Nobuhiro Nakamoto

Evaluation of liver fibrosis by transient elastography using acoustic radiation force impulse: comparison with Fibroscan ®

BackgroundAccurate evaluation of liver fibrosis in patients with chronic liver damage is required to determine the appropriate treatment. Various approaches, including laboratory tests and transient elastography, have been used to evaluate liver fibrosis. Recently, transient elastography with acoustic radiation force impulse (ARFI) has been developed and applied with conventional ultrasonography. The aim of this study was to evaluate the clinical utility of transient elastography with ARFI and to compare the results with this method and those of the Fibroscan® procedure.MethodsOne hundred and thirty-one patients with liver damage, who underwent liver biopsy at our department, were enrolled prospectively in this study. Elastography with ARFI (applied with ACUSON S2000®), and Fibroscan® was performed at the same time as liver biopsy. These measurements were compared with histological findings in liver biopsy specimens, and measurement accuracy was evaluated by receiver-operating characteristic analysis.ResultsElastography values with both procedures were significantly correlated with the stages of liver fibrosis and there was little difference in the results obtained using the 2 procedures. The accuracy of differential diagnosis between no fibrosis at F0 and more than F1 stage was insufficient with ARFI, but this procedure was sufficient for diagnosing advanced fibrosis. The accuracy of ARFI was almost equivalent to that of the Fibroscan® method. Moreover, both ARFI and Fibroscan® values increased in proportion to the severity of hepatic inflammation when fibrosis stage is low, but not in proportion to the severity of steatosis.ConclusionsTransient elastography with ARFI is simple, non-invasive and useful for diagnosing the stage of fibrosis in chronic liver disease. The utility of ARFI was almost equivalent to that of the Fibroscan® method.

Role of Toll-like receptors in immune activation and tolerance in the liver

Liver has a unique vascular system receiving the majority of the blood supply from the gastrointestinal tract through the portal vein and faces continuous exposure to foreign pathogens and commensal bacterial products. These gut-derived antigens stimulate liver cells and result in a distinctive immune response via a family of pattern recognition receptors, the Toll-like receptors (TLRs). TLRs are expressed on Kupffer cells, dendritic cells, hepatic stellate cells, endothelial cells, and hepatocytes in the liver. The crosstalk between gut-derived antigens and TLRs on immune cells trigger a distinctive set of mechanisms to induce immunity, contributing to various acute and chronic liver diseases including liver cirrhosis and hepatocellular carcinoma. Accumulating evidence has shown that TLRs stimulation by foreign antigens induces the production of immunoactivating and immunoregulatory cytokines. Furthermore, the immunoregulatory arm of TLR stimulation can also control excessive tissue damage. With this knowledge at hand, it is important to clarify the dual role of disease-specific TLRs as activators and regulators, especially in the liver. We will review the current understanding of TLR signaling and subsequent immune activation and tolerance by the innate immune system in the liver.

CCR9 Macrophages Are Required for Acute Liver Inflammation in Mouse Models of Hepatitis

BACKGROUND & AIMS Antigen-presenting cells (APCs) are involved in the induction of liver inflammation. We investigated the roles of specific APCs in the pathogenesis of acute liver injury in mice. METHODS We used concanavalin A (con A) or carbon tetrachloride to induce acute liver inflammation in mice and studied the roles of macrophages that express CCR9. RESULTS After injection of con A, we detected CCR9(+)CD11b(+)CD11c(-) macrophages that express tumor necrosis factor (TNF)-α in livers of mice, whereas CCR9(+)Siglec-H(+)CD11b(-)CD11c(low) plasmacytoid DCs (pDCs), which are abundant in normal livers, disappeared. The CCR9(+) macrophages were also detected in the livers of RAG-2(-/-) mice, which lack lymphocytes and natural killer T cells, after injection of con A. Under inflammatory conditions, CCR9(+) macrophages induced naive CD4(+) T cells to become interferon gamma-producing Th1 cells in vivo and in vitro. CCR9(-/-) mice injected with con A did not develop hepatitis unless they also received CCR9(+) macrophages from mice that received con A; more CCR9(+) macrophages accumulated in their inflamed livers than CCR9(+) pDCs, CCR9(-) pDCs, or CCR9(-) macrophages isolated from mice that had received injections of con A. Levels of CCL25 messenger RNA increased in livers after injection of con A; neutralizing antibodies against CCL25 reduced the induction of hepatitis by con A by blocking the migration of CCR9(+) macrophages and their production of TNF-α. Peripheral blood samples from patients with acute hepatitis had greater numbers of TNF-α-producing CCR9(+)CD14(+)CD16(high) monocytes than controls. CONCLUSIONS CCR9(+) macrophages contribute to the induction of acute liver inflammation in mouse models of hepatitis.

