Masaki Kaibori

Chronic inflammation associated with hepatitis C virus infection perturbs hepatic transforming growth factor β signaling, promoting cirrhosis and hepatocellular carcinoma†

Many patients with chronic hepatitis caused by hepatitis C virus (HCV) infection develop liver fibrosis with high risk for hepatocellular carcinoma (HCC), but the mechanism underling this process is unclear. Conversely, transforming growth factor beta (TGF‐β) activates not only TGF‐β type I receptor (TβRI) but also c‐Jun N‐terminal kinase (JNK), which convert the mediator Smad3 into two distinctive phosphoisoforms: C‐terminally phosphorylated Smad3 (pSmad3C) and linker‐phosphorylated Smad3 (pSmad3L). Whereas the TβRI/pSmad3C pathway suppresses epithelial cell growth by upregulating p21WAF1 transcription, JNK/pSmad3L‐mediated signaling promotes extracellular matrix deposition, partly, by upregulating plasminogen activator inhibitor 1 (PAI‐1). We studied the domain‐specific Smad3 phosphorylation in biopsy specimens representing chronic hepatitis, cirrhosis, or HCC from 100 patients chronically infected with HCV, and correlated Smad3 phosphorylation with clinical course. As HCV‐infected livers progressed from chronic hepatitis through cirrhosis to HCC, hepatocytic pSmad3L/PAI‐1 increased with fibrotic stage and necroinflammatory grade, and pSmad3C/p21WAF1 decreased. Of 14 patients with chronic hepatitis C with strong hepatocytic pSmad3L positivity, 8 developed HCC within 12 years; only 1 of 12 showing little pSmad3L positivity developed HCC. We further sought molecular mechanisms in vitro. JNK activation by the pro‐inflammatory cytokine interleukin‐1β stimulated the pSmad3L/PAI‐1 pathway in facilitating hepatocytic invasion, in the meantime reducing TGF‐β‐dependent tumor‐suppressive activity by the pSmad3C/p21WAF1 pathway. Conclusion: These results indicate that chronic inflammation associated with HCV infection shifts hepatocytic TGF‐β signaling from tumor‐suppression to fibrogenesis, accelerating liver fibrosis and increasing risk for HCC. (HEPATOLOGY 2007;46:48–57.)

Natural antisense transcript stabilizes inducible nitric oxide synthase messenger RNA in rat hepatocytes

During inflammation, inducible nitric oxide synthase (iNOS) is induced to generate the important mediator nitric oxide (NO). Interleukin 1β (IL‐1β) induces iNOS messenger RNA (mRNA), iNOS protein, and NO in rat hepatocytes. We found that the stability of iNOS mRNA changed during the induction and that the antisense (AS) strand corresponding to the 3′‐untranslated region (3′UTR) of iNOS mRNA was transcribed from the iNOS gene. Expression levels of the iNOS AS transcript correlated with those of iNOS mRNA. The 1.5‐kilobase region 3′‐flanking to iNOS gene exon 27 was involved in IL‐1β induction. Knockdown experiments suggest that sense oligonucleotides to iNOS mRNA significantly reduced iNOS mRNA levels in the hepatocytes by blocking the interaction between iNOS mRNA and the AS transcript. Overexpression of iNOS AS transcript stabilized the reporter luciferase mRNA through the fused iNOS mRNA 3′UTR. These results together with the data in a yeast RNA‐hybrid assay suggested that the iNOS AS transcript interacted with iNOS mRNA and stabilized iNOS mRNA. The iNOS mRNA colocalized with the AU‐rich element‐binding protein HuR, a human homolog of embryonic lethal‐abnormal visual protein, and heterogeneous nuclear ribonucleoprotein L (hnRNP L) in the cytoplasm of rat hepatocytes. Interaction assays further revealed that the iNOS AS transcript interacted with HuR, which interacted with hnRNP L, suggesting that iNOS mRNA, the AS transcript, and the RNA‐binding proteins may mutually interact. Conclusion: The natural AS transcript of the iNOS gene interacts with iNOS mRNA and may play an important role in the stability of iNOS mRNA. This RNA‐RNA interaction may be a new therapeutic target for NO‐mediating inflammatory diseases. (HEPATOLOGY 2008.)

