Prevalence of endothelial nitric oxide synthase gene polymorphisms in patients with atopic asthma

BACKGROUND: Asthma is a common multifactorial disease, the aetiology of which is attributable to both environmental and genetic factors. The endothelial nitric oxide synthase (NOS3) gene has been implicated in asthma pathogenesis. OBJECTIVE: This study investigated associations of 27 base-pair tandem repeat polymorphism in intron 4 and the Glu298Asp (G894T) variant of the NOS3 gene with atopic asthma in a Czech population. METHODS: Polymerase chain reaction was used to determine the NOS3 genotypes in subjects with atopic asthma (n = 163) and random controls (n = 209). RESULTS: The NOS3 allele or genotype distributions did not differ significantly between the control and asthma groups. However, the common genotype (bb) of the NOS3 polymorphism in intron 4 was found to be significantly associated with total IgE levels (P = 0.006), specific IgE levels for feathers (P = 0.0002) and a positive skin prick test for hay (P = 0.004). In one atopic patient, we identified an additional 27-bp repeat in the NOS3 gene (NOS3c), which occurred in heterozygous combination with the NOS3b allele (NOS3b/c genotype). In addition, we describe a new polymorphism (A5495G) in the NOS3 gene, which was in almost complete linkage disequilibrium with the NOS3 repeat polymorphism in intron 4. The Glu298Asp variant was not associated with asthma and/or related atopic phenotypes in our study. CONCLUSION: Neither the NOS3 'b' allele nor the NOS3 'b/b' genotype showed any general association with atopic asthma, but they were associated with atopy-related phenotypes. We conclude that the NOS3 gene polymorphisms may act as disease modifiers in atopic asthma phenotype in our population.     

Gene polymorphisms of endothelial nitric oxide synthase and angiotensin-converting enzyme in patients with asthma

BACKGROUND: Nitric oxide, including that produced by endothelial constitutive nitric oxide synthase (ecNOS), may regulate vascular and airway tone in the lungs and may influence various aspects of airway homeostasis. Angiotensin-converting enzyme (ACE) is expressed at high levels in the lungs and plays a role in the metabolism of angiotensin II, bradykinin, and substance P, all of which are potentially involved in the pathogenesis of asthma. An insertion-deletion polymorphism of the ACE gene has been shown to be associated with enzyme activity levels of ACE. To examine the possible involvement of the ecNOS and/or ACE genes as the genetic basis of bronchial asthma, we investigated whether there was any association between bronchial asthma and polymorphisms of the ecNOS and/or ACE genes. METHODS: A total of 310 patients with bronchial asthma and 121 healthy subjects took part in this study. The ecNOS and ACE genotypes were determined in all subjects by polymerase chain reaction. RESULTS: 1) The distribution of one genotype (bb) of ecNOS was significantly higher in the asthma group than in the control population. 2) The ACE genotype distribution was not significantly different between the control and the asthma groups. 3) In asthmatic patients, the ACE and ecNOS genotype distribution did not differ significantly among groups of patients with different severities of asthma. CONCLUSIONS: These results suggest that polymorphisms of the ecNOS gene, but not the ACE gene, may be associated with the development of asthma. However, the severity of asthma may not be influenced by polymorphisms of the ecNOS and ACE genes.     

Protective role of endothelial nitric oxide synthase

Nitric oxide is a versatile molecule, with its actions ranging from haemodynamic regulation to anti-proliferative effects on vascular smooth muscle cells. Nitric oxide is produced by the nitric oxide synthases, endothelial NOS (eNOS), neural NOS (nNOS), and inducible NOS (iNOS). Constitutively expressed eNOS produces low concentrations of NO, which is necessary for a good endothelial function and integrity. Endothelial derived NO is often seen as a protective agent in a variety of diseases.This review will focus on the potential protective role of eNOS. We will discuss recent data derived from studies in eNOS knockout mice and other experimental models. Furthermore, the role of eNOS in human diseases is described and possible therapeutic intervention strategies will be discussed.     

