Minoru Nagayama

Beneficial effects of inducible nitric oxide synthase inhibitor on reperfusion injury in the pig liver.

BACKGROUND Although inhibition of endothelial nitric oxide synthase (eNOS) has been reported to aggravate hepatic ischemia-reperfusion (I/R) injury, the role of inducible nitric oxide synthase (iNOS) has been still unknown. We investigated the role of NO produced by iNOS, and evaluated the effect of an iNOS inhibitor on prolonged warm I/R injury in the pig liver. METHODS Pigs were subjected to 120 min of hepatic warm I/R under the extracorporeal circulation. We investigated the time course of changes in serum and hepatic microdialysate NO2- + NO3- (NOx) and the cellular distribution of eNOS and iNOS by immunohistochemistry, including a double-immunofluorescence technique in combination with confocal laser scanning microscopy. The effect of iNOS inhibitor was also investigated. RESULTS Hepatic I/R induced new nitric oxide production in serum and hepatic microdialysate NOx after reperfusion and severe hepatic damage in the centrilobular region where nitrotyrosine was strongly expressed. Diffuse eNOS expression in sinusoidal endothelium did not differ before and after reperfusion. In contrast, strong iNOS expression in Kupffer cells and neutrophils appeared strongly in the centrilobular region after reperfusion. Pigs with intraportal administration of N(G)-nitro-L-arginine (10 mg/kg) died during the period of ischemia or early in the period of reperfusion with a high mortality rate (80.0%). Intraportal administration of aminoguanidine hemisulfate (10 mg/kg) significantly suppressed nitric oxide production and serum aspartate aminotransferase after reperfusion, inhibited nitrotyrosine expression, and attenuated hepatic damage. CONCLUSIONS These results indicate that hepatic I/R injury is triggered by centrilobular iNOS expression; and attenuated by inhibition of iNOS.

Reconsideration of Postoperative Oral Intake Tolerance After Pancreaticoduodenectomy: Prospective Consecutive Analysis of Delayed Gastric Emptying According to the ISGPS Definition and the Amount of Dietary Intake

Objective:A prospective consecutive study was planned to evaluate the postpancreaticoduodenectomy (PD) oral intake tolerance. The occurrence of delayed gastric emptying (DGE), as defined by the International Study Group of Pancreatic Surgery (ISGPS), and the amount of dietary intake were analyzed. The risk factors for low oral intake tolerance were additionally determined. Summary Background Data:The causation of DGE after PD is still unclear. Several possible factors have been discussed, such as reconstruction methods and other complications. However, none of them has followed the definition of ISGPS. Methods:Between 2003 and 2007, 101 consecutive patients underwent PD-related surgery, and standard operative procedure was performed on 85 patients. Perioperative data were prospectively collected in all patients, and the patients postoperative dietary intake was recorded for all meals until discharge. As an indicator of early postoperative oral intake tolerance, we added up the dietary intake from postoperative day 1 to 21 and called this value the total amount of dietary intake (TDI). The postoperative outcomes were compared between non-DGE and DGE. The high-low of TDI values was also analyzed. Multivariate analysis for factors associated with the occurrence of DGE and TDI was performed. Results:The occurrence of DGE as defined by ISGPS was 42%. The postoperative outcomes of DGE patients were significantly poor compared with those of non-DGE patients. TDI values were significantly low in DGE patients, and non-DGE patients with low TDI values showed a significantly extended duration of parenteral nutrition and postoperative hospitalization. Operative bleeding (>1,000 mL) and pancreatic fistulas were likely to be associated with DGE occurrence. Gender (women), BMI (>25 kg/m2), postoperative intraabdominal infection, and DGE were significantly associated with low TDI values. Conclusions:The ISGPS definition of DGE seemed feasible for patient management. TDI values provided additional information for analyzing postoperative oral intake tolerance, especially when describing the differences among non-DGE patients. Substantial risk factors for low oral intake tolerance were high BMI, postoperative intraabdominal infection, and DGE.

