Hiromitsu Nagata

Inhibition of c-Jun NH2-terminal kinase switches Smad3 signaling from oncogenesis to tumor- suppression in rat hepatocellular carcinoma†

Transforming growth factor beta (TGF‐β) signaling involves both tumor‐suppression and oncogenesis. TGF‐β activates the TGF‐β type I receptor (TβRI) and c‐Jun N‐terminal kinase (JNK), which differentially phosphorylate the mediator Smad3 to become COOH‐terminally phosphorylated Smad3 (pSmad3C) and linker‐phosphorylated Smad3 (pSmad3L). TβRI‐dependent pSmad3C transmits a tumor‐suppressive TGF‐β signal, while JNK‐dependent pSmad3L promotes carcinogenesis in human chronic liver disorders. The aim of this study is to elucidate how SP600125, a JNK inhibitor, affected rat hepatocellular carcinoma (HCC) development, while focusing on the domain‐specific phosphorylation of Smad3. The rats received subcutaneous injections of either SP600125 or vehicle 11 times weekly together with 100 ppm N‐diethylnitrosamine (DEN) administration for 56 days and were sacrificed in order to evaluate HCC development 28 days after the last DEN administration. The number of tumor nodules greater than 3 mm in diameter and the liver weight/body weight ratio were significantly lower in the SP600125‐treated rats than those in the vehicle‐treated rats (7.9 ± 0.8 versus 17.7 ± 0.9: P < 0.001; 6.3 ± 1.2 versus 7.1 ± 0.2%: P < 0.05). SP600125 significantly prolonged the median survival time in rats with DEN‐induced HCC (113 versus 97 days: log‐rank P = 0.0018). JNK/pSmad3L/c‐Myc was enhanced in the rat hepatocytes exposed to DEN. However, TβRI/pSmad3C/p21WAF1 was impaired as DEN‐induced HCC developed and progressed. The specific inhibition of JNK activity by SP600125 suppressed pSmad3L/c‐Myc in the damaged hepatocytes and enhanced pSmad3C/p21WAF1, acting as a tumor suppressor in normal hepatocytes. Conclusion: Administration of SP600125 to DEN‐treated rats shifted hepatocytic Smad3‐mediated signal from oncogenesis to tumor suppression, thus suggesting that JNK could be a therapeutic target of human HCC development and progression. (HEPATOLOGY 2009.)

Proliferative activity in hepatocellular carcinoma is closely correlated with glucose metabolism but not angiogenesis

BACKGROUND & AIMS This study investigated the relationship between tumor proliferative activity and the grade of tumor glucose metabolism or angiogenesis in hepatocellular carcinoma (HCC). METHODS The study was performed as a retrospective analysis of 63 patients with HCC who underwent fluorine-18 fluorodeoxyglucose positron emission tomography (FDG-PET) as a preoperative examination prior to liver resection. Tumor proliferative activity was evaluated by the Ki-67 labeling index (LI). The grade of tumor glucose metabolism was evaluated by measuring the protein expression level of glucose transporter (GLUT)-1, expression level of pyruvate kinase type M2 (PKM2) mRNA, and FDG uptake. The grade of tumor angiogenesis was evaluated by the protein expression level of vascular endothelial growth factor (VEGF) and tumor microvessel density. RESULTS All patients were divided into tertiles according to the Ki-67 LI: the low LI group (n = 21), the intermediate LI group (n = 21), and the high LI group (n = 21). The high LI group showed a tendency to have advanced tumor stage, and lower disease-free survival and overall survival rates than the low LI and the intermediate LI groups. The expression grade of GLUT-1, PKM2 mRNA, and FDG uptake gradually increased with the Ki-67 LI. On the other hand, the protein expression grade of VEGF and microvessel density was paradoxically decreased with the Ki-67 LI. CONCLUSIONS These data suggest that (1) the proliferative activity of a resected specimen predicted the prognosis in patients with HCC; (2) the proliferative activity was closely correlated with the glucose metabolism, but not with angiogenesis.

Sorafenib attenuates monocrotaline-induced sinusoidal obstruction syndrome in rats through suppression of JNK and MMP-9.

