Yasushi Tanoue

Autotaxin as a novel serum marker of liver fibrosis

BACKGROUND The clinical significance of autotaxin (ATX), a key enzyme for the production of the bioactive lysophospholipid lysophosphatidic acid remains unknown. Serum ATX enzymatic activity reportedly increases in parallel with liver fibrosis and exhibits a gender difference. METHODS Serum ATX antigen level, measured easier than the activity, was evaluated as a marker of liver fibrosis in 2 cohorts of chronic liver disease caused by hepatitis C virus. RESULTS In the first cohort, serum ATX level correlated significantly with liver fibrosis stage and was the best parameter for prediction of cirrhosis with an area under the receiver operating characteristic curve (AUROC) of 0.756 in male and 0.760 in female, when compared with serum hyaluronic acid and aminotransferase-to-platelet ratio index, an established marker of liver fibrosis. In another cohort, serum ATX level correlated significantly with liver stiffness, a novel reliable marker of liver fibrosis, being the second-best parameter in male (AUROC, 0.799) and in female (AUROC, 0.876) for prediction of significant fibrosis, and the best parameter in male (AUROC, 0.863) and the third-best parameter in female (AUROC, 0.872) for prediction of cirrhosis, both of which were judged by liver stiffness. CONCLUSIONS Serum ATX level may be a novel marker of liver fibrosis.

Plasma concentration of bioactive lipid mediator sphingosine 1-phosphate is reduced in patients with chronic hepatitis C

BACKGROUND Bioactive lipid mediator S1P has been suggested to play pathophysiological roles in various fields of clinical science as a circulating paracrine mediator. We previously established a reliable method of measuring plasma S1P concentration, and reported that the one in healthy subjects has a gender difference and a correlation with red blood cell (RBC)-parameters, however, the reports of S1P measurements in the blood in patients with a specific disease have been scarce. Because our previous evidence suggests that S1P is involved in liver pathophysiology, we examined plasma S1P concentration in chronic hepatitis C patients. METHODS S1P assay was performed using a high-performance liquid chromatography system. RESULTS Plasma S1P concentrations were reduced in chronic hepatitis C patients compared with in healthy subjects with the same hemoglobin concentration, irrespective of gender. Among the blood parameters, serum hyaluronic acid concentration, a surrogate marker for liver fibrosis, was most closely and inversely correlated with plasma S1P concentration. Furthermore, plasma S1P concentration decreased throughout the progression of carbon tetrachloride-induced liver fibrosis in rats. CONCLUSIONS Plasma S1P concentration was reduced in chronic hepatitis C patients, and liver fibrosis might be involved, at least in part, in the mechanism responsible for this reduction.

Antagonism of sphingosine 1‐phosphate receptor 2 causes a selective reduction of portal vein pressure in bile duct‐ligated rodents

Sinusoidal vasoconstriction, in which hepatic stellate cells operate as contractile machinery, has been suggested to play a pivotal role in the pathophysiology of portal hypertension. We investigated whether sphingosine 1‐phosphate (S1P) stimulates contractility of those cells and enhances portal vein pressure in isolated perfused rat livers with Rho activation by way of S1P receptor 2 (S1P2). Rho and its effector, Rho kinase, reportedly contribute to the pathophysiology of portal hypertension. Thus, a potential effect of S1P2 antagonism on portal hypertension was examined. Intravenous infusion of the S1P2 antagonist, JTE‐013, at 1 mg/kg body weight reduced portal vein pressure by 24% without affecting mean arterial pressure in cirrhotic rats induced by bile duct ligation at 4 weeks after the operation, whereas the same amount of S1P2 antagonist did not alter portal vein pressure and mean arterial pressure in control sham‐operated rats. Rho kinase activity in the livers was enhanced in bile duct‐ligated rats compared to sham‐operated rats, and this enhanced Rho kinase activity in bile duct‐ligated livers was reduced after infusion of the S1P2 antagonist. S1P2 messenger RNA (mRNA) expression, but not S1P1 or S1P3, was increased in bile duct‐ligated livers of rats and mice and also in culture‐activated rat hepatic stellate cells. S1P2 expression, determined in S1P  2LacZ/+ mice, was highly increased in hepatic stellate cells of bile duct‐ligated livers. Furthermore, the increase of Rho kinase activity in bile duct‐ligated livers was observed as early as 7 days after the operation in wildtype mice, but was less in S1P  2−/− mice. Conclusion: S1P may play an important role in the pathophysiology of portal hypertension with Rho kinase activation by way of S1P2. The S1P2 antagonist merits consideration as a novel therapeutic agent for portal hypertension. (HEPATOLOGY 2012)

Prediction of Hepatocellular Carcinoma Development by Plasma ADAMTS13 in Chronic Hepatitis B and C

Background: Chronic liver injury evokes a wound healing response, promoting fibrosis and finally hepatocellular carcinoma (HCC), in which hepatic stellate cells play an important role. Although a blood marker of hepatic stellate cells is not known, those cells importantly contribute to the regulation of plasma a disintegrin-like and metalloproteinase with thrombospondin type-1 motifs 13 (ADAMTS13) activity, a defect of which causes thrombotic thrombocytopenic purpura. Methods: Plasma ADAMTS13 was evaluated in chronic hepatitis B or C patients with or without HCC. Results: Plasma ADAMTS13 activity significantly correlated with serum aspartate aminotransferase and alanine aminotransferase, liver stiffness value, and aspartate aminotransferase-to-platelet ratio index, irrespective of the presence of HCC, suggesting that it may reflect hepatocellular damage and subsequent wound healing and fibrosis as a result of hepatic stellate cell action. During the three-year follow-up period for patients without HCC, it developed in 10 among 81 patients. Plasma ADAMTS13 activity was significantly higher in patients with HCC development than in those without and was a significant risk for HCC development by univariate and multivariate analyses. Furthermore, during the one-year follow-up period for patients with HCC treated with radiofrequency ablation, HCC recurred in 55 among 107 patients. Plasma ADAMTS13 activity or antigen level was significantly higher in patients with HCC recurrence than in those without and was retained as a significant risk for HCC recurrence by multivariate analysis. Conclusions: Higher plasma ADAMTS13 activity and antigen level was a risk of HCC development in chronic liver disease. Impact: Plasma ADAMTS13 as a potential marker of hepatic stellate cells may be useful in the prediction of hepatocarcinogenesis. Cancer Epidemiol Biomarkers Prev; 20(10); 2204–11. ©2011 AACR.