A novel inhibitor of inducible nitric oxide synthase (ONO-1714) prevents critical warm ischemia-reperfusion injury in the pig liver

Background. Recently, a novel inhibitor of inducible nitric oxide synthase, ONO-1714, was developed. We evaluated the effect of ONO-1714 on a critical warm I/R model of the pig liver. Methods. Pigs were subjected to 180 min of hepatic warm I/R under the extracorporeal circulation. We investigated the time course of changes in the serum NO2− + NO3− (NOx), the cellular distribution of endothelial and inducible nitric oxide synthase, thrombocyte-thrombi, and nitrotyrosine by immunohistochemistry. The hepatic tissue blood flow (HTBF) was measured continuously using a laser-Doppler blood flowmeter. Results. ONO-1714 at 0.05 mg/kg improved the survival rate from 54 (control group) to 100%. The serum NOx levels in the ONO-1714 group were significantly lower than those in the control group at 1, 1.5, 2, 3, and 6 hr after reperfusion. The serum aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) levels of the ONO-1714 group were significantly lower than the control group, and the HTBF of the ONO-1714 group was significantly higher than the control group. The formation of thrombocyte-thrombi and nitrotyrosine after reperfusion was significantly lower in the ONO-1714 group. Conclusions. These results indicated that ONO-1714 improved the survival rates and attenuated I/R injury in a critical hepatic warm I/R model of the pig. ONO-1714 will be beneficial for hepatectomy or liver transplantation in the clinical field.

Role of inducible nitric oxide synthase in pig liver transplantation

BACKGROUND Previously, we clarified the role of inducible nitric oxide synthase (iNOS) and the protective effect of an iNOS inhibitor in warm ischemia and reperfusion model. In this study, we investigated whether the same effects would be obtained by iNOS inhibitor in liver transplantation model. MATERIAL AND METHODS Orthotopic liver transplantation was performed in pigs in the usual manner after about 6 h of cold preservation in University of Wisconsin solution. Aminoguanidine hemisulfate (AG) was used as the iNOS inhibitor and AG was administered intraportally at the dose of 10 mg/kg just after reperfusion. Two experimental groups were subjected, control group (n = 10), and AG group (n = 10). We investigated changes of serum nitrite/nitrate (NOx) and aspartate aminotransferase (AST). Expression of iNOS was examined by immunohistochemistry, including a double immunofluorescnce technique in combination with cofocal laser scanning microscopy. RESULTS Serum NOx and AST were significantly lower in the AG group. Histological hepatic damage and thrombocyte thrombi were attenuated in the AG group. Expression of iNOS was recognized strongly at Kupffer cells and neutrophils in the centrilobular region of liver after reperfusion by cofocal laser scanning microscopy. Moreover, iNOS staining was attenuated in AG group compared with control group. CONCLUSIONS These results indicate that hepatic ischemia and reperfusion injury in liver transplantation might be triggered by iNOS expression of Kupffer cells and neutrophils, and attenuated by administration of an iNOS inhibitor. Moreover, AG showed down regulation of iNOS expression after reperfusion.

Transcriptional Control of Tight Junction Proteins via a Protein Kinase C Signal Pathway in Human Telomerase Reverse Transcriptase-Transfected Human Pancreatic Duct Epithelial Cells