BACKGROUND & AIMS Sinusoidal obstruction syndrome (SOS) is a drug-induced liver injury that occurs with oxaliplatin treatment and is associated with postoperative morbidity after hepatectomy. The aim of this study was to investigate the effects of sorafenib in a monocrotaline (MCT)-induced model of SOS in rats. METHODS Rats were divided into groups treated with sorafenib (2mg/kg) or vehicle, 36 h and 12h before MCT (90 mg/kg) administration by gavage. Liver tissues and blood were sampled 48 h after MCT administration to evaluate SOS. Survival after hepatectomy was examined and immunohistochemistry and electron microscopy were performed to assess sinusoidal injury. RESULTS In the vehicle group, liver histology showed sinusoidal dilatation, coagulative necrosis of hepatocytes, endothelial damage of the central vein, and sinusoidal hemorrhage. In the sorafenib group, these changes were significantly suppressed, total SOS scores were significantly decreased, and the elevation of serum transaminase levels observed in the vehicle group was significantly reduced. Survival after hepatectomy was significantly higher in the sorafenib group compared to the vehicle group (45% vs. 20%, p=0.0137). Immunohistochemistry and electron microscopy revealed a protective effect of sorafenib on sinusoidal endothelial cells at 6h after MCT treatment. Sorafenib also attenuated the activity of metallopeptidase-9 (MMP-9) and phosphorylation of c-Jun N-terminal kinase (JNK). CONCLUSIONS Sorafenib reduced the severity of MCT-induced SOS in rats through suppression of MMP-9 and JNK activity, resulting in improvement of survival after hepatectomy.

Dai-kenchu-to attenuates rat sinusoidal obstruction syndrome by inhibiting the accumulation of neutrophils in the liver.

Background and Aim:  Sinusoidal obstruction syndrome (SOS) is drug‐induced liver injury that occurs in patients who receive hematopoietic cell transplantation and oxaliplatin‐contained chemotherapy. The aim of study was to investigate the pharmacological treatment of SOS using a traditional Japanese medicine, Dai‐kenchu‐to (DKT).

Olprinone attenuates excessive shear stress through up‐regulation of endothelial nitric oxide synthase in a rat excessive hepatectomy model

After extended hepatectomy, excessive shear stress in the remnant liver causes postoperative liver failure. Olprinone (OLP), a selective phosphodiesterase inhibitor, has been reported to improve microcirculation and attenuate inflammation. The aim of this study was to investigate the effects of OLP on shear stress in rats with an excessive hepatectomy (EHx) model. In this study, EHx comprised 90% hepatectomy with ligation of the left and right Glissons sheaths in Lewis rats. OLP or saline was intraperitoneally administered with an osmotic pump 48 hours before EHx. To evaluate the shear stress, we measured the portal vein (PV) pressure. We also assessed sinusoidal endothelial cell injury by immunohistochemistry and electron microscopy. Furthermore, we assessed apoptosis in the liver with the terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick‐end labeling method. Treatment with OLP up‐regulated hepatic endothelial nitric oxide synthase (eNOS) expression. The increase in the PV pressure due to Glissons sheath ligation was attenuated in OLP‐treated rats during a 30‐minute period after ligation. Treatment with OLP preserved sinusoidal endothelial cells and reduced apoptosis in the remnant liver. The probability of survival in the OLP‐treated rats was significantly better than that in the controls (33.3% versus 13.3%). Furthermore, the postoperative eNOS activity in the OLP‐treated rats was higher than that in the controls. The administration of Nω‐nitro‐l‐arginine methyl ester to OLP‐treated rats eliminated the effects of OLP on PV pressure and survival after EHx. Therefore, we concluded that OLP attenuates excessive shear stress through the up‐regulation of eNOS and improves the survival rate after EHx. Liver Transpl 17:60–69, 2011.

A Phosphodiesterase Iii Inhibitor Protects Rat Liver From Sinusoidal Obstruction Syndrome Through Heme Oxygenase-1 Induction

Objective:The aim of study was to investigate pharmacological treatment for sinusoidal obstruction syndrome (SOS). Background:SOS is associated with oxaliplatin-based chemotherapy in patients with hepatic colorectal metastases. Patients with SOS have increased postoperative morbidity after major hepatectomy, but a method for effective prevention of SOS has not been established. Methods:Male Sprague-Dawley rats were treated with cobalt protoporphyrin IX (CoPP) or olprinone (OLP), a phosphodiesterase III inhibitor, and hepatic HO-1 expression and HO enzymatic activity were determined. Monocrotaline (MCT) was given to rats to induce SOS, and blockage of SOS by CoPP or OLP-induced hepatic HO-1 was examined in these rats. Zinc protoporphyrin IX (ZnPP), a competitive HO inhibitor, was given to MCT-treated rats together with OLP to clarify the mechanism of protection against SOS. We also examined if OLP preserved remnant liver function after 70% hepatectomy in MCT-treated rats. Results:OLP up-regulated hepatic HO-1 protein expression and HO enzymatic activity, and activated Akt protein. Administration of ZnPP to OLP-treated rats resulted in inhibition of HO activity and inactivation of Akt. Induction of HO-1 by pretreatment with CoPP led to amelioration of SOS in histologic findings and blockage of elevation of serum liver enzymes. Pretreatment with OLP gave a similar result and preserved remnant liver function, as indicated by improved survival after hepatectomy. ZnPP completely abolished the protective effects of OLP. Conclusions:Protection of the liver from drug-induced injury by prior up-regulation of HO-1 using OLP may have potential as a therapeutic strategy for prevention of SOS.

