不对称二甲基精氨酸与动脉粥样硬化

动脉粥样硬化 (AS)是一种多因素所致的动脉血管慢性疾病 ,集中表现为全身性血管内皮功能障碍(endothelialdysfunction)、血管内膜慢性炎症、纤维增生、管腔狭窄及血栓形成等。近来研究表明 ,以一氧化氮 (NO)生物利用度降低为主要生化表现的血管内皮功能障碍是促使AS发生的始动因素。AS高危人群如高胆固醇血症、高半胱氨酸血症、糖尿病、高血压、慢性肾衰患者均存在血管内皮功能障碍。不对称二甲基精氨酸 (ADMA)是新发现的一种内源性NO合成抑制剂 ,血浆ADMA水平升高是引起血管内皮功能障碍的重要原因。ADMA可能是一种前致动脉粥样硬…     

高尿酸血症与动脉粥样硬化的相关性及其诱发机制研究

目的 明确高尿酸血症是否通过诱导内皮型一氧化氮合酶(eNOS)脱偶联导致内皮细胞发生氧化应激造成内皮损伤.方法 (1)体外培养人脐静脉内皮细胞(HUVEC),以不同浓度的尿酸(0、200、400、600、800 μmol/L)作用24h、48h和72h,DHE荧光染色法测定ROS水平,NO试剂盒测定上清液NO含量,观察...     

芝麻素对2型糖尿病大鼠主动脉内皮功能的保护作用

目的探讨芝麻素改善2型糖尿病大鼠主动脉内皮功能损伤的作用及可能机制。方法采用长期高脂饮食加小剂量链脲佐菌素(streptozotocin,STZ)建立2型糖尿病大鼠模型。灌服不同剂量芝麻素(120、60 mg.kg-1.d-1)8周后处死动物。离体血管灌流法测大鼠主动脉内皮依赖性舒张反应及NO生物活性,测血清丙二醛(malondialdehyde,MDA)含量和总抗氧化能力(total antioxidative capacity,T-AOC),Western blot测主动脉内皮型一氧化氮合酶(endothelial nitricoxide synthase,eNOS)、硝基酪氨酸(nitrotyrosine,NT)和还原型辅酶Ⅱ(NADPH)氧化酶亚基P47phox蛋白表达。结果与模型组相比,芝麻素(120 mg.kg-1.d-1)组内皮依赖性血管舒张功能增强,NO活性升高;血清MDA含量降低,T-AOC水平升高;主动脉eNOS蛋白表达增高,NT和P47phox蛋白表达降低。结论芝麻素可改善糖尿病大鼠血管内皮功能,其机制与上调血管eNOS表达和减轻NO氧化失活有关。     

内皮型一氧化氮和酶及其抑制剂与2型糖尿病血管内皮功能

内皮型一氧化氮和酶(eNOS)是已知最重要的内源性血管舒张因子,其竞争性抑制剂非对称性二甲基精氨酸(ADMA),可抑制一氧化氮(NO)的合成,使NO/NOS通路发生障碍,NO合成减少.2型糖尿病(T2DM)内皮功能紊乱与氧化应激有关,内皮细胞的增殖及凋亡、缺氧/复氧损伤和NO介导的内皮舒张功能障碍均涉及eNOS和ADMA的变化.     

不对称性二甲基精氨酸与脑梗死的相关性研究

内皮依赖性血管舒张反应功能障碍是动脉粥样硬化的特征性表现.一氧化氮(nitric oxide,NO)对维持内皮正常功能有重要作用.不对称性二甲基精氨酸(asymmetric dimethylarginine,ADMA)为一氧化氮合酶(nictric oxide synthase,NOS)抑制剂,它能竞争性抑制NOS活性,减少NO生成.在多种病理生理状况下ADMA水平显著升高,内皮细胞NOS活性降低,NO合成减少,导致血管内皮功能不全~([1]).本研究旨在通过检测脑梗死患者ADMA水平,探讨ADMA与脑梗死的相关性.     