In human pancreatic cancer, integral membrane proteins of tight junction claudins are abnormally regulated, making these proteins promising molecular diagnostic and therapeutic targets. However, the regulation of claudin-based tight junctions remains unknown not only in the pancreatic cancer cells but also in normal human pancreatic duct epithelial (HPDE) cells. To investigate the regulation of tight junction molecules including claudins in normal HPDE cells, we introduced the human telomerase reverse transcriptase (hTERT) gene into HPDE cells in primary culture. The hTERT-transfected HPDE (hTERT-HPDE) cells were positive for the pancreatic duct epithelial markers such as CK7, CK19, and carbonic anhydrase isozyme 2 and expressed epithelial tight junction molecules claudin-1, -4, -7 and, -18, occludin, JAM-A, ZO-1, ZO-2, and tricellulin. By treatment with fetal bovine serum or 12-O-tetradecanoylphorbol 13-acetate (TPA), the tight junction molecules were up-regulated at the transcriptional level via a protein kinase C (PKC) signal pathway. A PKC-alpha inhibitor, Gö6976, prevented up-regulation of claudin-4 by TPA. Furthermore, a PKC-delta inhibitor, rottlerin, prevented up-regulation of claudin-7, occludin, ZO-1, and ZO-2 by TPA. By GeneChip analysis, up-regulation of the transcription factor ELF3 was observed in both fetal bovine serum- and TPA-treated cells. Treatment with small interfering RNAs of ELF3 prevented up-regulation of claudin-7 by TPA. These data suggest that tight junctions of normal HPDE cells were at least in part regulated via a PKC signal pathway by transcriptional control.

Apoptosis and necrosis after warm ischemia-reperfusion injury of the pig liver and their inhibition by ONO-1714.

Background. It is still controversial whether a major mode of cell death during hepatic ischemia-reperfusion (I/R) injuries is apoptosis or necrosis. Moreover, the correlation between these cell deaths and the effects of a novel inducible nitric oxide synthase inhibitor (ONO-1714) has not been studied before. Methods. Pigs were subjected to 180 min of hepatic warm I/R under extracorporeal circulation. The control group was not administered ONO-1714. In the ONO-1714 group, ONO-1714 was administered 5 min before ischemia at a dose of 0.05 mg/kg through a portal vein catheter. The apoptotic and necrotic changes after reperfusion were examined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and hematoxylin-eosin staining. Nitrotyrosine, active caspase-3, and cytochrome c were examined by immunohistochemistry. The plasma NO2- + NO3-, aspartate aminotransferase, and lactate dehydrogenase levels were also examined. Results. In the control group, the frequency of apoptotic cells was only 2.6%; nevertheless, that of necrotic cells was 37% at 24 hr after reperfusion. ONO-1714 significantly attenuated apoptosis and necrosis, the expression of nitrotyrosine, and the increases of the plasma aspartate aminotransferase, lactate dehydrogenase, and NO2- + NO3- levels in the reperfusion phase. Conclusions. A major mode of cell death during hepatic warm I/R injury was necrosis, and apoptosis was not dominant. These necrotic changes were caused by the excess production of peroxynitrite, and ONO-1714 greatly attenuated I/R injuries as the result of inhibition of the peroxynitrite production.

Changes in serum levels of apolipoprotein A-1 as an indicator of protein metabolism after hepatectomy

The clinical significance of serum apolipoprotein A‐1 levels as an indicator of hepatic protein synthesis after hepatectomy was investigated. A total of 50 patients who had undergone hepatectomy at our department from 1997 to 1999 were selected for this study. The serum levels of apolipoprotein A‐1, indocyanine green dye retention at 15 minutes, lectin‐cholesterol acyltransferase, prealbumin, and high‐density lipoprotein cholesterol were measured in these patients preoperatively and on postoperative days 7 and 14. The type of hepatic resection conducted was partial resection in 13 cases, subsegmentectomy in 13 cases, segmentectomy in five cases, and bisegmentectomy in 19 cases. All the patients tolerated the operation, and none of the cases had any severe complications, such as liver failure. In most cases, the serum apolipoprotein A‐1 levels decreased on postoperative day 7 and recovered by day 14. There were no significant differences in the changes in apolipoprotein A‐1 levels between patients with the individual types of operative procedures. The serum apolipoprotein A‐1 levels showed significant correlations with the serum high‐density lipoprotein cholesterol, lectin‐cholesterol acyltransferase and prealbumin levels on postoperative days 7 and 14; however, there was no significant correlation with the indocyanine green retention test. When the cases were divided into three groups according to the serum level of apolipoprotein A‐1 on postoperative day 7 (group A: over 81 mg/dl, group B: 61–80 mg/dl, group C: under 60 mg/dl), the serum indocyanine green retention, prealbumin, lectin‐cholesterol acyltransferase and high‐density lipoprotein cholesterol levels in group C were significantly lower than those in group A on postoperative day 7. On the basis of these results, it is suggested that the pattern of changes in the serum apolipoprotein A‐1 levels may be a good indicator of the hepatic protein synthetic ability during the perioperative period after hepatectomy.

