Expression of inducible nitric oxide synthase and cyclooxygenase-2 in pancreatic adenocarcinoma:Correlation with microvessel density

AIM:Cydooxygenases (COX) are key enzymes for conversionof arachidonic acid to prostaglandins.Nitric oxide synthase(NOS) is the enzyme responsible for formation of nitric oxide.Both have constitutive and inducible isoforms.The inducibleisoforms (iNOS and COX-2) are of great interest as regulatorsof tumor angiogenesis,tumorigenesis and inflammatoryprocesses.This study was to clarify their role in pancreaticadenocarcinomas.METHODS:We investigated the immunohistochemical iNOSand COX-2 expression in 40 pancreatic ductal adenocardnomasof different grade and stage.The results were comparedwith microvessel density and dinicopathological data.RESULTS:Twenty-one (52.5%) of the cases showed iNOSexpression,15 (37.5%) of the cases were positive for COX-2.The immunoreaction was heterogeneously distributed withinthe tumors.Staining intensity was different between thetumors.No correlation between iNOS and COX-2 expressionwas seen.There was no relationship with microvessel density.However,iNOS positive tumors developed more often distantmetastases and the more malignant tumors showed a higherCOX-2 expression.There was no correlation with otherclinicopathological data.CONCLUSION:Approximately half of the cases expressediNOS and COX-2.These two enzymes do not seem to bethe key step in angiogenesis or carcinogenesis of pancreaticadenocarcinomas.Due to a low prevalence of COX-2expression,chemoprevention of pancreatic carcinomas byCOX-2 inhibitors can only achieve a limited success.     

Increased hepatic expression of nitric oxide synthase type Ⅱ in cirrhotic rats

AIM: To determine the role and effect of nitric oxide synthase type Ⅱ (NOSII) in cirrhotic rats.METHODS: Expression of NOSⅡ mRNA was detected by real time RT-PCR. The activity of nitric oxide synthase and serum levels of NO, systemic and portal hemodynamics and degrees of cirrhosis were measured with high sensitive methods. Chinese traditional medicine tetrandrine was used to treat cirrhotic rats and to evaluate the function of NO.Double-blind method was applied during the experiment.RESULTS: The concentration of NO and the activity of NOS were increased markedly at all stages of cirrhosis, and iNOSmRNA was greatly expressed. Meanwhile the portalvenous-pressure (PVP), and portal-venous-flow (PVF) were significantly increased. NO, NOS and iNOSmRNA were positively correlated to the quantity of hepatic fibrosis.Tetrandrine significantly inhibited NO production and the expression of iNOSmRNA.CONCLUSION: Increased hepatic expression of NOSII is one of the important causes of hepatic cirrhosis and portal hypertension.     

Increased oxidative DNA damage, inducible nitric oxide synthase,nuclear factor κB expression and enhanced antiapoptosis-related proteins in Helicobacter pylori-infected non-cardiac gastric adenocarcinoma

AIM: Several epidemiological studies have demonstrated a dose association between Helicobacter pylori (H Pylon) infection and non-cardiac carcinoma of the stomach. H pylori infection induces active inflammation with neutrophilic infiltrations as well as production of oxygen free radicals that can cause DNA damage. The DNA damage induced by oxygen free radicals could have very harmful consequences,leading to gene modifications that are potentially mutagenic and/or carcinogenic. The aims of the present study were to assess the effect of H pyloriinfection on the expression of inducible nitric oxidative synthase (iNOS) and the production of 8-hydroxy-deoxyguanosine (8-OHdG), a sensitive marker of oxidative DNA injury in human gastric mucosa with and without tumor lesions, and to assess the possible factors affecting cell death signaling due tooxidative DNA damage.METHODS: In this study, 40 gastric carcinoma specimens and adjacent specimens were obtained from surgical resection. We determined the level of 8-OHdG formation by HPLC-ECD, and the expression of iNOS and mechanism of cell death signaling [including nuclear factor-κB(NFκB),MEKK-1, Caspase 3, B Cell lymphomal leukemia-2 (Bcl-2),inhibitor of apoptosis protein (IAP) and myeloid cellleukemia-1 (Mcl-1)] by Western-blot assay.RFSULTS: The concentrations of 8-OHdG, iNOS, NFx3, Mcl-1 and IAP were significantly higher in cancer tissues than in adjacent non-cancer tissues. In addition, significantly higher concentrations of 8-OHdG, iNOS, NFxB, Mcl-1 and lAP were detected in patients infected with H pyloricompared with patients who were not infected with HpylorL Furthermore,8-OHdG, iNOS, NFκB, Mcl-1 and IAP concentrations were significantly higher in stage 3 and 4 patients than in stage1 and 2 patients.CONCLUSION: Chronic Hpylori infection induces iNOS expression and subsequent DNA damage as well as enhances anti-apoptosis signal transduction. This sequence of events supports the rihypothesis that oxygen-free radical-mediated damage due to Hpyloriplays a pivotal role in the development of gastric carcinoma in patients with chronic gastritis.     