Neuronal nitric oxide synthase regulates endothelial inflammation

NO, produced by the endothelium, is a modulator of vascular inflammation. Traditionally, eNOS was believed to be the primary source of NO in the endothelium. However, recent data suggest an important role for nNOS in the endothelium, although little is known about factors regulating this novel eNOS. We examined the localization, regulation, and significance of endothelial nNOS in this study. Primary HUVECs were used as a model system. Inflammatory changes were induced by stimulation with TNF. We report that unlike eNOS, nNOS is predominantly localized to the nucleus of resting endothelial cells. This nNOS also contributed to basal NO production in the resting endothelium. Ablation of endothelial nNOS by pharmacological inhibition (using L-NPA) or siRNA further enhanced cytokine-mediated inflammatory responses, such as up-regulation of VCAM-1 and proinflammatory cytokines, as well as increased leukocyte recruitment. Based on these findings, we suggest a potential anti-inflammatory role of endothelial nNOS that can attenuate unopposed, proinflammatory cytokine actions. Our data indicate a novel location and an immunoregulatory role for nNOS in the endothelium.     

Association analysis of CA repeat polymorphism of the endothelial nitric oxide synthase gene with essential hypertension in Japanese

The nitric oxide synthase (NOS) gene is thought to be associated with essential hypertension (EH), because NO is implicated in endothelium-mediated vasodilation. We investigated the possible association between the alleles of simple tandem repeat DNA polymorphism of the endothelial constitutive NOS (cNOS) gene and EH in Japanese subjects. In all, 100 patients with EH and 123 subjects with normal blood pressure were studied. Polymerase chain reaction was used to amplify the CA repeat site in the endothelial cNOS gene and alleles based on the CA repeat number were determined. The allele frequencies in the hypertensive group and normotensive group were then compared. Twenty-three alleles were identified in this study of Japanese subjects. The overall distributions of allele frequencies in the two groups were not significantly different. However, comparing the allele frequencies in the EH group without left ventricular hypertrophy (LVH) and the normotensive group, the overall distributions were significantly different (p = 0.019). The 33-repeat allele was found more frequently in the EH group without LVH than in the normotensive group (p = 0.000047, Odds ratio = 3.71). In conclusion, the 33-repeat allele of the endothelial cNOS gene is associated with EH without LVH, and may be a genetic marker of EH in Japanese subjects.     

Polymorphisms of the endothelial nitric oxide synthase gene in premenopausal women with polycystic ovary syndrome

Objectives

To investigate the association of two common genetic polymorphisms of the gene encoding for endothelial nitric oxide synthase (Nos3), the enzyme catalyzing the production of nitric oxide (NO), with occurrence of the polycystic ovary syndrome (PCOS).

Methods

In a prospective case–control study, we analyzed 2 polymorphisms of the Nos3 gene cluster (Nos 3 exon 7 Glu298Asp and 27-base pair repeat in intron 4 of Nos3) in a series of 210 premenopausal Caucasian women with PCOS and 171 healthy controls using pyrosequencing and PCR, respectively. Women completed a detailed questionnaire and underwent a peripheral venous puncture, ultrasonography, and a standardized oral glucose tolerance test (OGTT).

Results

Genotype frequencies were not significantly different among women with PCOS and controls for the exon 7Nos3 and the intron 4Nos3 polymorphism (p = 0.3 and 0.2, respectively).

Conclusions

In our series, two common polymorphisms of the Nos3 gene cluster were not associated with occurrence of PCOS.     

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.     