Novel chemoimmunotherapeutic strategy for hepatocellular carcinoma based on a genome-wide association study

Pharmacotherapeutic options are limited for hepatocellular carcinoma (HCC). Recently, we identified the anti-tumor ligand MHC class I polypeptide-related sequence A (MICA) gene as a susceptibility gene for hepatitis C virus-induced HCC in a genome-wide association study (GWAS). To prove the concept of HCC immunotherapy based on the results of a GWAS, in the present study, we searched for drugs that could restore MICA expression. A screen of the FDA-approved drug library identified the anti-cancer agent vorinostat as the strongest hit, suggesting histone deacetylase inhibitors (HDACis) as potent candidates. Indeed, the HDACi-induced expression of MICA specific to HCC cells enhanced natural killer (NK) cell-mediated cytotoxicity in co-culture, which was further reinforced by treatment with an inhibitor of MICA sheddase. Similarly augmented anti-tumor activity of NK cells via NK group 2D was observed in vivo. Metabolomics analysis revealed HDACi-mediated alterations in energy supply and stresses for MICA induction and HCC inhibition, providing a mechanism for the chemoimmunotherapeutic actions. These data are indicative of promising strategies for selective HCC innate immunotherapy.

Induction of p53-Dependent p21 Limits Proliferative Activity of Rat Hepatocytes in the Presence of Hepatocyte Growth Factor

Background Hepatocyte growth factor (HGF), a potent mitogen for hepatocytes, enhances hepatocyte function without stimulating proliferation, depending on the physiological conditions. p53, a transcription factor, suppresses the cell proliferation by expressing p21WAF1/CIP1 in various tissues. Aim To investigate the mechanism through which the hepatocytes maintain mitotically quiescent even in the presence of HGF. Methods We studied the relationship between p53 and p21 expression and the effect of p53-p21 axis on hepatocyte proliferation in primary cultured rat hepatocytes stimulated by HGF. Hepatic p21 levels are determined serially after partial hepatectomy or sham operation in rats. Results DNA synthesis was markedly increased by HGF addition in rat hepatocytes cultured at low density but not at high density. Cellular p53 levels increased in the hepatocytes cultured at both the densities. p21 levels were increased and correlated with cellular p53 levels in hepatocytes cultured at high density but not at low density. When the activity of p53 was suppressed by a chemical inhibitor for p53, cellular p21 levels were reduced, and DNA synthesis was increased. Similarly, p21 antisense oligonucleotide increased the DNA synthesis. In rats after partial hepatectomy, transient elevation of hepatic p21 levels was observed. In contrast, in sham-operated rats, hepatic p21 levels were increased on sustained time scales. Conclusion p53-related induction of p21 may suppress hepatocyte proliferation in the presence of HGF in the setting that mitogenic activity of HGF is not elicitable.

Predominance of regorafenib over sorafenib: restoration of membrane-bound MICA in hepatocellular carcinoma cells

The multi‐kinase inhibitor regorafenib (REG) was recently demonstrated to be effective in patients with sorafenib (SOR)‐resistant hepatocellular carcinoma (HCC). Interestingly, SOR is known to enhance the accumulation of membrane‐bound MHC class I polypeptide‐related sequence A (mMICA) in HCC cells and to block the production of soluble MICA (sMICA), an immunological decoy. In addition, MICA is associated with HCC in patients with chronic hepatitis C. We have now compared the impact of REG and SOR on MICA in HCC cells, as well as the immunotherapeutic implications thereof.

Enzymatic inhibition of MICA sheddase ADAM17 by lomofungin in hepatocellular carcinoma cells: Lomofungin suppresses ADAM17 in hepatocellular carcinoma cells

In our previous study on hepatocellular carcinoma (HCC) susceptibility genes in chronic hepatitis patients, we identified the MHC class I polypeptide‐related sequence A (MICA). Natural killer cells eliminate various cancer cells, including HCC, by suppressing MICA shedding. Therefore, we investigated MICA sheddases and inhibitors for HCC immunotherapy. In this study, HepG2, PLC/PRF/5, and Hep3B were treated with the siRNA of a disintegrin and metalloproteases (ADAMs) and matrix metalloproteases to measure the concentration of soluble MICA (sMICA) by ELISA to detect the therapeutic target. Furthermore, an FDA‐approved drug library was tested for the enzymatic inhibition of the targeted enzyme in an in vitro drug screening assay system. ADAM17 knockdown reduced sMICA levels and increased membrane‐bound MICA (mMICA) expression in HCC cells. In an in vitro drug screen using an FDA‐approved drug library, lomofungin, an antifungal drug, was found to strongly decrease ADAM17 activity. In HCC cells, mMICA expression was induced and sMICA production was inhibited in a dose‐dependent manner. These effects were cancelled upon ADAM17 knockdown, suggesting that lomofungin targeted ADAM17. Analysis of lomofungin analogs revealed the responsible functional groups. In summary, we suggest lomofungin to be an attractive agent for the immunological control of HCC, via the suppression of ADAM17.