Kazuaki Tejima

Both plasma lysophosphatidic acid and serum autotaxin levels are increased in chronic hepatitis C.

Objectives Recent accumulating evidence indicates that lysophosphatidic acid (LPA) is a lipid mediator, abundantly present in blood, with a wide range of biologic actions including the regulation of proliferation and contraction in liver cells. Although it is speculated that LPA might play a role in pathophysiologic processes in vivo, not only its role but also even a possible alteration in its blood concentration under specific diseases is essentially unknown. Autotaxin (ATX), originally purified as an autocrine motility factor for melanoma cells, was revealed to be a key enzyme in LPA synthesis. We determined LPA and ATX levels in the blood of patients with liver disease. Methods ATX activity was measured by determining choline with the substrate of lysophosphatidylcholine, and the LPA level by an enzymatic cycling method in 41 patients with chronic hepatitis C. Results The serum ATX activity and plasma LPA level were significantly increased in patients, and were correlated positively with serum hyaluronic acid, and negatively with platelets, albumin, and prothrombin time. The plasma LPA level was strongly correlated with serum ATX activity. There were significant correlations between the histologic stage of fibrosis and both the serum ATX activity and plasma LPA level. Conclusions The serum ATX activity and plasma LPA level are increased in chronic hepatitis C in association with liver fibrosis. Our study may provide the first evidence showing a significant increase of both ATX and LPA in the blood under a specific disease.

Functional diversity between Rho-kinase- and MLCK-mediated cytoskeletal actions in a myofibroblast-like hepatic stellate cell line.

Using a rat myofibroblast-like hepatic stellate cell line, we studied the actomyosin-based cytoskeletal actions mediated by Rho-kinase and/or myosin light chain kinase (MLCK). Calmodulin/MLCK inhibitors W-7 and ML-7 attenuated cell migration dose-relatedly at concentrations from 10(-6) to 10(-4)M and collagen gel-contraction by the cells at 10(-4)M, respectively. Rho-kinase inhibitors Y-27632 and HA1077 attenuated the gel-contraction at concentrations from 10(-6) to 10(-4) M, respectively. These Rho-kinase inhibitors attenuated cell migration at 10(-7)M but enhanced the migration at 10(-4)M, respectively. They altered cell morphology showing prominent peripheral actin bundles and sparse central stress fibers, in comparison with the calmodulin/MLCK inhibitors. Both ML-7 and Y-27632 attenuated phosphorylation of myosin regulatory light chain and cell attachment to extracellular substrate. ML-7 attenuated the activation of GTP-binding protein Rac, while Y-27632 did not. These findings suggest that the actomyosin-based cytoskeletal actions can be functionally diverse depending on the Rho-kinase-mediated pathway and the MLCK-mediated pathway.

Leucine stimulates the secretion of hepatocyte growth factor by hepatic stellate cells

Branched-chain amino acids (BCAAs) modulate various cellular functions, in addition to providing substrates for the production of proteins. In this study, we examined the effect of BCAAs on the secretion of hepatocyte growth factor (HGF) by hepatic stellate cells. A hepatic stellate cell clone was cultured in medium supplemented with various concentrations of valine, leucine, or isoleucine. Of these BCAAs, leucine markedly induced an increase in the levels of HGF in the medium in a dose-dependent manner. The addition of valine or isoleucine had no significant effect on HGF levels in the medium. The difference in levels of HGF in the medium between leucine-treated and non-treated cells was enhanced by the incubation period. These results demonstrate that, among BCAAs, leucine stimulates the secretion of HGF by cultured hepatic stellate cells.

Hepatic stellate cell damage may lead to decreased plasma ADAMTS13 activity in rats.

ADAMTS13 is gaining attention, because its deficiency causes thrombotic thrombocytopenic purpura. Although its regulatory mechanism is not fully understood, we wondered if hepatic stellate cells (HSCs) play a role, because ADAMTS13 mRNA is exclusively expressed in the liver and primarily in HSCs. Plasma ADAMTS13 activity was markedly reduced in dimethylnitrosamine‐treated rats, where HSC apoptosis is an essential event, but not in carbon tetrachloride‐ or thioacetamide‐treated rats without HSC apoptosis. Furthermore, plasma ADAMTS13 activity was also reduced in 70% hepatectomized rats, where HSC loss occurs. These results suggest that HSC may be involved in the regulation of plasma ADAMTS13 activity.

Ischemic preconditioning in liver pathophysiology

Brief periods of tissue ischemia produced tissue resistance to prolonged ischemia and reperfusion, a phenomenon called ischemic preconditioning. The mechanisms of ischemic preconditioning were examined in a rat warm ischemia–reperfusion model as well as the effect of ischemic preconditioning on liver regeneration. Ischemic preconditioning decreased liver injury after warm ischemia–reperfusion, which was reversed by Kupffer cell depletion. Ischemic preconditioning stimulated Kupffer cells to produce reactive oxygen species. Scavengers of reactive oxygen species reversed the effect of ischemic preconditioning, and pretreatment with sublethal dose of hydrogen peroxide mimicked ischemic preconditioning effect. Rat livers were preconditioned by ischemia and subjected to 70% partial hepatectomy. Liver regeneration was then evaluated serially. Ischemic preconditioning promoted liver regeneration, which was reversed by adenosine A2 receptor antagonism and mimicked by adenosine A2 receptor agonism. Promotion of liver regeneration by ischemic preconditioning and adenosine A2 receptor agonism were reversed by Kupffer cell depletion. In conclusion, ischemic preconditioning stimulates Kupffer cells to produce reactive oxygen species, leading to hepatocyte protection against warm ischemia–reperfusion injury; and ischemic preconditioning promoted liver regeneration via adenosine A2 receptor pathway in Kupffer cells.