Improved prognosis of postoperative hepatocellular carcinoma patients when treated with functional foods: a prospective cohort study

BACKGROUND/AIMS Active hexose correlated compound (AHCC) is a newly developed functional food. In vitro experiments have shown that AHCC enhances natural killer cell activity, and may be considered a potent biological response modifier in the treatment of cancer patients. However, the effects of AHCC in a clinical setting have not been reported. We seek to determine whether AHCC can improve the prognosis of hepatocellular carcinoma (HCC) patients following surgical treatment. METHODS A prospective cohort study was performed from February 1, 1992 to December 31, 2001. A total of 269 consecutive patients with histologically confirmed HCC were studied. All of the patients underwent resection of a liver tumor. Time to treatment failure (disease recurrence or death) and ten parameters related to liver function after surgery were examined. RESULTS Of the 269 patients, 113 received AHCC orally after undergoing curative surgery (AHCC group). The AHCC group had a significantly longer no recurrence period (hazard ratio (HR), 0.639; 95% confidence interval (CI), 0.429-0.952; P=0.0277) and an increased overall survival rate (HR, 0.421; 95% CI, 0.253-0.701; P=0.0009) when compared to the control group by Coxs multivariate analysis. CONCLUSIONS This study suggests that AHCC intake can improve the prognosis of postoperative HCC patients.

Predictors of microvascular invasion before hepatectomy for hepatocellular carcinoma.

Microvascular invasion (MVI) is difficult to detect before resection of hepatocellular carcinoma (HCC).

Immunosuppressant FK506 inhibits inducible nitric oxide synthase gene expression at a step of NF-κB activation in rat hepatocytes

BACKGROUND/AIMS Recent evidence indicates that an increase in nitric oxide production after liver transplantation is associated with acute allograft rejection. Nitric oxide mediates cellular injury under various pathological conditions in the liver. Studies were performed to determine whether the immunosuppressants FK506 and cyclosporin A directly influence gene expression of inducible nitric oxide synthase by interleukin 1beta in hepatocytes. METHODS Primary cultures of rat hepatocytes were treated with interleukin 1beta in the presence and absence of FK506 or cyclosporin A. Release of nitrite (nitric oxide metabolite) into culture medium, levels of inducible nitric oxide synthase protein and mRNA, and activation of nuclear factor-kappaB were compared with the two drugs. RESULTS Interleukin 1beta increased levels of inducible nitric oxide synthase protein and inducible nitric oxide synthase mRNA, as well as nitric oxide production, in the cultured hepatocytes. Nuclear factor-kappaB, an important transcription factor in inducible nitric oxide synthase gene expression in response to inflammation, also appeared in the nuclear fraction of hepatocytes after addition of interleukin 1beta. FK506 markedly inhibited the nitric oxide formation, inducible nitric oxide synthase protein synthesis and inducible nitric oxide synthase mRNA expression induced by interleukin 1beta, but cyclosporin A had no effects. Furthermore, FK506 inhibited nuclear factor-kappaB activation and decreased mRNA levels of the p50/p65 subunits of nuclear factor-kappaB. CONCLUSIONS These results demonstrate that FK506, but not cyclosporin A, inhibits the induction of inducible nitric oxide synthase expression during nuclear factor-kappaB activation. FK506 may influence liver function during diseases by modulating the nitric oxide pathway, in addition to its immunosuppressive effect.

Vicinal dithiol-binding agent, phenylarsine oxide, inhibits inducible nitric-oxide synthase gene expression at a step of nuclear factor-kappaB DNA binding in hepatocytes.

Inflammatory cytokine interleukin 1β induces inducible nitric-oxide synthase (iNOS) mRNA and its protein, which are followed by increasing the production of nitric oxide, in primary cultures of rat hepatocytes. Nuclear factor-κB (NF-κB), an important transcription factor for iNOS gene expression, is also activated and translocated to the nucleus. In the present study, we found that vicinal dithiol-binding agent, phenylarsine oxide (PAO), inhibited the induction of iNOS protein and mRNA as well as the release of nitrite (nitric oxide metabolite) into the culture medium. Simultaneous addition of a vicinal dithiol compound, 2,3-dimercaptopropanol, with PAO completely abolished these inhibitions. PAO could not prevent either degradation of an inhibitory protein, IκB, of NF-κB or translocation of NF-κB to the nucleus. However, electrophoretic mobility shift assay demonstrated that PAO decreased the interaction between NF-κB and its binding consensus oligonucleotide. Transfection experiments with iNOS promoter-luciferase construct revealed that PAO inhibited NF-κB binding to DNA. These results indicate that PAO inhibits iNOS gene expression at a step of NF-κB binding to DNA by modifying its vicinal dithiol moiety, which may play a crucial role for the iNOS regulation in hepatocytes.

