非诺贝特上调血管内皮细胞一氧化氮合酶的表达

目的:观察PPARα激动剂非诺贝特对牛主动脉(BAECs)内皮细胞一氧化氮合酶(eNOS)活性和表达的影响。方法:制备5-9代BAECs,加入不同浓度的非诺贝特(0, 5, 10, 50, 100 μmol/L)后,用NOS Assay Kit测定eNOS活性,RT-PCR法检测eNOS mRNA表达,Western blot分析检测eNOS蛋白质表达。结果: 非诺贝特以浓度和时间依赖的方式增加eNOS活性,非诺贝特浓度10 μmol/L以上时,明显增加eNOS活性。50μmol/L非诺贝特处理48 h时eNOS活性最大(为对照组的2.32±0.47倍,P<0.01)。非诺贝特处理1 h和12 h不增加eNOS活性。RT-PCR分析表明,非诺贝特浓度大于5 μmol/L以上时,明显增加eNOS mRNA水平,在非诺贝特浓度为50 μmol/L时作用最大,为对照组的2.08±0.33倍(P<0.01)。此作用在6 h时出现,持续到48 h。Western blot显示,非诺贝特处理48 h,eNOS蛋白表达明显增加,在浓度为10,50 和100 μmol/L时,eNOS蛋白表达分别为对照组的1.80±0.45, 2.70±0.42 和 2.20±0.32 倍,均P<0.01。在非诺贝特处理12 h后出现,持续到48 h。结论:PPARα激动剂非诺贝特增加BAECs eNOS基因表达,提高eNOS活性及增加蛋白表达。     

Inducible nitric oxide synthase expression in coronary arteries of transplanted human hearts with accelerated graft arteriosclerosis.

Inducible nitric oxide synthase (iNOS) is a high-output isoform of NOS that produces nitric oxide (NO), a nonspecific immune effector molecule. In some animal models of autoimmunity, the induction of iNOS has been shown to lead to inflammation and tissue damage, and it has been suggested that iNOS is an immune mediator in humans as well. Using in situ hybridization and immunohistochemical techniques, we demonstrate that iNOS mRNA and protein are present in the coronary arteries of transplanted human hearts with accelerated graft arteriosclerosis (AGA). iNOS is expressed in cells morphologically consistent with macrophages in the neointima of 7 of 10 of the transplanted vessels with AGA that were examined. In serial sections, these same cells express the macrophage marker CD68. In contrast, iNOS is absent from five native coronary arteries with atherosclerosis and absent from two normal coronary arteries. Although iNOS is expressed in macrophages in AGA, its role in the pathogenesis of AGA is unknown.     

诱导型一氧化氮合酶与疾病

迟至 2 0世纪 80年代末 ,一氧化氮 (ｎｉｔｒｉｃｏｘｉｄｅ ,ＮＯ)才被认识到是人体内一种生物学活性分子。人体内的ＮＯ有两个来源。一为非酶生 (ｎｏｎ -ｅｎ ｚｙｍｓｔｉｇｅｎｅｓｅ) ,来自体表或者摄入的无机氮的化学降解 /转化 ;一为酶生 (ｅｎｚｙｍｓｔｉｇｅｎｅｓｅ) ,由ＮＯ合酶(ＮＯｓｙｎｔｈａｓｅＮＯＳ)所合成。现已认识到ＮＯ既是一个有细胞毒性效应器作用的分子 ,是组织损伤的诱发因子和各种病变的增强因子 ,有其病理上不好的一面 ;又是生物体许多部分的信号 (ｓｉｇｎａｌ)分子 ,是先天性免疫应答的调节性效应分子 ,作…     

Inducible and endothelial nitric oxide synthase expression in human atherogenesis: an immunohistochemical and ultrastructural study