人内皮型一氧化氮合酶基因在小鼠肺内的表达

目的 观察人内皮型一氧化氮合酶(heNOS)重组腺病毒感染小鼠肺组织后heNOS基因在小鼠肺组织内的表达.方法 采用反转录PCR(RT-PCR)法克隆heNOS基因,利用体外连接法构建复制缺陷型heNOS重组腺病毒表达载体;免疫组织化学分析heNOS在小鼠肺组织内的表达情况.结果 (1)序列分析表明,克隆所得heNOS基因片段含有完整开放阅读框架,与GenBank中heNOS DNA序列(*163729)同源性达99.93%.(2)该基因在转染细胞中能表达具有生物活性功能的NOS蛋白.(3)体外连接法成功构建出复制缺陷型heNOS重组腺病毒转移载体.(4)滴入heNOS重组腺病毒后,小鼠肺血管内皮细胞、平滑肌细胞、细支气管上皮细胞内heNOS阳性表达明显增加.结论 本实验成功构建的复制缺陷型heNOS重组腺病毒转移载体,能经呼吸道转移至肺组织,并在小鼠肺血管、支气管和肺泡上皮细胞表达所需的目的 蛋白heNOS.     

Association of plasma nitric oxide concentration and endothelial nitric oxide synthase T-786C gene polymorphism in coronary artery disease

Nitric oxide (NO) is synthesized from l-arginine by endothelium nitric oxide synthase (NOS3) and plays important roles in many physiologic and pathologic processes. NO involved in the pathogenesis of coronary atherosclerosis. In the present study we hypothesized that polymorphisms of NOS gene might be associated with increased risk of coronary artery disease (CAD) and plasma NO concentrations. The eNOS gene polymorphism was investigated in 241 unrelated CAD patients with positive coronary angiograms and 261 ages matched control subjects without a history of symptomatic CAD. The NOS3 gene polymorphisms were analyzed by RFLP. Plasma NO, lipid profile and other risk factors were also assessed. The genotype frequencies for T-786C polymorphism differed significantly between CAD patients and controls (p=0.041). The mean plasma NO(x) concentrations showed significant differences according to genotypes of T-786C polymorphism in total population only. The mean plasma NO(x) increased in those individuals that are homozygote for C allele in promoter compared with those individuals are heterozygote for this allele and homozygote for T allele in total population and Controls, but no in CAD patients. The present study provides evidences that T-786C polymorphism of the NOS3 gene is associated with CAD. T-786C polymorphism was not associated with increased plasma NO in CAD patients.     

Susceptible and protective endothelial nitric oxide synthase gene polymorphism in alopecia areata in the Kuwaiti population

Alopecia areata (AA) is a chronic inflammatory disease with evidence of T-cell involvement that causes hair follicle "immune privilege collapse". Nitric monoxide was shown to contribute in the pathogenesis of AA. We are investigating evidence for the association of eNOS gene polymorphism with AA. Genomic DNA was extracted from 176 subjects, 87 Kuwaiti AA patients and 89 matched (for ethnicity, gender and age) healthy controls. A variable number tandem repeat (VNTR) located in intron-4 of the eNOS gene consisting of either four or five (27-base pair) repeats was analyzed by polymerase chain reaction and electrophoresis fragment analysis using ABI 3100 genetic analyzer. Haploview and GenePOP software were used for data analysis. A significant association was found between the intron-4 27 bp-VNTR and AA, where 4b was identified as the risk allele had (chi(2) = 4.42, p = 0.035, OR = 2.03). Genotype (4b/4b) showed a significant association with susceptibility to AA and have a frequency of 22% higher in AA patients than in healthy controls (71 vs 49%) and a chi(2) = 6.39, (p = 0.011, OR = 2.63). We report a significant association of a polymorphism within the eNOS gene and susceptibility to AA.     