Intraoperative indocyanine green fluorescent imaging for prevention of bile leakage after hepatic resection

BACKGROUND Bile leakage is a common complication of hepatectomy, and is associated with an increase in sepsis and liver failure. There are no standard preventive methods against bile leakage after hepatic surgery. The aim of the present randomized clinical trial was to evaluate the application of indocyanine green (ICG) fluorescent cholangiography for preventing postoperative bile leakage. METHODS 102 patients who underwent hepatic resection without biliary reconstruction were divided into 2 groups. The control group (n = 50) underwent a leak test with ICG dye alone, and the experimental group underwent a leak test with ICG dye, followed by ICG fluorescent cholangiography using the Photodynamic Eye (PDE group, n = 52). RESULTS Among 42 patients with fluorescence in the PDE group, 25 patients had insufficient closure of bile ducts on the cut surface of the liver, which were closed by suture or ligation. There were 5 patients who developed postoperative bile leakage in the control group versus no bile leakage in the PDE group (10% vs 0%, P = .019). CONCLUSION ICG fluorescent cholangiography could detect insufficiently closed bile ducts that could not be identified by a standard bile leak test. ICG fluorescent cholangiography may have useful potential for prevention of bile leakage after hepatic resection.

Hepatic resection for hepatocellular carcinoma in the elderly

Aging of the population has significantly increased the number of elderly patients undergoing surgery for hepatocellular carcinoma (HCC). We aimed to compare the results of hepatectomy for HCC in patients ≥70 years old with those for younger patients.

Stimulation of liver regeneration and function after partial hepatectomy in cirrhotic rats by continuous infusion of recombinant human hepatocyte growth factor

BACKGROUND/AIMS Radical resection is accepted as one of the most curative treatments for hepatocellular carcinoma. However, most patients have coexisting cirrhosis and their liver function is usually impaired. It is therefore important to stimulate the regeneration and function of the remnant cirrhotic liver after hepatectomy. Hepatocyte growth factor is a potent mitogen that has been suggested to play a crucial role in liver regeneration. METHODS In this study, we performed 45% hepatectomy in rats with cirrhosis induced by thioacetamide, and administered recombinant human hepatocyte growth factor (rhHGF) with dextran sulfate by continuous infusion into the jugular vein with an infusion pump. RESULTS rhHGF stimulated an increase in the wet weight of the remnant liver compared with untreated control rats. The proliferating cell nuclear antigen labeling index showed that this increase resulted from the stimulation of DNA synthesis. Serum levels of liver enzymes increased after hepatectomy, but returned to the prehepatectomy level more rapidly in rhHGF-treated rats than in controls. rhHGF increased hepatic protein synthesis above prehepatectomy levels and also markedly increased the serum levels of hepatic lipid metabolites. CONCLUSIONS These results demonstrate that continuous intravenous infusion of rhHGF enhances the growth and function of the remnant liver in rats with cirrhosis after partial hepatectomy. Therefore, rhHGF may be useful after hepatic resection in patients with cirrhosis.

Risk factors and outcome of early recurrence after resection of small hepatocellular carcinomas

BACKGROUND This study aimed to clarify risk factors for early recurrence and examine the subsequent outcome in patients undergoing potentially R0 resection of small hepatocellular carcinomas (HCCs) (<or=2 cm in greatest dimension). METHODS Eighty-nine patients were divided into 2 groups as follows: 26 patients suffering from recurrence within 2 years of surgery (early recurrence group) and 63 patients who were disease-free for at least 2 years (disease-free 2Y group). RESULTS Only 7 of 63 patients (11%) from the group that was disease-free for at least 2 years died during the 5-year period after surgery, whereas 13 of 26 patients (50%) from the early recurrence group died. Multivariate analysis showed that the preoperative maximum removal rate of technetium-99m-diethylenetriamine pentaacetic acid-galactosyl human serum albumin and microscopic vascular invasion were independent predictors of the early recurrence of small HCC. CONCLUSIONS Early recurrence of small HCC is the leading cause of death within 5 years after R0 resection. The preoperative hepatic functional reserve influences early recurrence, even in patients with small tumors.