Hiroshi Munakata

Reactive oxygen species depolymerize hyaluronan: involvement of the hydroxyl radical

We have previously demonstrated that reactive oxygen species (ROS) are involved in cartilage degradation. A decrease in the size of hyaluronan (HA), which is the major macromolecule in synovial fluid and is responsible for imparting viscosity to it, is reported in arthritis patients. The purpose of this study is to determine the ROS that depolymerize HA. The luminol derivative, L-012, was used to determine the generation of ROS. To generate hydroxyl radicals, a mixture of hydrogen peroxide (H(2)O(2)) and ferrous ions (Fe(2+)) was added to HA. The antioxidants and the depolymerization of HA were studied in this system. The hydroxyl radical is one of the ROS, causing the depolymerization of HA, which reacts with L-01. These data suggest that hydroxyl radicals play an important role at the site of inflammation.

Hypoxia-induced hyaluronan synthesis by articular chondrocytes: the role of nitric oxide

Abstract.Objective: Articular cartilage is an avascular tissue in which chondrocytes are exposed to hypoxic conditions. We previously demonstrated that reactive oxygen species (ROS) induced apoptosis of chondrocytes. We also demonstrated that nitric oxide (NO) was induced when chondrocytes were exposed to hypoxia and that NO inhibited the ROS-induced apoptosis. Hyaluronan (HA) is a high molecular weight glycosaminoglycan whose antioxidative effects have been reported. The purpose of the present study was to determine whether HA synthesis was induced in chondrocytes exposed to hypoxia, and, if so, whether the hypoxia-induced HA synthesis is regulated by NO.n Methods: Bovine articular chondrocytes were used in this study. Levels of HA were determined by the sandwich enzyme-binding assay. Expression of HA synthase (HAS) was determined with reverse transcription-polymerase chain reaction. The production of NO was examined using the Griess reaction. We also determined inducible nitric oxide synthase (iNOS) enzyme synthesis using the histochemistry and Western blot analysis.n Results: Chondrocytes cultured under hypoxic conditions exhibited enhanced HA synthesis. When the NO inhibitors, L-NMMA and L-NAME, were added, the hypoxia-enhanced HA levels in the culture medium were significantly inhibited.n Conclusions: Endogenous NO synthesis plays an important role in hypoxia-enhanced HA synthesis.

Involvement of angiotensin II and reactive oxygen species in pancreatic fibrosis.

Background: Pancreatic cancers often develop in the context of pancreatic fibrosis caused by chronic pancreas inflammation, which also results in the accumulation of reactive oxygen species (ROS), pancreatic parenchymal cell death, and stellate cell activation. Angiotensin II, which is converted from angiotensin I by the angiotensin-converting enzyme (ACE), stimulates ROS production via NADPH oxidase. In stellate cells, angiotensin II activates the stress-activated protein kinase p38. However, the molecular mechanism by which angiotensin II regulates pancreatic inflammation and fibrosis remains to be determined. Methods: Wistar Bonn/Kobori (WBN/Kob) rats spontaneously develop chronic pancreatic inflammation. To examine whether blockade of the renin-angiotensin system affects the development of pancreatic fibrosis, WBN/Kob rats were given angiotensin II type 1 receptor (AT1R) blocker or ACE inhibitor (ACEI). Next, we assessed the role of angiotensin II and its possible downstream target p38α in stellate cell activation using primary stellate cells. Results: Treatment with AT1R blocker and ACEI prevented the development of chronic pancreatitis and fibrosis. In stellate cells, angiotensin II upregulated the expression of angiotensin II receptors, α-smooth muscle actin (SMA) and transforming growth factor-β. In addition, p38α was found to be essential to collagen type I production and α-SMA expression. ROS accumulation is enhanced in chronic pancreatic inflammation, which increases the risk of pancreatic cancer. Conclusions: Inhibition of the angiotensin II signaling pathway might be a promising strategy to prevent pancreatic fibrogenesis and subsequent carcinogenesis.

