Overexpression of miR-4669 Enhances Tumor Aggressiveness and Generates an Immunosuppressive Tumor Microenvironment in Hepatocellular Carcinoma: Its Clinical Value as a Predictive Biomarker.

Accumulating evidence suggests the involvement of tumor-derived exosomes in the development and recurrence of hepatocellular carcinoma (HCC). We previously identified miR-4669 as a highly expressed microRNA in circulating exosomes obtained from patients with post-transplant HCC recurrence. This study aimed to explore how overexpression of miR-4669 affects HCC development and recurrence. The impact of miR-4669 overexpression in Hep3B cells on tumor cell behavior and the tumor microenvironment was evaluated in vitro. In addition, the clinical value of exosomal miR-4669 for the prediction of treatment response to HCC downstaging therapies and following post-transplant HCC recurrence was explored. Overexpression of miR-4669 enhanced migration ability and led to acquired sorafenib resistance with an elevation of sirtuin 1 and long noncoding RNA associated with microvascular invasion. Active release of tumor-derived exosomes and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) contributed to generating an immunosuppressive tumor microenvironment through the induction of M2 macrophage polarization. The retrospective analysis demonstrated the clinical value of exosomal miR-4669 for predicting treatment response to HCC downstaging therapies and for risk assessment of post-transplant HCC recurrence. In summary, the present data demonstrate the impact of exosomal miR-4669 on HCC recurrence through the enhancement of tumor aggressiveness and generation of an immunosuppressive tumor microenvironment.

Circulating exosomal miR-92b: Its role for cancer immunoediting and clinical value for prediction of posttransplant hepatocellular carcinoma recurrence.

A recurrence of hepatocellular carcinoma (HCC) after living donor liver transplantation (LDLT) is one of the major concerns reflecting the higher mortality of HCC. This study aimed to explore the impact of circulating exosomes on HCC development and recurrence. One-shot transfusion of hepatoma serum to naïve rats induced liver cancer development with gradual elevation of alpha-fetoprotein (AFP), but exosome-free hepatoma serum failed to induce AFP elevation. The microarray analysis revealed miR-92b as one of the highly expressing microribonucleic acids in hepatoma serum exosomes. Overexpression of miR-92b enhanced the migration ability of liver cancer cell lines with active release of exosomal miR-92b. The hepatoma-derived exosomal miR-92b transferred to natural killer (NK) cells, resulting in the downregulation of CD69 and NK cell-mediated cytotoxicity. Furthermore, higher expression of miR-92b in serum exosomes was confirmed in HCC patients before LDLT, and its value at 1 month after LDLT was maintained at a higher level in the patients with posttransplant HCC recurrence. In summary, we demonstrated the impact of circulating exosomes on liver cancer development, partly through the suppression of CD69 on NK cells by hepatoma-derived exosomal miR-92b. The value of circulating exosomal miR-92b may predict the risk of posttransplant HCC recurrence.

A novel moonlight function of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for immunomodulation.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an energy metabolism-related enzyme, which generates NADH in glycolysis. Our previous study revealed a novel role of exogenous GAPDH in the amelioration of lipopolysaccharide (LPS)-induced sepsis-related, severe acute lung injury (ALI) in mice. Here, we show the effect of extracellular GAPDH on the physiological functions of macrophages, which play an important role in the onset of sepsis and ALI. GAPDH has no effect on cell viability, while it strongly suppressed cell adhesion, spreading, and phagocytic function of LPS-stimulated macrophages. GAPDH treatment significantly reduced tumor necrosis factor (TNF)-α, while it induced interleukin (IL)-10 production from LPS-stimulated macrophages in a dose-dependent manner. It is noteworthy that heat inactivation of GAPDH lost its immunomodulatory activity. Correspondingly, NADH significantly inhibited TNF-α and enhanced IL-10 production with elevation of both M1/M2 macrophage markers. These data suggest that extracellular GAPDH induces intermediate M1/M2 macrophages for termination of inflammation, partly through its enzyme activity for generation of NADH. © 2018 BioFactors, 44(6):597-608, 2018.

Hepatic miR-301a as a Liver Transplant Rejection Biomarker? And Its Role for Interleukin-6 Production in Hepatocytes.