BackgroundNitric oxide has been proven to play an important role in the maintenance of vascular tone and structure. Impairment of nitric oxide production is an early indicator of atherosclerosis, but not much is known about the real mechanisms underlying this phenomenon.MethodsIn the present study, immunocytochemical methods have been used to analyze the patterns of expression of endothelial nitric oxide synthase and inducible nitric oxide synthase proteins in healthy and atherosclerotic human aortae using both confocal laser scanning microscopy and electron microscopy.ResultsInduction of the expression of endothelial nitric oxide synthase and inducible nitric oxide synthase proteins was observed in smooth muscle cells of atherosclerotic human aortae. Altered nitric oxide synthase expression was reported in atheromatous plaques and in apparently normal vascular tissues adjacent to the lesions.ConclusionsOur data confirm and extend previous findings of a direct relationship between dysregulation of nitric oxide pathway and atherosclerosis, suggesting another possible mechanism by which nitric oxide synthase system abnormalities may promote vascular dysfunction during human atherogenesis. Changes in nitric oxide production might be the primary step in the development of atheroma.     

Inducible and endothelial nitric oxide synthase genes polymorphism in inflammatory bowel disease

To assess the influence of inducible and endothelial nitric oxide synthase gene (NOS2A and NOS3) polymorphisms in susceptibility to Crohn's disease (CD) and ulcerative colitis (UC). A total of 505 inflammatory bowel disease (IBD) patients (221 with UC and 284 with CD) and 332 ethnically matched controls were studied. Patients and controls were genotyped by polymerase chain reaction -based techniques for a multiallelic (CCTTT)(n) repeat and biallelic marker (TAAA)(n) in the promoter region of the NOS2A gene and for a T/C polymorphism at position -786 in the promoter region and a polymorphism in exon 7(298Glu/Asp) of the NOS3 gene. There was not association between NOS2A and NOS3 genotypes, alleles or haplotypes frequencies with UC, CD and controls. Our data suggest that NOS2A and NOS3 polymorphisms do not play a major role in IBD predisposition.     

Inducible nitric oxide synthase expression in human urinary bladder cancer

Nitric oxide (NO) is generated by a family of enzymes, nitric oxide synthases (NOS), in a wide range of mammalian cells. NO produced by the inducible NOS isoform (iNOS) has been suggested to play an important role in tumor biology with both tumor promoter and anti-tumor activity. Here, the cellular localization of iNOS in tissue of 100 cases of urinary bladder cancer was assessed immunohistologically using a commercially available antiserum. Positive iNOS immunostaining was detected in all samples of tumor tissue, whereas nonmalignant tissue adjacent to malignant areas did not show any iNOS positivity. The tumor tissue revealed a highly inhomogeneous staining pattern. In addition to uniformly stained tumor specimens, we also found markedly iNOS-positive tumor islets in the midst of unstained tumor tissue and scattered individual tumor cells expressing marked staining. In some cases, the tumor tissue showed no or only weak staining intensity. In some instances, the superficial epithelial layer of papillary carcinomas was extremely immunoreactive, in other cases it was not. Thus we were unable to show a clear correlation to tumor grade or stage. Further studies with a diversity of tumor markers including molecular genetics techniques will be necessary to elucidate how and to what extent NO and bladder cancer of different grades and stages are functionally interrelated.     

Inducible nitric oxide synthase in human diseases

Oestrogen has the capacity to suppress T cell-dependent DTH. To explore the mechanisms whereby oestrogen exerts its effects on the immune system we have used SCID mice which are largely devoid of functional T and B lymphocytes, hence being unable to raise DTH, but display intact antigen-presenting capacity. Transfer of lymphocytes to SCID mice restores the DTH capacity. In order to analyse if oestrogen down-regulates DTH by a direct action on T cells we reconstituted SCID mice with either splenocytes or thymocytes from congenic C.B-17 or allogeneic B6 donor mice. Either donor or recipient mice were exposed to estradiol before cell transfer. DTH response was registered in recipient SCID mice 1 and 3 weeks after challenge with oxazolone (OXA). SCID mice receiving estradiol-exposed spleen cells from congenic or allogeneic donor mice displayed lower DTH responses compared with control mice. In contrast, SCID mice receiving estradiol-exposed thymocytes from congenic donor mice showed no significant difference in DTH response compared with control mice. Estradiol-treated SCID mice, transferred with either spleen cells or thymocytes from congenic, hormonally non-treated donors, displayed a significantly lower DTH response compared with control mice. In contrast, estradiol-treated SCID mice receiving hormonally non-treated allogeneic spleen cells showed no difference in DTH response compared with control mice. The results show that T lymphocytes are not the target cell population for estradiol-mediated suppression of DTH in reconstituted female SCID mice.     