Susceptible and protective endothelial nitric oxide synthase gene polymorphism in alopecia areata in the Kuwaiti population

Alopecia areata (AA) is a chronic inflammatory disease with evidence of T-cell involvement that causes hair follicle “immune privilege collapse”. Nitric monoxide was shown to contribute in the pathogenesis of AA. We are investigating evidence for the association of eNOS gene polymorphism with AA. Genomic DNA was extracted from 176 subjects, 87 Kuwaiti AA patients and 89 matched (for ethnicity, gender and age) healthy controls. A variable number tandem repeat (VNTR) located in intron-4 of the eNOS gene consisting of either four or five (27-base pair) repeats was analyzed by polymerase chain reaction and electrophoresis fragment analysis using ABI 3100 genetic analyzer. Haploview and GenePOP software were used for data analysis. A significant association was found between the intron-4 27 bp-VNTR and AA, where 4b was identified as the risk allele had (χ² = 4.42, p = 0.035, OR = 2.03). Genotype (4b/4b) showed a significant association with susceptibility to AA and have a frequency of 22% higher in AA patients than in healthy controls (71 vs 49%) and a χ² = 6.39, (p = 0.011, OR = 2.63). We report a significant association of a polymorphism within the eNOS gene and susceptibility to AA.     

Endothelial nitric oxide synthase transgenic models of endothelial dysfunction

Endothelial production of nitric oxide is critical to the regulation of vascular responses, including vascular tone and regional blood flow, leukocyte–endothelial interactions, platelet adhesion and aggregation, and vascular smooth muscle cell proliferation. A relative deficiency in the amount of bioavailable vascular NO results in endothelial dysfunction, with conditions that are conducive to the development of atherosclerosis: thrombosis, inflammation, neointimal proliferation, and vasoconstriction. This review focuses on mouse models of endothelial dysfunction caused by direct genetic modification of the endothelial nitric oxide synthase (eNOS) gene. We first describe the cardiovascular phenotypes of eNOS knockout mice, which are a model of total eNOS gene deficiency and thus the ultimate model of endothelial dysfunction. We then describe S1177A and S1177D eNOS mutant mice as mouse models with altered eNOS phosphorylation and therefore varying degrees of endothelial dysfunction. These include transgenic mice that carry the eNOS S1177A and S1177D transgenes, as well as knockin mice in which the endogenous eNOS gene has been mutated to carry the S1177A and S1177D mutations. Together, eNOS knockout mice and eNOS S1177 mutant mice are useful tools to study the effects of total genetic deficiency of eNOS as well as varying degrees of endothelial dysfunction caused by eNOS S1177 phosphorylation.     

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。     

内皮型一氧化氮合酶基因转染对血管损伤后新生内膜增生的影响

背景：一氧化氮能够抑制血管平滑肌细胞的迁移和增殖,而一氧化氮合酶是其合成的关键酶,有关一氧化氮合酶基因体内转染对平滑肌细胞及动脉粥样硬化血管损伤后内膜增生影响少有报道。 目的：观察内皮型一氧化氮合酶 (endothelial nitric oxide synthase,eNOS)基因体内局部转染对动脉粥样硬化大鼠血管损伤后新生内膜增生的抑制作用。 方法：建立动脉粥样硬化Wistar大鼠颈动脉球囊损伤模型,建模后随机分成空白对照组、AdCMV-lacz对照组和AdCMV-eNOS组,分别将PBS,AdCMV-lacz和AdCMV-eNOS体内转染至以上3组大鼠的损伤血管壁。转染2周后培养并鉴定损伤局部平滑肌细胞,并用RT-PCR法检测各组损伤及转染后血管平滑肌细胞eNOS mRNA的表达,同时观察转染后不同时期新生内膜增生的影响。 结果与结论：AdCMV-eNOS组的颈总动脉血管平滑肌细胞可表达eNOS mRNA。3组大鼠转染后1和3个月,AdCMV-eNOS组内膜/中膜面积比值低于空白对照组和AdCMV-lacz对照组(P < 0.01)。结果显示,eNOS基因体内转染损伤后血管可以抑制血管新生内膜增生,减少再狭窄发生率。     