C-C motif chemokine receptor 9 positive macrophages activate hepatic stellate cells and promote liver fibrosis in mice†‡

Chemokine receptors mediate migration of immune cells into the liver, thereby promoting liver inflammation. C‐C motif chemokine receptor (CCR) 9+ macrophages are crucial in the pathogenesis of acute liver inflammation, but the role and underlying mechanisms of this macrophage subset in chronic liver injury and subsequent liver fibrosis are not fully understood. We confirmed that tumor necrosis factor alpha (TNF‐α)‐producing CCR9+ macrophages accumulated during the initiation of carbon tetrachloride (CCl4)‐induced liver injury, and CCR9 deficiency attenuated the degree of liver damage. Accumulation of CCR9+ macrophages persisted prominently during the process of liver fibrosis induced by repetitive CCl4 or thioacetamide (TAA)/leptin administration. Increased CCR9 expression was also found on activated hepatic stellate cells (HSCs). Importantly, experimental liver fibrosis was significantly ameliorated in CCR9−/− mice compared with wild‐type (WT) mice, assessed by α‐smooth muscle actin (α‐SMA) immunostain, Sirius red staining, and messenger RNA (mRNA) expression levels of α‐SMA, collagen 1α1, transforming growth factor (TGF)‐β1, and tissue inhibitor of metalloproteinase (TIMP)‐1. Accumulated CD11b+ macrophages in CCl4‐treated WT mice showed marked increases in TNF, NO synthase‐2, and TGF‐β1 mRNA expression compared with CCR9−/− mice, implying proinflammatory and profibrogenic properties. Hepatic CD11b+ macrophages from CCl4‐treated WT mice (i.e., CCR9+ macrophages), but not CD8+ T lymphocytes or non‐CD11b+ cells, significantly activated HSCs in vitro compared with those from CCR9−/− mice. TNF‐α or TGF‐β1 antagonism attenuated CCR9+ macrophage‐induced HSC activation. Furthermore, C‐C motif chemokine ligand (CCL) 25 mediated migration and, to a lesser extent, activation of HSCs in vitro. Conclusion: Accumulated CD11b+ macrophages are critical for activating HSCs through the CCR9/CCL25 axis and therefore promote liver fibrosis. CCR9 antagonism might be a novel therapeutic target for liver fibrosis. (HEPATOLOGY 2013;)

Rho/Rho kinase is a key enzyme system involved in the angiotensin II signaling pathway of liver fibrosis and steatosis.

Background and Aim:  The molecular mechanisms underlying the involvement of the renin‐angiotensin system in hepatic fibrosis are unclear. Recently, it was reported that a Rho kinase inhibitor prevented fibrosis of various tissues and that the Rho/Rho kinase pathway was involved in the renin‐angiotensin system of vascular smooth muscle cells. In this study, the involvement of the Rho/Rho kinase pathway on angiotensin II signaling in liver fibrogenesis and generation of steatosis was investigated.

Reduction of telomerase activity in human liver cancer cells by a histone deacetylase inhibitor

The presence of telomerase has been demonstrated recently in many different malignancies. Several reports documented that in human hepatocellular carcinoma, the level of telomerase activity parallels its differentiation stage. In the present study, the effect of the differentiation‐inducing agent sodium butyrate on telomerase activity in four human liver cancer cell lines was investigated using the telomeric repeat amplification protocol. We assayed telomerase activity before and after butyrate treatment and in cell cycle synchronized non‐dividing quiescent cells. In addition, telomerase reverse transcriptase levels were measured at the mRNA level. All four cell lines possessed high but not identical levels of telomerase activity. Telomerase activity was significantly reduced by treatment with sodium butyrate as well as trichostatin A in a dose‐ and time‐dependent fashion, paralleling the reduction of cell proliferation. Although methotrexate, hydroxyurea, and colchicine synchronized the cell cycle at G1, S, and G2/M, respectively, and thereby also caused proliferating cells to cease dividing and become quiescent, in this case telomerase activity remained essentially unaltered compared to the control cultures. Moreover, levels of mRNA encoding telomerase reverse transcriptase were not always significantly altered by either sodium butyrate treatment or cell cycle synchronization. These results suggest that sodium butyrate, as a histone deacetylase inhibitor, effectively reduces telomerase activity without affecting transcription levels of the reverse transcriptase component.

DNMT1 and DNMT3b silencing sensitizes human hepatoma cells to TRAIL-mediated apoptosis via up-regulation of TRAIL-R2/DR5 and caspase-8.

