF was inversely correlated with Model for End-Stage Liver Disease and controlling nutritional status (CONUT) scores, but had a weak positive correlation with skeletal muscle mass index (SMI). Multivariate analysis revealed that serum BDNF was independently associated with sepsis. Preoperative serum BDNF was a better predictor of sepsis in LDLT patients than CONUT score or SMI. Serum BDNF was positively correlated with lymphocyte counts, especially T cells. In vitro, T cells and skeletal muscle cells produced BDNF. CONCLUSIONS: Preoperative serum BDNF could be a predictive biomarker for sepsis after LDLT, by reflecting the systemic condition including hepatic function, nutritional status, and immune status.

Dried blood spot-based host genome analysis technique targeting pathological associations with hepatitis B: Development and clinical application in the Cambodian population.

AIM: Reports of patients with hepatitis B have highlighted associations between polymorphisms in the human leukocyte antigen (HLA)-DPB1, CXCL13, and CXCR5 genes and disease pathology. Owing to its potential to contribute to the development of new diagnostic and therapeutic methods, we aimed to establish a reliable host genome analysis technique that can be used in countries with inadequate infrastructure. METHOD: We compared multiple commercially available kits for dried blood spot (DBS)-based sample collection to develop a basic DBS-based host genome analysis technique. We then collected blood samples from Cambodian patients with hepatitis  B and performed single-nucleotide polymorphism genotyping and HLA allele typing by the DBS system. RESULT: We were able to perform single-nucleotide polymorphism genotyping and HLA allele typing with host DNA samples obtained using a combination of a HemaSpot™ filter paper-based device and a SMITEST® EX-R&D DNA extraction kit. The accuracy of genotyping using samples obtained by this method was not inferior to one using samples obtained by venipuncture. In the Cambodian population, significant associations of HLA-DPB1*04:01 with protection against chronic hepatitis B virus (HBV) infection, and HLA-DPB1*05:01 and HLA-DPB1*13:01 with susceptibility to chronic HBV infection were identified. CONCLUSION: Based on the DBS system, we clarified the associations of HLA-DPB1 alleles with chronic HBV infection in the Cambodian population for the first time. Because the DBS is a low-cost, durable, transportable, and easy-to-handle modality, genetic analysis based on the DBS system is a feasible strategy for obtaining a deeper understanding of HBV epidemiology, especially in middle- or low-income countries.

The impact of the COVID-19 pandemic on hepatocellular carcinoma diagnosis and treatment in Japan: A multicenter collaborative observational study.

AIM: Coronavirus disease 2019 emerged in December 2019 and spread worldwide. This study aimed to clarify the impact of the coronavirus disease 2019 pandemic on the diagnosis and treatment of hepatocellular carcinoma (HCC) in Japan. METHODS: First, we collected the monthly numbers of HCC-related general medical practices from January 2019 to December 2021 at liver disease-specific medical institutions in Japan. Next, we collected individual clinical information from patients with newly diagnosed HCC during this period. RESULTS: There was a decrease in the number of HCC-related medical practices, including referrals, enhanced abdominal ultrasonography and radiofrequency ablation, in Japan's first state of emergency (SOE; April-May 2020) compared with 2019. Fewer patients were diagnosed with new HCC during the first SOE than before or after it. There was no difference in tumor diameter, number of tumors or Barcelona Clinic Liver Cancer stage between patients diagnosed before the first SOE and those diagnosed during or after the first SOE. The median waiting times for treatment of patients diagnosed during and after the first SOE were 31 and 37 days, which were significantly shorter and not longer than that of patients diagnosed before the first SOE (36 days), respectively. CONCLUSION: The number of HCC-related general medical practices decreased during the first SOE. However, the coronavirus disease 2019 pandemic did not lead to HCC progression by diagnostic delays or cause HCC treatment delays in Japan.

Effects of sodium glucose cotransporter 2 inhibitors and pioglitazone on FIB-4 index in metabolic-associated fatty liver disease.