扇贝裙边糖胺聚糖对OX-LDL致血管内皮细胞损伤的保护作用

目的　研究扇贝裙边提取物糖胺聚糖 (SS GAG)对血管内皮细胞的保护作用 ,进而探讨其抗动脉粥样硬化 (AS)作用机制。方法　本研究采用氧化低密度脂蛋白 (OX LDL)建立体外培养的人脐静脉血管内皮细胞株 (HUVEC ,CRL 2 4 80 )损伤模型 ,用MTT法和化学方法分别从细胞和分子水平观察SS GAG对血管内皮细胞增殖活性及细胞内一氧化氮 (NO)水平和内皮型一氧化氮合酶 (eNOS)活性的影响。结果　OX LDL能明显抑制血管内皮细胞的增殖活性 (P <0 0 1) ,降低血管内皮细胞内NO水平及eNOS的活性 (P <0 0 1) ;用SS GAG(终浓度为 5 0 ,10 0 ,2 0 0mg·L-1)预处理后 ,能逆转上述效应 (P <0 0 1)。结论　OX LDL抑制血管内皮细胞的增殖 ,降低内皮型eNOS的活性 ,减少细胞内NO的生成 ;SS GAG对血管内皮细胞的脂质过氧化物损伤有保护作用。     

乐卡地平对原发性高血压大鼠动脉粥样硬化血管内皮功能的影响

目的观察乐卡地平对原发性高血压大鼠动脉粥样硬化血管内皮的保护作用,探讨乐卡地平抗动脉粥样硬化的机制。方法采用大剂量维生素D3联合高脂饲料饲养的方法建立原发性高血压大鼠动脉粥样硬化模型。将20只8周龄雄性原发性高血压大鼠分为原发性高血压大鼠动脉粥样硬化模型组(模型组)和乐卡地平治疗组(治疗组),10只8周龄WKY大鼠设为空白对照组(对照组)。于实验结束时,取血检测一氧化氮(NO)水平和一氧化氮合酶(NOS)活性,并取胸主动脉下段做HE和Vonkossa染色,进行病理形态学观察。结果模型组大鼠的血清NO水平及诱导型NOS(iNOS)、内皮型NOS(eNOS)活性与对照组间差异均有统计学意义(P<0.01);治疗组大鼠的血清iNOS、eNOS活性与模型组间差异有统计学意义(P<0.05)。结论乐卡地平可升高eNOS活性,降低iNOS活性,促进内皮源性NO释放,改善内皮依赖性舒张功能,这可能是发挥其抗动脉粥样硬化作用的机制之一。     

普罗布考对高血压合并脑梗死患者氧化型低密度脂蛋白水平的影响*

【摘要】 目的 探讨急性脑梗死患者氧化型低密度脂蛋白(ox-LDL)水平的特点及ox-LDL致脑梗死的发病机制,评价普罗布考对ox-LDL及血管功能的改善作用。方法 120例急性脑梗死患者根据是否合并高血压分为血压正常组和合并高血压组各60例,再将2个病例组分别随机分为干预组和未干预组,每组30例,干预组患者在常规治疗的基础上加用普罗布考治疗,所有患者分别于治疗前,治疗后2周、12周检测ox-LDL、内皮型一氧化氮合酶(eNOS)及NO水平。结果脑梗死合并高血压组的ox-LDL高于正常血压组,eNOS、NO水平低于正常血压组。对于脑梗死正常血压患者,普罗布考干预治疗12周后,TC、HDL、ox-LDL水平降低,eNOS升高(P<0.05)。对于脑梗死合并高血压患者,普罗布考干预治疗12周后,TC、TG、LDL、ox-LDL水平降低,eNOS、NO升高(P<0.05)。结论 普罗布考除具有调脂作用外,还可降低ox-LDL水平,改善血管内皮功能,稳定斑块,有利于防止发生动脉粥样硬化。     