Hypoxia-induced nitric oxide protects chondrocytes from damage by hydrogen peroxide

AbstractObjective:Because articular cartilage has no vascular supply, chondrocytes are hypoxic under normal physiological conditions. Nitric oxide (NO) plays an important role in chondrocyte damage, such as apoptosis. Although oxygen stress with hydrogen peroxide was found to cause chondrocyte damage, these data were obtained under normoxic (21% O2) conditions. We investigated the effects of hypoxia on hydrogen peroxide-induced chondrocyte damagen Methods:Bovine articular chondrocytes were used in this study. Proteoglycan (PG) synthesis and the induction of apoptosis were analyzed with [35S]-sulfate incorporation and annexin V staining, respectively. The induction of NO was examined using a fluorescent probe and RT-PCR.n Results:Cells maintained at 5% O2 had the maximum PG synthesis. Under normoxic conditions, hydrogen peroxide inhibited PG synthesis and induced annexin V positive cells in a dose-dependent fashion. However, in those cells cultured under hypoxic (5%) conditions, the hydrogen peroxide-induced annexin V expression was attenuated. Chondrocytes exposed to hypoxia showed induction of NO. When the hypoxia-induced NO was inhibited, the hypoxia-enhanced PG synthesis was abolished and hydrogen peroxide clearly induced cell damage.n Conclusions:Endogenous NO induced by hypoxia protects chondrocytes from apoptosis induced by an oxidative stress.

The cancer stem cell marker CD133 is a predictor of the effectiveness of S1+ pegylated interferon α-2b therapy against advanced hepatocellular carcinoma

BackgroundCombination therapy with the oral fluoropyrimidine anticancer drug S1 and interferon is reportedly effective for the treatment of advanced hepatocellular carcinoma (HCC), but selection criteria for this therapy have not been clarified. In this study, we attempted to identify factors predicting the effectiveness of this combination therapy.MethodsPathological specimens of HCC were collected before treatment from 31 patients with advanced HCC who underwent S1+ pegylated-interferon (PEG-IFN) α-2b therapy between January 2007 and January 2009. In these pathological specimens, the expression levels of CD133, thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), and interferon-receptor 2 (IFNR2) proteins were determined by Western blot assay. The presence or absence of p53 gene mutations was determined by direct sequencing. The relationships between these protein expression levels and the response rate (RR), progression-free survival (PFS), and overall survival (OS) were evaluated.ResultsThe CD133 protein expression level was significantly lower in the responder group than in the nonresponder group. Comparing the PFS and OS between high- and low-level CD133 expression groups (nxa0=xa013 and 18, respectively) revealed that both parameters were significantly prolonged in the latter group. The expression levels of TS, DPD, and IFNR2 protein and the presence of p53 gene mutations did not correlate with the RR.ConclusionsCD133 was identified as a predictor of the therapeutic effect of S1+ PEG-IFN α-2b therapy against advanced HCC.

1′-Acetoxychavicol Acetate Inhibits Adipogenesis in 3T3-L1 Adipocytes and in High Fat-Fed Rats

Alpinia galanga and Languas galanga, which are plants belonging to the ginger family, are frequently used for cooking, especially in Thai and Indonesian cuisine. The compound 1-acetoxychavicol acetate (ACA), which is naturally obtained from the rhizomes and seeds of these gingers, has antioxidant and anti-inflammatory properties. We investigated the anti-obesity effects of ACA in 3T3-L1 adipocytes and in high fat diet (HFD)-induced rat models of obesity. ACA caused a significant decrease in the activity of GPDH in 3T3-L1 adipocytes without eliciting cell cytotoxicity, and it inhibited cellular lipid accumulation through the down-regulation of transcription factors such as PPARγ and C/EBPα. ACA also induced a dose-dependent phosphorylation of AMP-activated protein kinase (AMPK). In the animal model, rats fed an HFD containing 0.05% ACA gained less weight than rats fed an HFD alone. The visceral fat mass in rats fed an HFD containing 0.05% ACA tended to be lower than that in rats fed an HFD alone. Furthermore, a histological examination of livers from rats fed an HFD showed steatohepatitis. However, rats fed an HFD containing 0.05% ACA showed no histopathological changes in the liver tissue. Our results show that ACA exerts anti-obesity activities both in vitro and in vivo and suggests that ACA may have a novel preventive activity against obesity and possibly other metabolic diseases.

Subcellular localization of human heparanase and its alternative splice variant in COS‐7 cells

Heparanase, the enzyme that degrades heparan sulfate, has been implicated to play important and characteristic roles in organogenesis, tissue organization, cell migration, and tumor metastasis. Clarification of its expression, its intracellular sorting, and its secretion is, therefore, of much importance to understand its role in cell biology. In addition to the 1.7 Kb transcript previously reported, we detected a 1.5 Kb transcript of human heparanase by RT‐PCR. The smaller transcript was shown to be an alternatively spliced variant lacking exon 5, which contains the essential glutamic acid residue required for enzyme activity. When expressed in COS‐7 cells this variant did not show any heparanase activity. Full‐length heparanase and the exon 5‐deleted splice variant were expressed in COS‐7 cells and examined by confocal laser scanning microscopy. Both proteins co‐localized with calnexin, a marker protein for the endoplasmic reticulum, and they co‐immunoprecipitated with calnexin. Both proteins were postulated to be precursors based upon the results of SDS–PAGE analyses. Treatment with endoglycosidases revealed that all potential N‐glycosylation sites in the proteins were glycosylated. Tunicamycin treatment of transfected COS‐7 cells inhibited N‐glycosylation but did not change the subcellular localization. These results indicate that overexpressed heparanase and its splice variant localize to the endoplasmic reticulum independent of glycosylation in COS‐7 cells. Copyright