Acute rejection (AR) of liver transplantation remains a formidable challenge for diagnostic medicine and biomarker discovery. We characterized AR-related microRNAs (miRNAs) and the underlying AR mechanisms in liver transplantation. Using a rat model of orthotopic liver transplantation (OLT) as well as microarrays, we compared the miRNA expression profiles between naive and AR livers on day 7 after OLT with short- (<14 days, donor Dark Agouti [DA] liver into Lewis [LEW] recipient) and long-term (>60 days, donor DA liver into Piebald Virol Glaxo [PVG] recipient) survival fates. The microarray analysis revealed that the levels of miR-301a in the lethal AR livers were significantly higher than in naive and tolerogenic AR livers. The reduced expression of miR-301a in inflamed livers suggested a difference between AR and inflammation in terms of miR-301a-mediated molecular events. Overexpression of hepatic miR-301a induced IL-6 production in rat primary hepatocytes. Hepatocytes overexpressing miR-301a were capable of transferring miR-301a to cocultured splenocytes through exosomes. These splenocytes then showed overexpression of miR-301a and downregulation of protein inhibitor of activated STAT3 (PIAS3) expression, resulting in the induction of T helper 17 cell differentiation. In conclusion, this report raises the possibility that hepatic miR-301a might potentially prove value as a biomarker of liver transplant rejection. We call for future research on this molecular target and the attendant pathways as liver transplant rejection and its early diagnosis continue to be veritable healthcare challenges.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) prevents lipopolysaccharide (LPS)-induced, sepsis-related severe acute lung injury in mice.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an energy metabolism-related enzyme in the glycolytic pathway. Recently, it has been reported that GAPDH has other physiological functions, such as apoptosis, DNA repair and autophagy. Some in vitro studies have indicated immunological aspects of GAPDH function, although there is no definite study discussing the advantage of GAPDH as a therapeutic target. Here, we show that GAPDH has an anti-inflammatory function by using a lipopolysaccharide (LPS)-induced, sepsis-related severe acute lung injury (ALI) mouse model, which is referred to as acute respiratory distress syndrome (ARDS) in humans. GAPDH pre-injected mice were protected from septic death, and their serum levels of proinflammatory cytokines were significantly suppressed. In lung tissue, LPS-induced acute injury and neutrophil accumulation were strongly inhibited by GAPDH pre-injection. Pulmonary, proinflammatory cytokine gene expression and serum chemokine expression in GAPDH pre-injected mice were also reduced. These data suggest the therapeutic potential of GAPDH for sepsis-related ALI/ARDS.

Biological interactions of CYP2C19 genotypes with CYP3A4*18, CYP3A5*3, and MDR1-3435 in living donor liver transplantation recipients.

BACKGROUND: Polymorphisms in CYP2C19 are related to the metabolic oxidation of drugs to varying degrees. The CYP3A4*18, CYP3A5*3, and MDR1-3435 variant alleles are very important, particularly in tacrolimus metabolism in organ transplant rejection. AIM: The aim of this study is o explore possible interactions among different CYP2C19 genotypes, namely, between homozygous extensive metabolizers (HomEM), heterozygous extensive metabolizers (HetEM), and poor metabolizers (PM), and the CYP3A4*18, CYP3A5*3, and MDR1-3435 variants in living donors and patients who received a living donor liver transplant (LDLT). METHODS: This prospective study enrolled 133 living donors and 133 corresponding recipients. On the basis of the HomEM, HetEM, and PM CYP2C19 genotypes, the distributions of CYP3A4*18 (exon 10; T878C), CYP3A5*3 (intron 3; A6986G), and MDR1-3435 (exon 26; C3435T) genotypes were analyzed for single nucleotide polymorphisms among donors and recipients. RESULTS: Among 102 HomEM genotypes, including 56 donors and 46 recipients, 91.2% of individuals harbored the T/T genotype of CYP3A4*18; 53.9% possessed G/G, and 34.3% had A/G genotypes of CYP3A5*3; and 38.2% had C/C and 50.0% had C/T genotypes at MDR1-3435. Among 130 HetEM genotypes, including 58 donors and 72 recipients, 97.7% of individuals possessed T/T genotype at CYP3A4*18; 50.0% harbored G/G and 41.5% had A/G genotypes at CYP3A5*3; and 40.0% had C/C and 49.2% had C/T genotypes at MDR1-3435. In 34 PMs, including 19 donors and 15 recipients, 88.2% had T/T genotypes at CYP3A4*18; 41.2% had G/G and 58.8% had A/G genotypes at CYP3A5*3; and 47.1% possessed C/C and 47.1% had C/T genotypes at MDR1-3435. On the basis of the CYP2C19 genotypes, no statistically significant distribution of genotypes were observed between donors and recipients for all genotypes of CYP3A4*18, CYP3A5*3, and MDR1-3435 (P >0.05). CONCLUSIONS: In conclusion, the CYP2C19 genotypes do not affect the expression of CYP3A4*18, CYP3A5*3, or MDR1-3435 variants, which are independently distributed among donors and recipients during LDLT.