BACKGROUND: The antidiabetic drugs sodium glucose cotransporter 2 inhibitors (SGLT2is) and thiazolidinediones have beneficial effects on the liver dysfunction of patients with nonalcoholic fatty liver disease and type 2 diabetes mellitus (T2DM). We aimed to determine the efficacy of these drugs for the treatment of liver disease in patients with metabolic dysfunction-associated fatty liver disease (MAFLD) and T2DM. METHODS: We undertook a retrospective study of 568 patients with MAFLD and T2DM. Of these, 210 were treating their T2DM with SGLT2is (n = 95), 86 with pioglitazone (PIO), and 29 with both. The primary outcome was the change in Fibrosis-4 (FIB-4) index between baseline and 96 weeks. RESULTS: At 96 weeks, the mean FIB-4 index had significantly decreased (from 1.79 ± 1.10-1.56 ± 0.75) in the SGLT2i group, but not in the PIO group. The aspartate aminotransferase to platelet ratio index, serum aspartate and alanine aminotransferase (ALT), hemoglobin A1c, and fasting blood sugar significantly decreased in both groups (ALT: SGLT2i group, -17 ± 3 IU/L; PIO group, -14 ± 3 IU/L). The bodyweight of the SGLT2i group decreased, but that of the PIO group increased (-3.2 kg and +1.7 kg, respectively). When the participants were allocated to two groups according to their baseline ALT (>30 IU/L), FIB-4 index significantly decreased in both groups. In patients taking pioglitazone, the addition of SGLT2i improved liver enzymes but not FIB-4 index for 96 weeks. CONCLUSIONS: Treatment with SGLT2i causes a larger improvement in FIB-4 index than PIO in patients with MAFLD over 96 weeks.

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.

Establishment of monoclonal antibodies broadly neutralize infection of hepatitis B virus.

Antibodies against hepatitis B virus S protein can protect against hepatitis B virus (HBV) infection. Therefore, hepatitis B immunoglobulin (HBIG), which contains HBsAb, is used clinically as a therapy for HBV infection. In this study, a series of monoclonal antibodies that recognize multiple HBV genotypes was obtained. All the antibodies recognized conformational epitopes of S protein, but not linear epitopes. Several antibodies neutralized HBV infection and exhibited strong affinities and neutralizing activities. Antigenic epitope analysis demonstrated that they recognized residue Ile152 of S protein, which is localized outside the "a" determinant. Ile152 is highly conserved, and a mutation in this residue resulted in reduced expression of large hepatitis B surface proteins (L protein), suggesting that the amino acid at this position is involved in the expression of L protein. In addition, the antibodies neutralized the infection of hepatitis D virus possessing a Gly145 mutation to Arg in S protein, which is a well-known escape mutation against HBIG treatment. Using mouse monoclonal antibodies, a humanized antibody possessing affinities and neutralizing activities similar to those of the original mouse antibody was successfully established. The antibodies generated in this study may have the potential for use in alternative antibody therapies for HBV infection.

Impact of antiviral therapy for disease progression and non-invasive liver fibrosis index in patients with chronic hepatitis C: Markov chain model analysis.

BACKGROUND/AIM: Antiviral therapy advancements resulted in an era in which eradication of hepatitis C has become a goal, however, there are few reports on the long-term course of liver disease progression with antiviral therapy. The aim of this study was to use the Markov model to analyze disease progression and non-invasive liver fibrosis index in hepatitis C Patients. METHODS: Patients with chronic hepatitis C (n = 1432) were diagnosed between January 2012 and May 2021 in the Musashino Red Cross Hospital. Patients with other hepatitis virus co-infection, chronic liver disease, and hepatocellular carcinoma (HCC) at the beginning of the study were excluded. A total of 618 patients with a 1-year or longer observation period were studied. The liver disease state was defined as chronic hepatitis (CH), compensated liver cirrhosis (CLC), decompensated liver cirrhosis (DLC), and HCC. RESULTS: Cirrhosis and high FIB-4 index (≥3.61) were 42 cases (6.8%) and 208 cases (33.6%), respectively at the start of the study. The 40 years estimated transition analysis of 40-year-old CH low FIB-4 level (<3.61) revealed that the proportion of CH low/high, CLC low/high, DLC low/high, and HCC were 10.83%/10.86%, 0.35%/2.64%, 0%/3.21% 72.11% in untreated unit and 47.83%/9.21%, 6.69%/1.32%, 0.70%/0.99%, 33.27% in treated unit, respectively. Antiviral therapy suppressed liver fibrosis, disease progression, and HCC development significantly. CONCLUSION: Markov model analysis of hepatitis C virus patients showed the impact of antiviral therapy on the suppression of disease progression in the order of CH, CLC, and DLC.