Downregulation of the Wnt antagonist Dkk2 links the loss of Sept4 and myofibroblastic transformation of hepatic stellate cells

BACKGROUND/AIMS Sept4, a subunit of the septin cytoskeleton specifically expressed in quiescent hepatic stellate cells (HSCs), is downregulated through transdifferentiation to fibrogenic and contractile myofibroblastic cells. Since Sept4(-/-)mice are prone to liver fibrosis, we aimed to identify the unknown molecular network underlying liver fibrosis by probing the association between loss of Sept4 and accelerated transdifferentiation of HSCs. METHODS We compared the transcriptomes of Sept4(+/+) and Sept4(-/-) HSCs undergoing transdifferentiation by DNA microarray and quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis. Because Dickkopf2 (Dkk2) gene expression is reduced in Sept4(-/-) HSCs, we tested whether supplementing Dkk2 could suppress myofibroblastic transformation of Sept4(-/-) HSCs. We tested the involvement of the canonical Wnt pathway in this process by using a lymphoid enhancer-binding factor/transcription factor-luciferase reporter assay. RESULTS We observed consistent upregulation of Dkk2 in primary cultured HSCs and in a carbon tetrachloride liver fibrosis in mice, which was decreased in the absence of Sept4. Supplementation with Dkk2 suppressed the induction of pro-fibrotic genes (α-smooth muscle actin and 2 collagen genes) and induced an anti-fibrotic gene (Smad7) in Sept4(-/-) HSCs. In human liver specimens with inflammation and fibrosis, Dkk2 immunoreactivity appeared to be positively correlated with the degree of fibrotic changes. CONCLUSIONS Pro-fibrotic transformation of HSCs through the loss of Sept4 is, in part, due to reduced expression of Dkk2 and its homologues, and the resulting disinhibition of the canonical Wnt pathway.

Effects of oral intake of hydrogen water on liver fibrogenesis in mice

Liver fibrosis is the universal consequence of chronic liver diseases. Sustained hepatocyte injury initiates an inflammatory response, thereby activating hepatic stellate cells, the principal fibrogenic cells in the liver. Reactive oxygen species are involved in liver injury and are a promising target for treating liver fibrosis. Hydrogen water is reported to have potential as a therapeutic tool for reactive oxygen species‐associated disorders. This study aimed to investigate the effects of hydrogen water on liver fibrogenesis and the mechanisms underlying these effects.

Effect of olprinone on liver microstructure in rat partial liver transplantation

BACKGROUND Donor safety is a major concern in living-donor liver transplantation. However, partial grafts do not meet the functional demands of recipients and lead to small-for-size syndrome (SFSS). In a previous study, we showed that olprinone (OLP), a selective phosphodiesterase ІІІ inhibitor, up-regulates endothelial nitric oxide synthase level in the liver and attenuates shear stress, sinusoidal endothelial cell injury, and hepatocyte apoptosis after excessive liver resection in a rat model. We aimed to examine whether OLP treatment has beneficial effects on SFSS in a rat model of partial liver transplantation (PLT). METHODS We performed experiments in a rat model of 30% PLT. In the OLP group, we inserted an osmotic pump with OLP into the peritoneal cavity 48 h before liver graft sampling. Recipient rats were not treated with OLP. We examined the liver microstructure by electron microscopy and biochemical examination, and determined the 7-d survival of recipients. RESULTS In the OLP group 1 h after PLT, the sinusoidal endothelial cells of the liver were well preserved and we observed few vacuolar structures in hepatocytes. The total serum bilirubin level 1 wk after PLT tended to be lower in the OLP group than in the controls, and the liver microstructures were also well preserved in the OLP group. The probability of survival in the OLP group (100%; 14 of 14 rats) was significantly higher than that in the control group (75%; 15 of 20 rats). CONCLUSIONS Olprinone treatment was demonstrated to have therapeutic potential to overcome SFSS.

Prophylactic respiratory management after liver resection with bilevel positive airway pressure ventilation: Report of three cases

Pulmonary complications after hepatectomy occur with relative frequency and are associated with increased morbidity and mortality. Moreover, their prevention is often difficult. We report using prophylactic bilevel positive airway pressure ventilation, initiated just after the operation, for the successful postoperative respiratory management of three patients predisposed to the development of pulmonary complications. One patient had insufficient pulmonary function (forced expiratory volume in 1 s (FEV1) 0.95 l; FEV1/forced vital capacity 40.8%) caused by previous thoracoplasty. The other two patients were obese and had obstructive sleep apnea syndrome (OSAS). None of the patients required endotracheal intubation after hepatectomy, although the two with severe OSAS suffered pulmonary thromboembolism postoperatively. Bilevel positive airway pressure ventilation was well tolerated and there were no adverse effects, suggesting its effectiveness for preventing pulmonary complications after hepatectomy.