哮喘模型大鼠肺组织一氧化氮合酶的分布

研究一氧化氮 (NO)在哮喘大鼠肺组织中的作用。采用组化法观察一氧化氮合酶 (NOS)在大鼠哮喘模型肺组织中的分布 ,应用免疫组化法观察大鼠哮喘模型气道mIL 2R+ 细胞变化。结果显示 ,哮喘大鼠肺NADPH染色呈强阳性 ,并波及肺泡膈。肺组织中NOS含量明显高于对照组 [哮喘组 (37 44± 0 77)pmol/mg,对照组 (8 73± 0 79)pmol/mg],气道炎性细胞增多 ,特别是mIL 2R+ 细胞 [哮喘组mIL 2R+ 细胞为 (2 3 8± 7 9)个 ,对照组为 0个 ],而NOS抑制剂DMA组气道炎性细胞少 ,NADPH呈阴性。提示NO是哮喘大鼠的炎性效应分子。     

Tissue-engineered blood vessels with endothelial nitric oxide synthase activity

Nondegradable synthetic polymer vascular grafts used in cardiovascular surgery have shown serious shortcomings, including thrombosis, calcification, infection, and lack of growth potential. Tissue engineering of vascular grafts with autologous stem cells and biodegradable polymeric materials could solve these problems. The present study is aimed to develop a tissue-engineered vascular graft (TEVG) with functional endothelium using autologous bone marrow-derived cells (BMCs) and a hybrid biodegradable polymer scaffold. Hybrid biodegradable polymer scaffolds were fabricated from poly(lactide-co-epsilon-caprolactone) (PLCL) copolymer reinforced with poly(glycolic acid) (PGA) fibers. Canine bone marrow mononuclear cells were induced in vitro to differentiate into vascular smooth muscle cells and endothelial cells. TEVGs (internal diameter: 10 mm, length: 40 mm) were fabricated by seeding vascular cells differentiated from BMCs onto PGA/PLCL scaffolds and implanted into the abdominal aorta of bone marrow donor dogs (n = 7). Eight weeks after implantation of the TEVGs, the vascular grafts remained patent. Histological and immunohistochemical analyses of the vascular grafts retrieved at 8 weeks revealed the regeneration of endothelium and smooth muscle and the presence of collagen. Western blot analysis showed that endothelial nitric oxide synthase (eNOS) was expressed in TEVGs comparable to native abdominal aortas. This study demonstrates that vascular grafts with significant eNOS activity can be tissue-engineered with autologous BMCs and hybrid biodegradable polymer scaffolds.     

一氧化氮合酶3基因多态性与复发性早期自然流产的关系

目的探讨一氧化氮合酶3(nitric oxide synthase 3,NOS3)基因第4内含子27bp数目可变串联重复序列(variable number of tandem repeat,VNTR)多态性和第7外显子894(G/T)多态性与复发性早期自然流产(recurrent early spontaneous abortion,RESA)的相关性。方法选取140例RESA患者和140名健康妇女,应用聚合酶链反应-琼脂糖凝胶电泳法检测NOS3基因第4内含子VNTR多态性,聚合酶链反应-限制性片段长度多态性分析技术检测第7外显子894(G/T)多态性。结果RESA组aa ba基因型频率和a等位基因频率与正常对照组相比差异有统计学意义(χ2=4.51,P<0.05;χ2=4.29,P<0.05)。与bb基因型相比,携带a等位基因的妇女与RESA显著相关(OR为1.8,95%CI:1.04~3.24)。RESA组TT GT基因型频率和T等位基因频率与正常对照组相比差异无统计学意义(χ2=1.16,P>0.05;χ2=1.12,P>0.05)。与GG基因型相比,携带T等位基因的妇女与RESA无相关。结论NOS3基因第4内含子27bp数目可变的串联重复序列多态性与复发性自然流产密切相关,NOS3基因第7外显子894G/T多态性与RESA无明显相关性,a等位基因是RESA重要的遗传易感基因。