Inducible nitric oxide synthase expression in benign and malignant cutaneous melanocytic lesions

Nitric oxide (NO) is synthesized by nitric oxide synthases (NOS) and plays an important role in tumour growth. In this study, inducible NOS (iNOS) expression was evaluated by immunohistochemistry in 34 melanocytic naevi (13 common melanocytic naevi, six Spitz naevi, and 15 so-called 'dysplastic naevi'), ten cutaneous melanomas in situ, 50 stage I invasive melanomas, and eight subcutaneous metastases of melanoma. In addition, four samples of melanocytic naevi and four samples of invasive melanomas were collected in order to perform western blot and northern blot analysis. By immunohistochemistry, melanocytic naevi never expressed iNOS. Among cases of melanoma in situ, two were negative, seven displayed staining in less than 20% of melanoma cells, and positivity was observed in 21-50% of melanoma cells in only one case. iNOS expression was detected in 46 out of 50 invasive melanomas (92%). Among these cases, 18 showed positivity in less than 20% of melanoma cells, 18 showed positivity in 21-50% of melanoma cells, and ten showed iNOS expression in more than 50% of cells. Statistical analysis revealed a significant difference in iNOS expression between melanocytic naevi and cutaneous melanomas (p<0.001). In addition, iNOS expression was significantly higher in invasive melanomas than in melanomas in situ (p=0.01). Among primary cutaneous melanomas, no significant correlation was found between iNOS expression and histopathological parameters (histotype, level, thickness and presence of regression/inflammatory infiltrate) and disease-specific survival. In subcutaneous melanoma metastases, iNOS expression was diffuse in more than 50% of cells. Statistical analysis revealed that subcutaneous melanoma metastases showed greater iNOS immunoreactivity than invasive melanomas (p=0.02). Molecular analyses confirmed that iNOS mRNA and protein were highly expressed in melanoma samples. In conclusion, iNOS was constantly absent in melanocytic naevi, whereas it was frequently expressed in melanomas, with up-regulation of the enzyme paralleling tumour progression. These data suggest that iNOS may play a role in the malignant transformation of melanocytes and in tumour growth. In addition, iNOS may be useful as an immunohistochemical marker for malignant melanocytic lesions.     

Inducible nitric oxide synthase expression and its prognostic significance in colorectal cancer

Zafirellis K, Zachaki A, Agrogiannis G, Gravani K. Inducible nitric oxide synthase expression and its prognostic significance in colorectal cancer. APMIS 2010; 118: 115–24. Nitric oxide synthases (NOS) are expressed in colorectal cancer. The aim of this study was to examine the inducible NOS (iNOS) expression in colorectal cancer and to investigate its prognostic relevance. Tissue sections of primary tumors from 132 patients undergoing curative resection for colorectal cancer were immunohistochemically examined for iNOS expression. The expression pattern of iNOS was correlated with various clinicopathological characteristics and survival. iNOS immunoreactivity was observed in the cytoplasm of tumor epithelial cells in 60 patients (45.5%) and positively correlated with lymph node involvement (p = 0.019). No significant correlation was found between iNOS expression and various clinicopathological characteristics, including age, gender, tumor location, tumor size, tumor grade, T stage, and Union International Contra la Cancrum (UICC) stage. Survival analysis showed a significant correlation between iNOS‐positive tumors and poor disease‐specific survival (p < 0.0001), with independent prognostic significance in multivariate analysis (HR = 4.42; p < 0.0001). Patients with stage II disease and iNOS‐positive tumors had significantly worse disease‐specific survival than those with iNOS‐negative tumors (p < 0.0001). In addition, patients with stage III disease and iNOS‐positive tumors had significantly worse disease‐specific survival than those with iNOS‐negative tumors (p = 0.001). The ability of iNOS to predict outcome in colorectal cancer patients may be independent of other known prognostic factors, providing a new molecular marker with significant potential for clinical utility.     