Induction of antinuclear antibodies by de novo autoimmune hepatitis regulates alloimmune responses in rat liver transplantation.

Concanavalin A (Con A) is a lectin originating from the jack-bean and well known for its ability to stimulate T cells and induce autoimmune hepatitis. We previously demonstrated the induction of immunosuppressive antinuclear autoantibody in the course of Con A-induced transient autoimmune hepatitis. This study aimed to clarify the effects of Con A-induced hepatitis on liver allograft rejection and acceptance. In this study, we observed the unique phenomenon that the induction of transient de novo autoimmune hepatitis by Con A injection paradoxically overcomes the rejection without any immunosuppressive drug and exhibits significantly prolonged survival after orthotopic liver transplantation (OLT). Significantly increased titers of anti-nuclear Abs against histone H1 and high-mobility group box 1 (HMGB1) and reduced donor specific alloantibody response were observed in Con A-injected recipients. Induction of Foxp3 and IL-10 in OLT livers of Con A-injected recipients suggested the involvement of regulatory T cells in this unique phenomenon. Our present data suggest the significance of autoimmune responses against nuclear histone H1 and HMGB1 for competing allogeneic immune responses, resulting in the acceptance of liver allografts in experimental liver transplantation.

Western blotting analysis for quantitative detection of CYP2C19 expression in liver tissues in the setting of living donor liver transplantation.

BACKGROUND: The cytochrome P450 (CYP) drug-metabolizing enzymes play an important role in cellular metabolism. Therapeutic failure or drug toxicity in the period after liver transplantation (LT) is influenced by the drug metabolizing capacity of the graft. The expression levels of CYP2C19 enzyme are often used as an indicator of the functioning of the CYP system. The aim of the present study was to assess the CYP2C19 protein expression in the setting of living donor liver transplantation (LDLT) by using western blotting analysis. METHODOLOGY: We performed CYP2C19 genotyping of liver biopsy samples obtained from 24 donors and 8 recipients each in the pre- and post-LT periods, after which we analyzed the CYP enzyme activity by using western blotting analysis. RESULTS: The CYP2C19/ß-actin ratio, which was an indicator of CYP expression, was 61.75% (23-100%) in donors, 59.13% (15-100%) in pre-LT recipients and 46.71% (12-67%) in post-LT recipients (p>0.05). The CYP2C19 expression levels associated with different genotypes were as follows: homozygous extensive metabolizers (HomEMs; n=24), 56.63±24.74%; heterozygous extensive metabolizers (HetEMs; n=15), 63.0±25.14% and poor metabolizers (PMs; n=1), 82.0% (p>0.05). CONCLUSIONS: Western blotting analysis showed low CYP2C19 protein expression not only in samples from the pre- and post-LT recipients but also from the donors.

Homogenous phenomenon of graft liver CYP2C19 genotypes after living donor liver transplantation.

BACKGROUND: The donor liver grafts with different allelic patterns do not affect CYP2C19 genotypes in the peripheral blood of living donor liver transplantation (LDLT) recipients. AIM: This study investigated the influence of graft liver CYP2C19 genotypes on recipients who received the same or different CYP2C19 genotypes from donors after LDLT. METHODS: There were 30 donors and 30 recipients with the same CYP2C19 genotypes and 47 donors and 47 recipients with different CYP2C19 genotypes. Genomic DNA was isolated from the liver tissue of recipients. The CYP2C19 haplotypes were determined by polymerase chain reaction. RESULTS: A homogenous phenomenon in the sequences of graft liver CYP2C19 genotypes was indicated because the recipients showed mixed patterns that were similar to that of the original donor after LDLT. A significant decrease in homozygous extensive metabolizer (HomEM) and an increase in poor metabolizer (PM) distribution were observed in recipients with different CYP2C19 genotypes from their donors compared with recipients with the same CYP2C19 genotype as their donors (P < 0·05). CONCLUSIONS: Homogenous phenomenon of sequence changes in graft liver CYP2C19 from the different genotypes between the donors and the recipients may play a role in graft stability by causing decreased HomEM and increased PM after LDLT.