Role of inducible nitric oxide synthase expression in aberrant crypt foci－adenoma－carcinoma sequence

AIM: To investigate the expression of inducible nitric oxidesynthase (iNOS) in aberrant crypt foci (ACF) -adenomacarcinoma sequence and its relation with tumor cellapoptosis, proliferation and angiogenesis. METHODS: The expression of iNOS, proliferating cellnuclear antigen (PCNA) and microvessel density (MVD) indifferent stages of colorectal cancer were studied by immunohistochemical method from 30 normal tissues, 30nonhyperplastic ACF, 30 hyperplastic ACF, 30 dysplastic ACF,30 adenomas and 60 carcinomas. The apoptotic cells were detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method using an Apop Tag in situ detection kit. RESULTS: The immunoreactivity of iNOS significantly increased in the transition from hyperplastic ACF to dysplastic ACF. This transition was associated with a significant decrease in the apoptotic index (AI) (0.73+0.37 vs0.61±0.35, P＜0.05)and significant increases in the PCNA labeling index (LI)(27.3+2.80 vs 40.3+3.11, P＜0.01) and microvessel density (MVD) (55±11.5 vs 70±13.2, P＜0.01). The expression of iNOS was in low levels and positively correlated with PCNALI (r=0.812, P＜0.01) and MVD (r=0.863, P＜0.01) during transition from normal mucosa to nonhyperplastic ACF and hyperplastic ACF. The expression of iNOS was in high levels and positively correlated with AI (r=0.901, P＜0.01) after transition from hyperplastic ACF to dysplastic ACF, adenoma and carcinoma. CONCLUSION: The results suggest that the transition from hyperplastic ACF to dysplastic ACF may be a crucial step in the ACF-adenoma-carcinoma sequence, in which iNOS plays an important role by regulating tumor cell apoptosis,proliferation and angiogenesis.     

Effect of lipopolysaccharide on diarrhea and gastrointestinal transit in mice： Roles of nitric oxide and prostaglandin E2

AIM: To investigate the effect of lipopolysaccharide (LPS) on the diarrheogenic activity, gastrointestinal transit (GIT), and intestinal fluid content and the possible role of nitric oxide (NO) and prostaglandin E2 (PGE2) in gastrointestinal functions of endotoxin-treated mice. METHODS: Diarrheogic activity, GIT, and intestinal fluid content as well as nitric oxide and PGE2 products were measured after intraperitoneal administration of LPS in mice. RESULTS: LPS dose-dependently accumulated abundant fluid into the small intestine, induced diarrhea, but decreased the GIT. Both nitric oxide and PGE2 were found to increase in LPS-treated mice. Western blot analysis indicated that LPS significantly induced the protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 in mice intestines. Pretreatment with NG-nitro-L-arginine-methyl ester (L-NAME, a non-selective NOS inhibitor) or indomethacin (an inhibitor of prostaglandin synthesis) significantly attenuated the effects of LPS on the diarrheogenic activity and intestine content, but reversed the GIT. CONCLUSION: The present study suggests that the pathogenesis of LPS treatment may mediate the stimulatory effect of LPS on nitric oxide and PGE2 production and NO/ prostaglandin pathway may play an important role on gastrointestinal function.     