Effect of COVID-19 on hepatitis B and C virus countermeasures: Hepatologist responses from nationwide survey in Japan.

AIM: Achieving hepatitis B virus (HBV) and hepatitis C virus (HCV) elimination requires continuous and sustained high volumes of diagnosis and treatment, which have been affected by the ongoing COVID-19 pandemic. This study assessed the effects of COVID-19 on hepatitis-related services in Japan and compared Japan's situation with a global survey. METHODS: We conducted an online cross-sectional questionnaire survey of hepatologists from the Japan Society of Hepatology from August to October 2021 by using the same questionnaire from which a survey was conducted globally to address the effects of COVID-19 on hepatitis-related services. Hepatologists responded based on own impressions of their affiliated institutions. RESULTS: In total, 196 hepatologists participated from 35 prefectures including 49.5% in managerial positions. Approximately 40% survey participants reported a 1%-25% decline in HBV and HCV screening and confirmatory testing. In addition, 53.6% and 45.4% reported no decline in HBV and HCV treatment initiation, respectively. Comparing any level of decrease with the global survey, there was less of a decline observed in Japan for screening (HBV: 51% vs. 56.3%, HCV: 51% vs. 70.9%) and treatment initiation (HBV: 32.7% vs. 52.4%, HCV: 41.8% vs. 66%). However, patient anxiety/fear (67.4%) and loss of staff due to COVID-19 (49.0%) were reported as challenges for resuming services to pre-COVID-19 levels. CONCLUSION: Although in Japan all-inclusive decline in HBV- and HCV-related services were lower than in other countries, a greater decline was observed in HBV and HCV screening and diagnosis than in treatment initiation. Prolonged anxiety/fear among patients, and loss of staff and facilities from the COVID-19 response activities must be addressed to achieve elimination of hepatitis by 2030.

Impact of Immune Reconstitution-Induced Hepatic Flare on Hepatitis B Surface Antigen Loss in Hepatitis B Virus/Human Immunodeficiency Virus-1 Coinfected Patients.

BACKGROUND: Hepatitis B surface antigen (HBsAg) loss is an ideal goal for chronic hepatitis B patients. Antiretroviral therapy (ART) in hepatitis B virus/human immunodeficiency virus-1 (HBV/HIV-1)-coinfected patients can lead to hepatic flare (HF) caused by immune reconstitution-induced inflammatory syndrome (IRIS). Here, we investigated the impact of IRIS-HF on HBsAg loss. METHODS: This was a retrospective study of 58 HBV/HIV-1-coinfected subjects HBsAg-positive for ≥6 months before ART initiation and followed for ≥1 year (median 9.9 years) after ART initiation. We examined humoral factors in sera from healthy volunteers, HIV-monoinfected patients, and HBV/HIV-1-coinfected patients with IRIS-HF or acute hepatitis B infection. RESULTS: During ART, HBsAg loss was observed in 20 of 58 HBV/HIV-1-coinfected patients (34.5%). Of the 58 patients, 15 (25.9%) developed IRIS-HF within 12 months of ART initiation. HBsAg loss was more frequent among patients who developed IRIS-HF (11/15, 73.3%) than those who did not (9/43, 20.9%). Multivariate analysis showed IRIS-HF was an independent predictor of subsequent HBsAg loss. Younger age and higher baseline HBV DNA titer were associated with IRIS-HF. Elevation of sCD163, not CXCL9, CXC10, CXCXL11, or CXCL13, was observed at IRIS-HF. CONCLUSIONS: IRIS-HF was associated with HBsAg loss in HBV/HIV-1-coinfected patients.