Influence of CYP2C19 genotypes on graft pathological findings and postoperative liver function in recipients after living-donor liver transplantation.

BACKGROUND: New graft cytochrome P450 2C19 (CYP2C19) should have different characteristics after living-donor liver transplantation (LDLT). We prospectively investigated the influence of genotypes of CYP2C19 in liver graft pathological finding (GPF) and postoperative liver function (POLF) of recipients after LDLT. MATERIAL/METHODS: Among 60 consecutive patients who underwent LDLT, 36 recipients developed sudden-onset abnormal liver function and required liver biopsy, while the remaining 24 recipients did not require biopsy. LDLT recipient blood samples were genotyped for CYP2C19 variants, and recipients were categorized as homozygous extensive metabolizers (HomEM), heterozygous extensive metabolizers (HetEM), and poor metabolizers (PM). RESULTS: The acute rejection rate was 15.0% (9/60). There were 4 characteristics of GPF when abnormal POLF occurred. Logistic regression analysis showed that the risk estimate of abnormal POLF was 26.7 times higher for LDLT recipients with the CYP2C19 HomEM genotype as compared to the PM genotype, and 5.0 times higher for LDLT recipients with the HomEM genotype as compared to the HetEM genotype. A linear trend relationship was found between the incidence of abnormal POLF and the CYP2C19 genotype. CONCLUSIONS: These results suggest that the speed of drug metabolism as characterized by the CYP2C19 genotype (HomEM > HetEM > PM) may affect the outcome after LDLT; thus, CYP2C19 genotyping may be valuable for predicting abnormal POLF with different GPF after LDLT.

Donor graft does not affect the P450 2C19 genotype expressed in peripheral blood in recipients of living donor liver transplantation.

The function of cytochrome P450 2C19 (CYP2C19) is altered in patients with end-stage liver disease (ESLD) that require liver transplantation (LT). The status of CYP2C19 is of considerable interest because the transplanted healthy donor livers are perfused with the blood of the recipient with ESLD. This study aims to clarify the changes in CYP2C19 in the peripheral blood before and after LT. Thirty pairs of living donors and recipients were enrolled in this study. The CYP2C19 genotype in peripheral blood mononuclear cells (PBMCs) was studied immediately before operation in donors, on the day preceding the operation in the unstable recipients, and one month after LT in stable recipients. Limited data suggest that the post-LT genotype in liver biopsy is the same as donor's original genotype in most cases (80.0%) and that only 2 patients in the study cohort had the same liver tissue genotype as the respective recipient PBMCs. However, expression of the CYP2C19 genotype after living donor LT (LDLT) was identical to pre-transplant expression in 100% (30/30) of recipients, i.e., CYP2C19 genotypes in recipient PBMCs did not change after LDLT, suggesting that the donor liver did not render any mutations to the CYP2C19 genotypes after LT.

Prolongation of heart allograft survival of rats treated by a Th2 inhibitor.

In terms of Th1/Th2 balance in response to signals given during donor antigen presentation, induction of tolerance is more often correlated with Th2-type than with Th1-type reactions. However, in our study, heart allograft survival was prolonged by treatment of rats with a Th2 inhibitor. Suplatast tosilate (IPD; Taiho; Tokyo, Japan) is a novel immunoregulator that suppresses IgE production and eosinophil infiltration through selective inhibition of interleukin (IL)-4 and IL-5 synthesis by Th2-like cells but not IFN-gamma production in Th1 cells. Five LEW rats of DA heart grafts were treated with IPD (100 microg/day, p.o.) for 10 days. Heart allograft survival of all IPD-treated cases was prolonged more than 14 days while the beating of heart grafts in control groups was stopped within 9 days. In an in vitro study, the cell proliferation both in Con A blast and in mixed lymphocyte reaction assay was suppressed by IPD in dose-dependent manner. We could at least in part conclude that Th2 inhibition might temporarily suppress heart allograft rejection.