Effects of acupuncturing Tsusanli （ST36） on expression of nitric oxide synthase in hypothalamus and adrenal gland in rats with cold stress ulcer

AIM: To study the protective effect of acupuncturing Tsusanli (ST36) on cold stress ulcer, and the expression of nitric oxide synthase (NOS) in hypothalamus and adrenal gland. METHODS: Ulcer index in rats and RT-PCR were used to study the protective effect of acupuncture on cold stress ulcer, and the expression of NOS in hypothalamus and adrenal gland. Images were analyzed with semi-quantitative method. RESULTS: The ulcer index significantly decreased in rats with stress ulcer. Plasma cortisol concentration was up regulated during cold stress, which could be depressed by pre-acupuncture. The expression of NOS1 in hypothalamus increased after acupuncture. The increased expression of NOS2 was related with stress ulcer, which could be decreased by acupuncture. The expression of NOS3 in hypothalamus was similar to NOS2, but the effect of acupuncture was limited. The expression of NOS2 and NOS3 in adrenal gland increased after cold stress, only the expression of NOS1 could be repressed with acupuncture. There was no NOS2 expression in adrenal gland in rats with stress ulcer. CONCLUSION: The protective effect of acupuncturing Tsusanli (ST36) on the expression of NOS in hypothalamus and adrenal gland can be achieved.     

Correlation between expression of cyclooxygenase-2 and the presence of inflammatory cells in human primary hepatocellular carcinoma： Possible role in tumor promotion and angiogenesis

AIM: To investigate the association of cyclooxygenase-2 (COX-2) expression with angiogenesis and the number and type of inflammatory cells (macrophages/Kupffer cells; mast cells) within primary hepatocellular carcinoma (HCC) tissues and adjacent non-tumorous (NT) tissues. METHODS: Immunohistochemistry for COX-2, CD34, CD68 and mast cell tryptase (MCT) was performed on 14 well-characterized series of liver-cirrhosis-associated HCC patients. COX-2 expression and the number of inflammatory cells in tumor lesions and surrounding liver tissues of each specimen were compared. Moreover, COX-2, CD34 staining and the number of inflammatory cells in areas with different histological degrees within each tumor sample were comparatively analyzed. RESULTS: The percentage of COX-2 positive cells was significantly higher in NT tissues than in tumors. COX-2 expression was higher in well-differentiated HCC than in poorly-differentiated tissues. Few mast cells were observed within the tumor mass, whereas a higher number was observed in the surrounding tissue, especially in peri-portal spaces of NT tissues. Abundant macrophages/ Kupffer cells were observed in NT tissues, whereas the number of cells was significantly lower in the tumor mass. However, a higher cell number was observed in the well-differentiated tumor and progressively decreased in relation to the differentiation grade. Within the tumor, a positive correlation was found between COX-2 expression and the number of macrophages/Kupffer cells and mast cells. Moreover, there was a positive correlation between CD34 and COX-2 expression in tumor tissues. Comparison between well- and poorly-differentiated HCC showed that the number of CD34-positive cells decreased with dedifferentiation. However, COX-2 was the only independent variable showing a positive correlation with CD34 in a multivariate analysis. CONCLUSION: The presence of inflammatory cells and COX-2 expression in liver tumor suggests a possible relationship with tumor angiogenesis. COX-2 expressing cells and the number of macrophages/Kupffer cells and mast cells decrease with progression of the disease.     

Predictive value of Ki67 and p53 in locally advanced rectal cancer：Correlation with thymidylate synthase and histopathological tumor regression after neoadjuvant 5-FU-based chemoradiotherapy