Increased production of ADAMTS13 in hepatic stellate cells contributes to enhanced plasma ADAMTS13 activity in rat models of cholestasis and steatohepatitis

Although hepatic stellate cells, endothelial cells, glomerular podocytes and plateles were reported to be a source of ADAMTS13, it is not clarified which source is involved in the regulation of plasma ADAMTS13 activity. It was demonstrated previously that selective hepatic stellate cell damage in rats caused decreased plasma ADAMTS13 activity. To further elucidate the potential contribution of hepatic stellate cells to the regulation of plasma ADAMTS13 activity, this study examined plasma ADAMTS13 activity when hepatic stellate cells proliferate during the process of liver fibrosis by employing rat models of liver fibrosis due to cholestasis, bile duct ligation, and steatohepatitis, a choline-deficient L-amino acid-defined-diet. ADAMTS13 expression was increased with co-localisation with smooth muscle alpha-actin, a marker of hepatic stellate cells, in bile duct-ligated livers up to four weeks, in which a close correlation between ADAMTS13 and smooth muscle alpha-actin mRNA expressions was determined. Plasma ADAMTS13 activity, measured by a sandwich ELISA involving a specific substrate to ADAMTS13, was increased in bile duct-ligated rats with a significant correlation with ADAMTS13 mRNA expression levels in the liver. Furthermore, ADAMTS13 mRNA expression was increased with enhanced mRNA expression in smooth muscle alpha-actin in the livers of rats fed a choline-deficient L-amino acid-defined-diet for 16 weeks, in which increased plasma ADAMTS13 activity was determined. Thus, increased plasma ADAMTS13 activity in cholestasis and steatohepatitis in rats may be due, at least in part, to enhanced ADAMTS13 production in the liver, suggesting a significant role of hepatic stellate cells in the regulation of plasma ADAMTS13 activity.

Effect of Rapamycin on Hepatocyte Function and Proliferation Induced by Growth Factors

Background: Rapamycin is a specific inhibitor of the mammalian target of rapamycin (mTOR). The effect of rapamycin on proliferation and cellular function was studied in hepatocytes stimulated by hepatocyte growth factor (HGF) or transforming growth factor-α (TGFα). Methods and Results: When isolated rat hepatocytes were cultured at low density, the addition of HGF or TGFα increased DNA synthesis but did not affect albumin or fibrinogen concentrations in the medium. In contrast, in hepatocytes cultured at high density, the albumin and fibrinogen concentrations, but not DNA synthesis, were increased by HGF or TGFα. The HGF- or TGFα-induced increase in DNA synthesis and in albumin or fibrinogen concentrations was suppressed by the addition of rapamycin, as well as wortmannin, a phosphatidylinositol-3 kinase inhibitor. Conclusion: HGF and TGFα stimulate proliferation and function of hepatocytes depending upon the conditions, and rapamycin inhibited these stimulatory effects, possibly by inhibiting the mTOR-dependent signaling pathway.

HMG-COA reductase inhibitor modulates collagen GEL-contraction by hepatic myofibroblast-like stellate cell line: involvement of geranylgeranylated proteins

Department of Internal Medicine, Kawakita General Hospital, Tokyo, JapanEmail: Mikio Yanase* - yanase-1IM@h.u-tokyo.ac.jp; Hitoshi Ikeda- ikeda-1IM@h.u-tokyo.ac.jp; Atsushi Matsui - atsushim@saitama-med.ac.jp; Eisei Noiri - noiri-1IM@h.u-tokyo.ac.jp; Tomoaki Tomiya -tomiya-1IM@h.u-tokyo.ac.jp; Masahiro Arai - arai-1IM@h.u-tokyo.ac.jp; Yukiko Inoue - yanase-1IM@h.u-tokyo.ac.jp; Kazuaki Tejima - yanase-1I M@h.u-tokyo.ac.jp; Kayo Nagashima- yanase-1IM@h.u-tokyo.ac.jp; Takako Nishikawa - yanase-1IM@h.u-tokyo.ac.jp; Satoshi Kimura - yanase-1IM@h.u-tokyo.ac.jp; Kenji Fujiwara - yanase-1IM@h.u-tokyo.ac.jp; Marcos Rojkind - marcos.rojkind@na.amedd.army.mil; Itsuro Ogata - itsuro@viola.ocn.ne.jp* Corresponding author

Reply to the letter by H. Mawatari et al. regarding “Thrombocytopenia is more severe in patients with advanced chronic hepatitis C than B with the same grade of liver stiffness and splenomegaly”

We thank Mawatari et al. for their interest in our article [1] and their presenting data regarding the platelet count in patients with hepatitis C virus-related chronic liver disease (CLD-C) and in those with nonalcoholic fatty liver disease (NAFLD) (please see Letter to the editor). The usefulness of transient elastography for the detection of liver fibrosis in NAFLD has been well established [2, 3], although the controversy still exits [4]. We had an impression in the clinical setting that thrombocytopenia might be less severe in patients with NAFLD than in those with CLD-C, and this point has been clearly determined in their study with adjustment for the degree of liver stiffness. Considering the mechanism of the decrease in the platelet count, we are interested in the splenomegaly in their patients with CLD-C and NAFLD. If the spleen size is not different in patients with the same degree of liver stiffness, the impaired platelet production rather than the enhanced platelet destruction may explain the distinct thrombocytopenia between CLD-C and NAFLD as well as hepatitis B virus-related chronic liver disease and CLD-C [1]. Nonetheless, it has become more likely that thrombocytopenia in chronic liver disease may vary according to its etiology. Thrombocytopenia is still a big problem in chronic liver disease, considering the interferon therapy for CLD-C or the treatment for hepatocellular carcinoma frequently arisen from chronic liver disease. Its mechanism should be further investigated.