Blood angiopoietin-2 predicts liver angiogenesis and fibrosis in hepatitis C patients.

BACKGROUND: Pathological angiogenesis is involved in the development of hepatocellular carcinoma. In patients with chronic hepatitis C (CHC), the level of angiogenic factor angiopoietin (ANGP)-2 is reported to be increased in the blood, correlating with fibrosis. In this study, we aimed to clarify whether blood ANGP-2 is useful as a biomarker for liver angiogenesis and fibrosis in CHC patients and to further reveal the relationship between such pathology in a carbon tetrachloride (CCl4)-treated liver fibrosis mouse model. METHODS: Plasma levels of ANGP-2, expression of a liver sinusoidal endothelial cell (LSEC) marker (CD31), collagen deposition (Sirius Red staining) in the liver, clinical fibrosis markers (Mac-2 binding protein glycosylation isomer, virtual touch quantification, and liver stiffness measurement), and liver function (albumin bilirubin score) were examined in CHC patients. To determine the effects of an anti-angiogenic agent on liver fibrosis in vivo, sorafenib was administered to the CCl4-treated mice (BALB/c male). RESULTS: The plasma levels of ANGP-2 were increased in CHC patients compared to healthy volunteers and decreased by the eradication of hepatitis C with direct-acting antivirals. In addition, plasma ANGP-2 levels were correlated with CD31 expression, collagen deposition, clinical fibrosis markers, and liver function. Sorafenib inhibited liver angiogenesis and fibrosis in the CCl4-treated mice and was accompanied by decreased ANGP-2 expression in LSECs. CONCLUSIONS: ANGP-2 may serve as a useful biomarker for liver angiogenesis and fibrosis in CHC patients. In addition, angiogenesis and fibrosis may be closely related.

Effects of a low-intensity resistance exercise program on serum miR-630, miR-5703, and Fractalkine/CX3CL1 expressions in subjects with No exercise habits: A preliminary study.

AIMS: Exercise is effective for the prevention of liver cancer. Exercise exerts biological effects through the regulation of microRNAs (miRNAs) and cytokines/myokines. We aimed to investigate the effects of low-intensity resistance exercise on serum miRNA and cytokine/myokine expressions in subjects with no exercise habits. METHODS: We enrolled seven male subjects with no exercise habits in this prospective before-after study. All subjects performed a low-intensity resistance exercise program (three metabolic equivalents, approximately 20 min/session). Serum miRNA expressions were evaluated using microarrays. We performed Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of differentially expressed miRNAs before and after exercise. Serum cytokine/myokine expressions were evaluated using a multiplex panel. RESULTS: All subjects completed the exercise program with no adverse events. In the microarray analysis, seven miRNAs showed a significant change between before and after exercise. Of these, microRNA (miR)-630 and miR-5703 showed a >1.5-fold increase (miR-630: 40.7 vs. 69.3 signal intensity, p = 0.0133; miR-5703: 30.7 vs. 55.9 signal intensity, p = 0.0051). KEGG pathway enrichment analysis showed that miR-630- and miR-5703-related genes were enriched in 37 and 5 pathways, including transforming growth factor-beta and Wnt signaling pathways, respectively. In the multiplex analysis, 12 cytokines/myokines showed significant alteration after exercise compared to before exercise. Of these, fractalkine/CX3CL1 showed the most significant up-regulation by exercise (94.5 vs. 109.1 pg/ml, p = 0.0017). CONCLUSIONS: A low-intensity resistance exercise program was associated with upregulation of serum miR-630, miR-5703, and fractalkine/CX3CL1 expressions in subjects with no exercise habits. Thus, even low-intensity exercise may alter miRNA and cytokine/myokine expressions in humans.