DNA methylation plays a critical role in chromatin remodeling and gene expression. DNA methyltransferases (DNMTs) are hypothesized to mediate cellular DNA methylation status and gene expression during mammalian development and in malignant diseases. In this study, we examined the role of DNA methyltransferase 1 (DNMT1) and DNMT3b in cell proliferation and survival of hepatocellular carcinoma (HCC) cells. Gene silencing of both DNMT1 and DNMT3b by targeted siRNA knockdown reduces cell proliferation and sensitizes the cells to tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL)‐mediated cell death. The proapoptotic protein caspase‐8 demonstrated promoter hypermethylation in HCC cells and was up‐regulated by knockdown of DNMT1 and DNMT3b both at mRNA and protein levels. In addition, death receptor TRAIL‐R2/DR5 (TRAIL receptor 2/death receptor 5) did not exhibit promoter hypermethylation in HCC cells but was also up‐regulated by knockdown of DNMT1 and DNMT3b both at mRNA and protein levels. Consistent with this observation, the combined transfection of DNMT1‐siRNA plus DNMT3b‐siRNA enhanced formation of the TRAIL‐death‐inducing signaling complex formation in HCC cells. In conclusion, our data suggest that DNA methylation of specific genomic regions maintained by DNMT1 and DNMT3b plays a critical role in survival of HCC cells, and a simultaneous knockdown of both DNMT1 and DNMT3b may be a novel anticancer strategy for the treatment of HCC. (Cancer Sci 2010)

Clinical usefulness of edaravone for acute liver injury

Background: Edaravone, a newly synthesized radical scavenger, has shown an excellent effect on treating stroke patients. The effect of edaravone on carbon tetrachloride (CCl4)‐induced acute liver injury was examined.

Reduction of c-myc expression by an antisense approach under Cre/loxP switching induces apoptosis in human liver cancer cells.

c‐Myc has been documented to be both a positive and a negative signal for the induction of apoptosis. It is well known that overexpression of the c‐myc gene induces apoptosis of normal cells, but the result of a reduction in its expression is not fully understood. We examined whether a reduction in c‐myc expression would induce apoptosis in human liver cancer cells. Specifically, antisense and sense oligodeoxynucleotides (oligos) against the human c‐myc mRNA were synthesized, mixed with a liposome reagent at various ratios, and were applied to the liver cancer‐derived cell lines, HCC‐T, HepG2, and PLC/PRF/5. To exclude effects resulting from using oligos, plasmid vectors expressing the full‐length c‐myc cDNA in both sense and antisense orientations under the control of the Cre/loxP system were generated. Monoclonal cell lines including these plasmid vectors were produced and Cre was supplied by adenovirus infection. Apoptosis was determined morphologically and c‐Myc and Bcl‐2 expression was examined by Western blotting. The antisense myc significantly inhibited the proliferation of the cells within two days, while neither the liposome reagent alone nor sense myc did so. Most of the cells were rounded up by the antisense‐treatment and nuclear fragmentation and DNA ladder formation were detected after two days in antisense c‐myc‐treated cells. Antisense c‐myc largely reduced c‐Myc and partially Bcl‐2 expression; overexpression of Bcl‐2 partially rescued from apoptosis in HCC‐T and HepG2 cells. These results suggest that the massive reduction in c‐myc mRNA induces apoptosis in liver cancer cell lines and consequent decrease in Bcl‐2 enhances the cell death. c‐Myc reduction under the Cre/loxP switching system may be a useful tool for the clarification of c‐myc‐related cellular mechanisms in differentiation and proliferation.

MyD88-dependent pathway accelerates the liver damage of Concanavalin A-induced hepatitis

We have explored the pathological role of the MyD88 signaling pathway via Toll-like receptors (TLRs) that mediate the recognition of pathogen-associated molecular patterns (PAMPs) in a murine model of autoimmune hepatitis induced by administering Concanavalin A (ConA). We first found that various TLRs and MyD88 molecules were expressed in liver of Con A-treated and untreated wild-type (WT) mice including liver macrophages. Flowcytometric analysis revealed that liver CD11b(+)CD11c(-) and CD11b(+)CD11c(+) antigen-presenting cells express TLR2, although NK and NKT cells did not. When WT and MyD88(-/-) mice were intravenously administered with Con A, the severity of hepatitis was significantly lower in Con A-injected MyD88(-/-) mice than in WT mice in terms of the histopathology, the levels of serum transaminase and pro-inflammatory cytokines (TNF-alpha, IFN-gamma, and IL-6), and upregulation of CD80/CD86 and TNF-alpha on/in liver macrophages. The results provide evidence of a possible contribution of the TLRs-MyD88 signaling pathway in activating TLR-expressing liver macrophages in the autoimmune hepatitis model, and thus indicate that the strategy of blockade of pathological pathogens via the intestinal lumen may be feasible for the treatment of the disease.