诱导型一氧化氮合酶与动脉血压的调节

目的:探讨诱导型一氧化氮合酶在血液动力学调控中的作用。方法:本实验所用动物为健康、雄性、Dahl 盐敏感(DS)及Dahl盐不敏感(DR)大鼠,体重(190±4) g。通过慢性血液动力学实验观察静脉输入诱导型一氧化氮合酶(iNOS)抑制剂AG对大鼠平均动脉压的影响。此外,本实验还测定了一氧化氮(NO)终产物NO₂^-及NO₃^-含量以及钙依赖及钙不依赖的NOS活性以了解iNOS的功能。结果:(1)iNOS特异抑制剂AG对正常血压(包括高盐或低盐输入的Dahl盐抵抗大鼠及低盐输入的Dahl盐敏感大鼠)没有明显影响,但能明显放大高钠引起的DS大鼠的血压上升效应;(2)高盐在导致的DS大鼠血压升高的同时,能引起iNOS活性的明显升高。结论:iNOS是参与血液动力学调控的重要组成部分,尤其是在血压处于较高水平时,iNOS具有不容忽视的调节作用。     

低水平铅暴露人胎盘组织诱导型和内皮型一氧化氮合酶的表达及意义

目的探讨孕期低水平铅暴露对胎盘组织诱导型和内皮型一氧化氮合酶的表达及其与铅水平的关系。方法2005年2月至2006年12月孕期外周血铅水平大于30μg/l的67例孕妇,根据铅水平分组,血铅水平30μg/l-60μg/l的孕妇35例为A组;血铅水平在60μg/l-100μg/l的孕妇32例为B组。检测胎盘中一氧化氮合酶系统表达。结果低水平铅暴露下诱导型和内皮型一氧化氮合酶在各组胎盘中的表达均分布在合体滋养细胞、细胞滋养细胞、微小血管内皮细胞、蜕膜细胞、绒毛间纤维细胞(villus fibrocyte)的胞浆,定位显示无显著差异;高血铅组的表达水平与强度显著高于低铅水平组(P<0.05)。结论金属铅可诱导发育中胎盘组织诱导型和内皮型一氧化氮合酶产生,胎盘NOS酶活性上升,能够改善子宫-胎盘血循环障碍,抵御铅毒性,维持正常妊娠的进行。     

Adenovirus mediated endothelial nitric oxide synthase gene transfer prevents restenosis of vein grafts

The possibility of endothelial nitric oxide synthase (eNOS) gene transfer, which prevents restenosis of vein grafts, was explored in 16 goats. The recombinant adenoviral vector coding endothelial nitric oxide synthase (AdCMVeNOS) and adenoviral vector (AdCMV) were constructed. A total of 6 cm jugular vein was removed, cut into two equal lengths for vein grafts, and infected with AdCMVeNOS or AdCMV in vitro. One segment (2 cm) of each carotid artery was removed. The vein graft that had been infected with AdCMVeNOS was anastomosed to the right carotid artery, and the vein graft that had been infected with AdCMV was anastomosed to the left. The functional expression of eNOS in vein grafts was assessed by the immunohistochemical staining and measurement of NO concentration. The inhibition of intimal hyperplasia in vein grafts was evaluated by the assay of 3H-TDR incorporation, histologic analysis, measurement of intimal thickness, and percent area stenosis. Adenovirus mediated eNOS gene transfer to goat vein grafts resulted in functional transgene expression with increased NO release. Increased local NO production could inhibit intimal hyperplasia and increase the patent rate of vein grafts.     