AIM： To investigate the predictive value of Ki67 and p53 and their correlation with thymidylate synthase （TS） gene expression in a rectal cancer patient cohort treated according to a standardized recommended neoadjuvant treatment regimen.METHODS： Formalin fixed, paraffin embedded pre-therapeutical tumor biopsies （n = 22） and post-therapeutical resection specimens （n = 40） from patients with rectal adenocarcinoma （clinical UICC stage Ⅱ/Ⅲ） receiving standardized neoadjuvant 5-fluorouracil （5-FU） based chemoradiotherapy were studied for Ki67 and p53 expression by immunohistochemistry and correlated with TS mRNA expression by quantitative TaqMan real-time PCR after laser microdissection. The results were compared with histopathological tumor regression according to a standardized semiquantitative score grading system.RESULTS： Responders （patients with high tumor regression） showed a significantly lower Ki67 expression than non-responders in the pre-therapeutical tumor biopsies （81.2% vs 16.7%; P 〈 0.05） as well as in the post-therapeutical resection specimens （75.8% vs 14.3%; P 〈 0.01）. High TS mRNA expression was significantly correlated with a high Ki67 index and low TS mRNA expression was significantly correlated with a low Ki67 index in the pre-therapeutical tumor biopsies （corr. coef. = 0.46; P 〈 0.01） as well as in the post-therapeutical resection specimens （corr. coef. = 0.40; P 〈 0.05）. No significant association was found between p53 and TS mRNA expression or tumor regression.CONCLUSION： Ki67 has, like TS, predictive value in rectal cancer patients after neoadjuvant 5-FU based chemoradiotherapy. The close correlation between Ki67 and TS indicates that TS is involved in active cell cycle processes.     

Expression and significance of Tie-1 and Tie-2 receptors, and angiopoietins-1, 2 and 4 in colorectal adenocarcinoma： Immunohistochemical analysis and correlation with clinicopathological factors

AIM: There is strong evidence that tyrosine kinases are involved in the regulation of tumor progression, cellular growth and differentiation. Recently, many kinds of tyrosine kinase receptors have been reported, among them Tie-1 and Tie-2 receptors constitute a major class. Angiopoietin (Ang)-1 is known as a ligand of Tie-2 tyrosine kinase receptor. The objective of this study was to establish a comprehensive Tie-1 and Tie-2 and Ang-1, 2 and 4 expression profile in human colorectal adenocarcinomas. METHODS: We examined 96 cases of surgically resected human colorectal adenocarcinoma by immunohistochemistry and investigated the statistical correlation between the expressions of Ties and Angs and clinicopathological factors. RESULTS: Among the 96 cases of adenocarcinoma, 87 (90.6%), 92 (95.8%), 83 (86.5%), 89 (92.7%), and 76 cases (79.2%) showed positive staining in the cytoplasm of carcinoma cells for the Tie-1 and Tie-2 and Ang-1, 2 and 4 proteins, respectively. Histologically, the expressions of Ties and Angs were variable. The expressions of Ties and Angs were correlated with several clinicopathological factors, but did not correlate with the presence of lymph node metastasis. Ties and Angs were highly expressed in human colorectal adenocarcinoma cells. CONCLUSION: These findings suggest that the Tie-Ang receptor-ligand complex is one of the factors involved in the cellular differentiation and progression of human colorectal adenocarcinoma.     

β-catenin up-regulates the expression of cyclinD1, c-myc and MMP-7 in human pancreatic cancer： Relationships with carcinogenesis and metastasis