Myostatin as a fibroblast-activating factor impacts on postoperative outcome in patients with hepatocellular carcinoma.

AIM: In patients with liver cirrhosis, high levels of serum myostatin are associated with poor prognosis. We aimed to clarify the influence of myostatin on the prognosis of patients with non-alcoholic fatty liver disease-hepatocellular carcinoma (NAFLD-HCC) without cirrhosis and on the progression of liver fibrosis. METHODS: Serum myostatin levels were evaluated in 234 patients who underwent primary surgical resection for single HCC. To clarify the impact of myostatin on liver fibrosis, we established human primary liver fibroblasts from resected livers, and cultured them in the presence of myostatin. RESULTS: The median age was 67.4 years, the median L3 skeletal muscle mass index was 44.4 cm2 /m2 , and the median body mass index was 23.4 kg/m2 . Eighty-two (35.0%) patients had sarcopenia (L3 skeletal muscle mass index: men <42, women <38 cm2 /m2 ). The etiologies of liver disease were hepatitis B virus (n = 61), hepatitis C virus (n = 86), and non-B non-C hepatitis (n = 87) including NAFLD (n = 74). High preoperative serum myostatin and vascular invasion were independent predictors of poor overall survival (OS). High serum myostatin was associated with poor OS in patients with no sarcopenia (n = 152). In patients without advanced liver fibrosis (Fibrosis stage, 0-2; n = 58), high levels of serum myostatin were also associated with poor OS, regardless of sarcopenia. Serum myostatin levels were increased with the progression of liver fibrosis. Liver fibroblasts were activated and produced collagen following stimulation with myostatin. CONCLUSIONS: In patients with NAFLD-HCC without advanced liver fibrosis, high levels of serum myostatin were associated with poor OS. Myostatin activated primary fibroblasts and stimulated collagen production.

USP15 Participates in Hepatitis C Virus Propagation through Regulation of Viral RNA Translation and Lipid Droplet Formation.

Hepatitis C virus (HCV) utilizes cellular factors for efficient propagation. Ubiquitin is covalently conjugated to the substrate to alter its stability or to modulate signal transduction. In this study, we examined the importance of ubiquitination for HCV propagation. We found that inhibition of deubiquitinating enzymes (DUBs) or overexpression of nonspecific DUBs impaired HCV replication, suggesting that ubiquitination regulates HCV replication. To identify specific DUBs involved in HCV propagation, we set up RNA interference (RNAi) screening against DUBs and successfully identified ubiquitin-specific protease 15 (USP15) as a novel host factor for HCV propagation. Our studies showed that USP15 is involved in translation of HCV RNA and production of infectious HCV particles. In addition, deficiency of USP15 in human hepatic cell lines (Huh7 and Hep3B/miR-122 cells) but not in a nonhepatic cell line (293T cells) impaired HCV propagation, suggesting that USP15 participates in HCV propagation through the regulation of hepatocyte-specific functions. Moreover, we showed that loss of USP15 had no effect on innate immune responses in vitro and in vivo We also found that USP15-deficient Huh7 cells showed reductions in the amounts of lipid droplets (LDs), and the addition of palmitic acids restored the production of infectious HCV particles. Taken together, these data suggest that USP15 participates in HCV propagation by regulating the translation of HCV RNA and the formation of LDs.IMPORTANCE Although ubiquitination has been shown to play important roles in the HCV life cycle, the roles of deubiquitinating enzymes (DUBs), which cleave ubiquitin chains from their substrates, in HCV propagation have not been investigated. Here, we identified USP15 as a DUB regulating HCV propagation. USP15 showed no interaction with viral proteins and no participation in innate immune responses. Deficiency of USP15 in Huh7 cells resulted in suppression of the translation of HCV RNA and reduction in the amounts of lipid droplets, and the addition of fatty acids partially restored the production of infectious HCV particles. These data suggest that USP15 participates in HCV propagation in hepatic cells through the regulation of viral RNA translation and lipid metabolism.