Role of inducible nitric oxide synthase on hepatic ischemia and reperfusion injury

PROTECTION of hepatocytes from ischemia/reperfusion (I/R) injury is an important issue in the field of hepatic surgery, which includes liver resection and transplantation. Nitric oxide (NO), a well-known endotheliumderived relaxing factor, has been implicated in tissue injury as a result of its capacity to react with superoxides and hydroxyl radical, suggesting that it may influence the pathogenesis of I/R injury. However, little is known about the role of inducible nitric oxide synthase (iNOS) in hepatic I/R injury. In the present study, we investigate the role of iNOS on hepatic I/R injury and study the cellular distribution of endothelial nitric oxide synthase (eNOS) and iNOS by immunohistochemistry, including a double-immunofluorescence technique in combination with confocal laser scanning microscopy. The effect of intraportal administration of aminoguanidine (AG), a selective iNOS inhibitor, is also investigated.

New strategy for the antifibrotic therapy with oral administration of FR260330 (a selective inducible nitric oxide synthase inhibitor) in rat experimental liver cirrhosis

Inducible nitric oxide synthase (iNOS) activity is significantly elevated in viral hepatitis, alcoholic cirrhosis, and cholestasis. However, there are few reports on the relationship between iNOS and cirrhosis. Here, we investigated the effects of a new iNOS inhibitor that has been developed for oral administration in an experimental rat liver cirrhosis model. A cirrhotic rat model was developed by long‐term administration of thioacetamide injections. FR260330 is a new, rationally designed, selective iNOS inhibitor that can be administered orally. After 12 weeks of treatment with FR260330, the rats showed inhibition of progressions of cirrhosis, ascites, and splenomegaly as well as a significant reduction in the proportions of connective tissue in the liver. The expression of nitrotyrosine, which indicates the existence of peroxynitrite and nuclear factor‐κB activation, was remarkably decreased in the FR260330 treatment group. In addition, immunohistochemical and Western blot analyses showed that the expression of transforming growth factor‐β1 was remarkably decreased in this group. The present study demonstrates that FR260330 reduces liver fibrosis by the inhibition of transforming growth factor‐β1 and retards the development of cirrhosis. This oral iNOS inhibitor will be a new strategy for the treatment of cirrhosis.

Changes in hepatic venous oxygen saturation in hepatic warm ischemia/reperfusion injury in pigs.

Abstract: To clarify the changes that occur in hepatic venous oxygen saturation (Shvo 2 ) during hepatic ischemia/reperfusion (I/R) injury, we examined the relationship between Shvo 2 , hepatic tissue blood flow (HTBF), and portal vein pressure (PVP) in a warm I/R model using pig livers. Female pigs weighing 18–23 kg were subjected to warm I/R under extracorporeal circulation between the superior mesentric vein and the left jugular vein to avoid portal congestion. The warm ischemic times were 120 min ( n = 4), 180 min ( n = 14), and 240 min ( n = 4). Shvo 2 , HTBF, and PVP were measured after reperfusion. The survival rates of the pigs 3 days after reperfusion were 100% in the 120-min group, 57% in the 180-min group, and 25% in the 240-min group. In the 180-min group, the Shvo 2 was lower in the pigs that died than in those that survived. There was a significant correlation between Shvo 2 and both PVP and HTBF after reperfusion. Histological examination revealed findings of severe I/R injury in pigs with a low Shvo 2 , and mild I/R injury in pigs with a stable Shvo 2 . These observations suggest that the changes in Shvo 2 could reflect the degree of hepatic I/R injury, especially that related to microcirculatory disturbances occurring at the sinusoid levels.