AIM: To investigate whether abnormal expression of β-catenin in conjunction with overexpression of cyclin D1, c-myc and matrix metalloproteinase-7 (MMP-7) correlated with the carcinogenesis, metastasis and prognosis of pancreatic cancer, and to analyze the relationship of β-catenin expression with cyclinD1, c-myc and MMP-7 expression.METHODS: Using immunohistochemistry, we examined the expression of β-catenin, cyclinD1, c-myc and MMP-7 in 47 pancreatic adenocarcinoma tissues, 12 pancreatic intraepithelial neoplasia (PanIN) and 10 normal pancreases,respectively. Proliferation cell nuclear antigen was also tested as the index of proliferative activity of pancreatic cancer cells.RESULTS: In 10 cases of normal pancreatic tissues,epithelial cells showed equally strong membranous expression of βcatenin protein at the cell-cell boundaries,but the expression of cyclin D1, c-myc and MMP-7 was negative. The expression of βcatenin, cyclinD1, c-myc and MMP-7 in PanIN and pancreatic adenocarcinoma tissues had no significant difference [6/12 and 32/47 (68.1%),6/12 and 35/47 (74.5%), 5/12 and 33/47 (70.2%), 7/12 and 30/47 (63.8%), respectively]. The abnormal expression of β-catenin was significantly correlated to metastasis and one-year survival rate of pancreatic cancer, but had no relation with size, differentiation and cell proliferation.The expression of cyclinD1 was correlated with cell proliferation and extent of differentiation, but not with size,metastasis and one-year survival rate of the pancreatic ancer. The expression of c-myc was not correlated with ize, extent of differentiation, metastasis and 1-year urvival rate, but closely with cell proliferation of pancreatic ancer. The overexpression of MMP-7 was significantly ssociated with metastasis and 1-year survival rate of ancreatic cancer, but not with size, extent of differentiation and cell proliferation. There was a highly significant positive association between abnormal expression of β-catenin and overexpression of cyclinD1, c-myc and MMP-7 not only in PanIN (r = 1.000, 0.845, 0.845), but also in pancreatic cancer (r = 0.437, 0.452, 0.435).CONCLUSION: The abnormal expression of β-catenin plays a key role in the carcinogenesis and progression of human pancreatic carcinoma by up-regulating the expression of cyclinD1, c-myc and MMP-7, resulting in the degradation of extracellular matrix and uncontrolled cell proliferation and differentiation, β-catenin abnormal expression and MMP-7 overexpression may be considered as two useful markers for determining metastasis and prognosis of human pancreatic cancer.     

Role of cyclooxygenase-2 and inducible nitric oxide synthase in pancreatic cancer

BACKGROUND AND AIM: Recently, it has been recognized that both cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) produce important endogenous factors of human tumor progression. However, the clinicopathological and biological significance of the expression of COX-2 and iNOS in pancreatic cancer remains unclear. The objective of this study is to find the possible roles and clinical significance of COX-2 and iNOS expression in pancreatic cancer. METHODS: Seventy-two pancreatic adenocarcinoma tissue specimens were obtained through surgical resection. We investigated the immunohistochemical expression of COX-2 and iNOS in respect to variable clinicopathological characteristics, proliferation activity (by Ki-67 expression), apoptosis (by terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling stain), and microvessel density (by CD34 expression; angiogenesis). RESULTS: Immunohistochemical investigations demonstrated immunolabeling of tumor cells with the primary antibodies, bovine anti-iNOS and anti-COX-2 antibodies. The COX-2 and iNOS positive rates were 41.7 and 66.7%, respectively. There was significant correlation between positive COX-2 and positive iNOS expression (P = 0.043). The proliferation index (Ki-67 labeling index) was higher in COX-2 positive specimens compared to COX-2 negative specimen (P = 0.015). The apoptotic index of positive iNOS expressions was significantly higher than negative expressions (P < 0.001). The expression of COX-2 and iNOS proteins did not correlate with age, sex, serum bilirubin, CA-19-9, location, size, American Joint Committee on Cancer stage, differentiation, distant metastasis, patient survival, or microvessel density. CONCLUSIONS: Although the pattern of positive expression was similar in both enzymes, the effect on tumor progression differed; iNOS expression may play a role in apoptosis of tumor cell, while COX-2 expression may contribute to tumor proliferation. However, COX-2 and iNOS expression is not related to prognosis in patients with pancreatic cancer.     

Interaction between inducible nitric oxide synthase and cyclooxygenase-2 after cerebral ischemia