Neuroprotective effect of (–)-epigallocatechin-3-gallate in rats when administered pre- or post-traumatic brain injury

Our previous study indicated that consuming (–)-epigallocatechin gallate (EGCG) before or after traumatic brain injury (TBI) eliminated free radical generation in rats, resulting in inhibition of neuronal degeneration and apoptotic death, and improvement of cognitive impairment. Here we investigated the effects of administering EGCG at various times pre- and post-TBI on cerebral function and morphology. Wistar rats were divided into five groups and were allowed access to (1) normal drinking water, (2) EGCG pre-TBI, (3) EGCG pre- and post-TBI, (4) EGCG post-TBI, and (5) sham-operated group with access to normal drinking water. TBI was induced with a pneumatic controlled injury device at 10xa0weeks of age. Immunohistochemistry and lipid peroxidation studies revealed that at 1, 3, and 7xa0days post-TBI, the number of 8-Hydroxy-2′-deoxyguanosine-, 4-Hydroxy-2-nonenal- and single-stranded DNA (ssDNA)-positive cells, and levels of malondialdehyde around the damaged area were significantly decreased in all EGCG treatment groups compared with the water group (Pxa0<xa00.05). Although there was a significant increase in the number of surviving neurons after TBI in each EGCG treatment group compared with the water group (Pxa0<xa00.05), significant improvement of cognitive impairment after TBI was only observed in the groups with continuous and post-TBI access to EGCG (Pxa0<xa00.05). These results indicate that EGCG inhibits free radical-induced neuronal degeneration and apoptotic death around the area damaged by TBI. Importantly, continuous and post-TBI access to EGCG improved cerebral function following TBI. In summary, consumption of green tea may be an effective therapy for TBI patients.

Characteristic pattern of reactivation of hepatitis B virus during chemotherapy for solid cancers.

Objective: A number of studies have reported reactivation of hepatitis B during intensive immunosuppressive therapy such as cases of hematological malignancy, whereas little has been reported for characteristics of reactivation triggered by chemotherapy for solid cancer. Methods: A total of 130 patients underwent chemotherapy for treatments of common solid cancer between May 2011 and May 2012 at Kinki University Hospital. Among them, 27 patients were suspected for a past infection of hepatitis B virus (HBV), showing positive for hepatitis B core antibody or surface antibody but negative for hepatitis B surface antigen, and were eligible for this study. Results: Hepatitis B reactivation was observed in 2 of 27 cases (7.4%). The duration between the start of chemotherapy and increase of serum HBV load was 30 days in both cases. Conclusions: We reported the 2 cases of hepatitis B reactivation receiving chemotherapy for solid cancer in terms of patterns and characteristics of reactivation. Accumulation of such cases will help in clarifying the clinical importance of hepatitis B reactivation during treatment of solid malignancies.

Effects of decorin on the expression of α-smooth muscle actin in a human myofibroblast cell line

Myofibroblasts are metabolically and morphologically distinctive fibroblasts expressing α-smooth muscle actin (α-SMA), and their activation plays a key role in development of the fibrotic response. In an activated state, myofibroblasts cease to proliferate and start to synthesize large amounts of extracellular component proteins. The expression of α-SMA correlates with the activation of myofibroblasts. Decorin, a member of the small leucine-rich proteoglycan gene family, has been implicated in the negative control of cell proliferation primarily by upregulating the expression of p21, a potent inhibitor of cyclin-dependent kinase. In order to examine the effect of decorin on myofibroblast cell growth, we rendered a human lung myofibroblast cell line, MRC-5, quiescent by either cell–cell contact or serum starvation, and examined the relationship between decorin and α-SMA expression in these cells. The expression of decorin in cells made quiescent by serum starvation was lower than that in cells made quiescent by cell–cell contact. In contrast, the expression of α-SMA in cells made quiescent by cell–cell contact was lower than that in cells made quiescent by serum starvation. Furthermore, forced expression of decorin was accompanied by a suppression of α-SMA expression, whereas knocking down of decorin expression by RNA interference increased the expression of α-SMA.