Inducible nitric oxide synthase expression is increased in the brain in fatal cerebral malaria

AIMS: Nitric oxide (NO) has been hypothesized to play a major role in the pathogenesis of cerebral malaria caused by P. falciparum infection. NO may act as a local neuroactive mediator contributing to the coma of cerebral malaria (CM). We hypothesized that increased expression of inducible nitric oxide synthase (iNOS) may cause increased release of NO, and examined the expression and distribution of iNOS in the brain during CM. MATERIAL AND RESULTS: Brain tissues from fatal cases of cerebral malaria in Thai adults were examined using immunohistochemical staining to detect iNOS. The distribution and strength of staining was compared between 14 patients with CM, three of whom were recovering from coma, and controls. iNOS expression was found in endothelial cells, neurones, astrocytes and microglial cells in CM cases. There was also strong staining in macrophages surrounding ring haemorrhages. iNOS staining was decreased in recovering malaria cases compared to acute CM, and was low in controls. Quantification showed a significant association between the intensity and number of iNOS positive vessels with the severity of malaria related histopathological changes, although the total number of cells staining was not increased compared to recovering CM cases. CONCLUSIONS: This study indicates that an acute induction of iNOS expression occurs in the brain during CM. This occurs in a number of different cells types, and is increased in the acute phase of CM compared to cases recovering from coma. As NO may activate a number of secondary neuropathological mechanisms in the brain, including modulators of synaptic function, induction of iNOS expression in cerebral malaria may contribute to coma, seizures and death.     

Inducible nitric oxide synthase activity correlates with lymphangiogenesis and vascular endothelial growth factor-C expression in head and neck squamous cell carcinoma

Nitric oxide (NO) is a diatomic free radical molecule that has been implicated in tumour angiogenesis and progression of head and neck squamous cell carcinoma (HNSCC). However, the mechanism underlying the effect of NO on tumour spread remains largely unknown. Tumour lymphangiogenesis has recently received considerable attention and there is increasing evidence that it is relevant for metastasis to lymph nodes in HNSCC. Here, we study the correlation between inducible NOS synthase (iNOS) activity and lymphangiogenesis in a series of 60 HNSCCs and the possible involvement of the lymphangiogenic factor vascular endothelial growth factor (VEGF)-C. HNSCC presenting with lymph node metastasis had a significantly higher lymphatic vessel density in both the tumour mass and the peritumour area (p = 0.006 and p = 0.001, respectively). Similarly, tumours with lymph node metastasis showed greater lymphatic vessel area than tumours with no lymph node involvement (p = 0.001 for intratumour lymphatics and p < 0.001 for peritumour lymphatics). iNOS activity measured in specimens from the tumour periphery correlated strongly with both lymphatic vessel density and lymphatic vessel area (p = 0.01, rs = 0.45 and p < 0.001, rs = 0.725, respectively). Conversely, these correlations were not observed in specimens from the tumour core. In addition, VEGF-C mRNA expression was significantly elevated in tumours with high iNOS activity (p = 0.008, rs = 0.563), and VEGF-C expression correlated positively with the presence of lymph node metastases (p = 0.03). In vitro, in the A431 human squamous carcinoma cell line, exogenous and endogenous stimulation of the iNOS pathway led to up-regulation of VEGF-C, which was blocked by the NOS inhibitor L-NNA. Taken together, our results indicate that iNOS activity may promote lymphangiogenesis and spread to lymph nodes in HNSCC, with the possible involvement of VEGF-C.     