Focal cerebral ischemia is associated with expression of both inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), enzymes whose reaction products contribute to the evolution of ischemic brain injury. We tested the hypothesis that, after cerebral ischemia, nitric oxide (NO) produced by iNOS enhances COX-2 activity, thereby increasing the toxic potential of this enzyme. Cerebral ischemia was produced by middle cerebral artery occlusion in rats or mice. Twenty-four hours after ischemia in rats, iNOS-immunoreactive neutrophils were observed in close proximity (<20 μm) to COX-2-positive cells at the periphery of the infarct. In the olfactory bulb, only COX-2 positive cells were observed. Cerebral ischemia increased the concentration of the COX-2 reaction product prostaglandin E₂ (PGE₂) in the ischemic area and in the ipsilateral olfactory bulb. The iNOS inhibitor aminoguanidine reduced PGE₂ concentration in the infarct, where both iNOS and COX-2 were expressed, but not in the olfactory bulb, where only COX-2 was expressed. Postischemic PGE₂ accumulation was reduced significantly in iNOS null mice compared with wild-type controls (C57BL/6 or SV129). The data provide evidence that NO produced by iNOS influences COX-2 activity after focal cerebral ischemia. Pro-inflammatory prostanoids and reactive oxygen species produced by COX-2 may be a previously unrecognized factor by which NO contributes to ischemic brain injury. The pathogenic effect of the interaction between NO, or a derived specie, and COX-2 is likely to play a role also in other brain diseases associated with inflammation.     

Estrogen up-regulates inducible nitric oxide synthase, nitric oxide, and cyclooxygenase-2 in splenocytes activated with T cell stimulants: role of interferon-gamma

Estrogen is implicated in many autoimmune diseases and is a robust immunomodulator. For example, it regulates interferon (IFN)-gamma, a cytokine believed to up-regulate inducible nitric oxide synthase (iNOS). A notable gap in the literature is a lack of information on the regulation of nitric oxide in immune tissues by estrogen. We now show that activation of splenocytes with T cell stimulants [concanavalin-A (Con-A) or anti-CD3 antibodies] results in copious release of nitric oxide in splenocyte cultures from estrogen-treated but not placebo-treated mice. Moreover, even a low dose of T cell stimulants induced nitric oxide in splenocytes from estrogen-treated, but not placebo-treated, mice. Con-A-activated splenocytes from estrogen-treated mice also have up-regulated iNOS mRNA, iNOS protein, and cyclooxygenase-2 (a nitric oxide-regulated downstream proinflammatory protein) when compared with controls. Our studies suggest that the induction of nitric oxide by activated splenocytes from estrogen-treated mice is mediated in part by IFNgamma. First, blocking costimulatory signals mediated through interactions of CD28 and B7 molecules by CTLA-4Ig markedly decreased not only IFNgamma but also nitric oxide. Second, estrogen treatment of IFNgamma-knockout (IFNgamma(-)/(-)) mice did not induce iNOS protein or nitric oxide. Finally, in vitro addition of recombinant IFNgamma to Con-A-activated splenocytes from IFNgamma((-)/(-)) mice induced iNOS protein primarily in estrogen-treated mice. Overall, this is the first report to show that estrogen treatment up-regulates IFNgamma-inducible-iNOS gene expression, iNOS protein, nitric oxide, and cyclooxygenase-2 as an indirect consequence of activation of T cells. These findings may have wide implications to immunity and inflammatory disorders including female-predominant autoimmune diseases.     

Heterogeneous expression of cyclooxygenase-2 and inducible nitric oxide synthase within colorectal tumors: Correlation with tumor angiogenesis

BackgroundRecent studies have shown that the cyclooxygenase (COX) and the inducible nitric oxide synthase (iNOS) pathways are involved in the development of tumor angiogenesis in human cancers.AimsTo investigate whether a different pattern of COX-2 and iNOS expression/activity exists within different areas of colorectal tumors and to analyze the relationship between these two enzymes and tumor angiogenesis.MethodsMicrovessel density (MVD) and COX-2, iNOS, vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR-2) protein expression were evaluated at both the invasive front (IF) and the tumor center (TC) in 46 human colorectal cancer specimens. We also investigated the concentration of PGE₂ and NO at the same sites.ResultsCOX-2 and iNOS protein expression and activity were significantly higher within the IF than the TC of the tumor specimens. Similarly, MVD and VEGF/VEGFR-2 expression significantly increased from the TC to the IF. Only COX-2 expression was significantly correlated with MVD and VEGF/VEGFR-2 expression at both the TC and the IF.ConclusionOur study shows a heterogeneous expression of COX-2 and iNOS in colorectal cancer. The up-regulation of COX-2 at the IF parallels an increase in vessel density and VEGF/VEGFR-2 expression, thus supporting the hypothesis that the tumor periphery is the most aggressive portion of a colorectal tumor.     

Expression of endothelial nitric oxide synthase and inducible nitric oxide synthase in synovium of rheumatoid arthritis]

OBJECTIVES: To examine the localization and distribution of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS), which participate in nitric oxide (NO) production, in synovium of rheumatoid arthritis (RA). MATERIALS AND METHODS: Immunohistochemical analysis for eNOS and iNOS in synovial tissues obtained from 10 patients with RA who were underwent total knee replacement. Synovial tissues of osteoarthritis (OA) were used as control. The percentage of cells that were positive for eNOS and iNOS was estimated in five hundred endothelial cells, synovial lining cells and interstitial cells, respectively. And mRNA expression of NOS was confirmed by in situ hybridization. In addition, to test NO production, nitration of tyrosines was assessed by immunohistochemistry. RESULTS: Not only endothelial cells but also synovial lining cells and interstitial cells exhibited immune-reactive both eNOS and iNOS. Cells which were seemed immune-reactive eNOS and iNOS expressed nitrotyrosin. By in situ hybridization, we detected mRNA expression for eNOS and iNOS. CONCLUSIONS: Endothelial cells, synovial lining cells and interstitial cells expressed both eNOS and iNOS with high frequency in RA synovium compared with OA synovium. It seemed to correlate with NO production. These results suggest that expression of iNOS may be involved in the induction of arthritis and eNOS may be participated in augmentation of inflammation in RA.     

Expression of inducible nitric oxide synthase in human gastric cancer

INTRODUCTIONInduciblenitricoxidesynthase(iNOS)isanenzymethatcatalyzestheformationofnitric0xide(N0)fromL-arginine.iNOSexpressionandactivityresultsintheproduction0fhighlevelsofNO[1].ThegenerationofphysiologicallevelsofNOisimp0rtantformucosalfunctionanditalsoexertsacytoprotectiveeffectonthegastr0intestinalmucosa.However,increasediNOSexpressionhasbeenobservedinpatientswithchronicinflammatorydiseasesofthegastr0intestinaltract,suchasulcerativec0litis[2'3],andgastritis['Jandithasbeenspecul…     

Immunocytochemical evidence for inducible nitric oxide synthase and cyclooxygenase-2 expression with nitrotyrosine formation in human hibernating myocardium

Background: Myocardial hibernation may result from repetitive episodes of transient ischaemia leading to prolonged dysfunction. Inducible nitric oxide synthase (iNOS) expression has been demonstrated in animals following brief, non-lethal ischaemia-reperfusion injury. We therefore, hypothesised that in human hibernating myocardium: 1) iNOS would be present; 2) the reaction of nitric oxide and superoxide would form the strong oxidant peroxynitrite; 3) that this process would be accompanied by the expression of cyclooxygenase-2 (Cox-2) which interacts with NOS and whose products could further affect myocardial function. Method and results: In sixteen patients with coronary artery disease (CAD), left ventricular biopsies were obtained from chronically dysfunctional segments subtended by a stenotic artery (> 75 %) and shown to be viable by ¹⁸F-fluorodeoxyglucose positron emission tomography. Comparison was made with myocardial biopsies (n = 8) from normally contracting myocardium in patients undergoing coronary surgery, from unused transplant donors and at post-mortem. Regional wall motion score improved in all patients 6 months post-revascularisation (from 2.7 ± 0.7 to 1.5 ± 0.5; p < 0.001), confirming hibernation. Immunocytochemistry localized reactivity to iNOS, Cox-2 and nitrotyrosine (a marker of peroxynitrite formation) to cardiomyocytes from hibernating segments. No difference in reactivity to endothelial NOS was seen between hibernating and control cardiomyocytes. Conclusion: Cox-2 and iNOS are co-expressed in hibernating myocardium with nitrotyrosine suggesting nitric oxide production and peroxynitrite formation. We propose that this is secondary to ischaemia-reperfusion and that the products of these enzymes may have consequences for myocardial contractile function and survival. Received: 11 February 2002, Returned for revision: 14 February 2002, Revision received: 4 March 2002, Accepted: 11 March 2002