脑动脉内皮细胞缺氧时内皮型一氧化氮合酶基因表达的变化及蛋白激酶C的作用

目的：研究缺氧时脑动脉内皮细胞(CAECs)内皮型一氧化氮合酶(eNOS)基因表达的变化，并探讨可能的分子机制。方法： 分别采用RT-PCR和蛋白质免疫印迹技术检测原代培养的猪脑动脉内皮细胞缺氧2、6、12、24、48 h后eNOS mRNA和蛋白质表达的变化，并观察蛋白激酶C(PKC)抑制剂对缺氧24 h引起的eNOS mRNA和蛋白质变化的影响。加入转录抑制剂放线菌素D后观察缺氧24 h对eNOS mRNA稳定性的影响。结果：缺氧2 h后脑动脉内皮细胞eNOS mRNA和蛋白质表达均增加，12 h达到高峰，约分别为常氧组的2.5倍和2.0倍，缺氧48 h仍高于常氧组。缺氧对eNOS mRNA稳定性无明显影响。选择性PKC抑制剂BIM I(1 μmol/L)、G6983(1 μmol/L)均能降低缺氧24 h所引起的eNOS基因表达的上调。结论： 脑动脉内皮细胞缺氧时可通过PKC信号途径上调eNOS基因的表达，并可能由此介导缺氧时脑血管的扩张反应，发挥其神经保护作用。     

Inducible nitric oxide synthase expression in human colorectal cancer: correlation with tumor angiogenesis

To investigate the potential involvement of the nitric oxide (NO) pathway in colorectal carcinogenesis, we correlated the expression and the activity of inducible nitric oxide synthase (iNOS) with the degree of tumor angiogenesis in human colorectal cancer. Tumor samples and adjacent normal mucosa were obtained from 46 surgical specimens. Immunohistochemical expression of iNOS, vascular endothelial growth factor (VEGF), and CD31 was analyzed on paraffin-embedded tissue sections. iNOS activity and cyclic GMP levels were assessed by specific biochemical assays. iNOS protein expression was determined by Western blot analysis. iNOS and VEGF mRNA levels were evaluated using Northern blot analysis. Both iNOS and VEGF expressions correlated significantly with intratumor microvessel density (r(s) = 0.31, P = 0.02 and r(s) = 0.67, P < 0.0001, respectively). A significant correlation was also found between iNOS and VEGF expression (P = 0.001). iNOS activity and cyclic GMP production were significantly higher in the cancer specimens than in the normal mucosa (P < 0.0001 and P < 0.0001, respectively), as well as in metastatic tumors than in nonmetastatic ones (P = 0.002 and P = 0.04, respectively). Western and Northern blot analyses confirmed the up-regulation of the iNOS protein and gene in the tumor specimens as compared with normal mucosa. NO seems to play a role in colorectal cancer growth by promoting tumor angiogenesis.     

Inducible nitric oxide synthase is expressed in synovial fluid granulocytes

The objective of the study was to evaluate the NO-producing potential of synovial fluid (SF) cells. SF from 15 patients with arthritis was compared with blood from the same individuals and with blood from 10 healthy controls. Cellular expression of inducible nitric oxide synthase (iNOS) was analysed by flow cytometry. High-performance liquid chromatography was used to measure l-arginine and l-citrulline. Nitrite and nitrate were measured colourimetrically utilizing the Griess' reaction. Compared to whole blood granulocytes in patients with chronic arthritis, a prominent iNOS expression was observed in SF granulocytes (P < 0.001). A slight, but statistically significant, increase in iNOS expression was also recorded in lymphocytes and monocytes from SF. l-arginine was elevated in SF compared to serum (257 +/- 78 versus 176 +/- 65 micro mol/l, P = 0.008), whereas a slight increase in l-citrulline (33 +/- 11 versus 26 +/- 9 micro mol/l), did not reach statistical significance. Great variations but no significant differences were observed comparing serum and SF levels of nitrite and nitrate, respectively, although the sum of nitrite and nitrate tended to be elevated in SF (19.2 +/- 20.7 versus 8.6 +/- 6.5 micro mol/l, P = 0.054). Synovial fluid leucocytes, in particular granulocytes, express iNOS and may thus contribute to intra-articular NO production in arthritis.