同型半胱氨酸对内皮细胞一氧化氮合酶活力及基因表达的影响

目的 观察同型半胱氨酸(Hcy)对培养的人脐静脉内皮细胞(HUVEC)一氧化氮合酶(eNOS)活力及其基因表达的动态影响.方法 10、30、100、300 μmol · L-1Hcy与HUVEC分别培养24、48、72 h后,用HPLC测定细胞内不对称二甲基精氨酸(ADMA)的含量,反转录聚合酶链反应(RT-PCR)检测细胞内eNOS mRNA的表达,并分别测定细胞二甲基精氨酸二甲基氨基水解酶(DDAH)、eNOS的活力和NO的含量.结果 HUVEC经不同浓度Hcy分别处理24、48、72 h后,其细胞内ADMA聚积增多,DDAH活性和eNOS活力降低,NO生成减少,且呈时间和浓度依赖性.但只有100 μmol · L-1 Hcy与HUVEC作用72 h时,才引起eNOS mRNA表达的减少.结论 Hcy对内皮功能的损伤可能通过抑制DDAH活性,引起ADMA聚积,从而降低eNOS活力,导致NO生成减少.此外,eNOS mRNA表达的抑制也是Hcy诱导的内皮功能障碍的机制之一.     

乐卡地平对原发性高血压大鼠动脉粥样硬化血管内皮功能的影响

目的 观察乐卡地平对原发性高血压大鼠动脉粥样硬化血管内皮的保护作用,探讨乐卡地平抗动脉粥样硬化的机制.方法 采用大剂量维生素D3联合高脂饲料饲养的方法建立原发性高血压大鼠动脉粥样硬化模型.将20只8周龄雄性原发性高血压大鼠分为原发性高血压大鼠动脉粥样硬化模型组(模型组)和乐卡地平治疗组(治疗组),10只8周龄WKY大鼠设为空白对照组(对照组).于实验结束时,取血检测一氧化氮(NO)水平和一氧化氮合酶(NOS)活性,并取胸主动脉下段做HE和Von kossa染色,进行病理形态学观察.结果 模型组大鼠的血清NO水平及诱导型NOS(iNOS)、内皮型NOS(eNOS)活性与对照组间差异均有统计学意义(P＜0.01);治疗组大鼠的血清iNOS、eNOS活性与模型组间差异有统计学意义(P＜0.05).结论 乐卡地平可升高eNOS活性,降低iNOS活性,促进内皮源性NO释放,改善内皮依赖性舒张功能,这可能是发挥其抗动脉粥样硬化作用的机制之一.     

Nitric oxide therapies in vascular diseases.

Endothelial dysfunction defined as the impaired ability of vascular endothelium to stimulate vasodilation plays a key role in the development of atherosclerosis and in various pathological conditions which predispose to atherosclerosis, such as hypercholesterolemia, hypertension, type 2 diabetes, hyperhomocyst (e) inemia and chronic renal failure. The major cause of the endothelial dysfunction is decreased bioavailability of nitric oxide (NO), a potent biological vasodilator produced in vascular endothelium from L-arginine by the endothelial NO synthase (eNOS). In vascular diseases, the bioavailability of NO can be impaired by various mechanisms, including decreased NO production by eNOS, and/or enhanced NO breakdown due to increased oxidative stress. The deactivation of eNOS is often associated with elevated plasma levels of its endogenous inhibitor, N(G) N(G)-dimethyl-L-arginine (ADMA). In hypercholesterolemia, a systemic deficit of NO may also increase the levels of low density lipoproteins (LDL) by modulating its synthesis and metabolism by the liver, as suggested by recent in vivo and in vitro studies using organic NO donors. Therapeutic strategies aiming to reduce the risk of vascular diseases by increasing bioavailability of NO continue to be developed. Cholesterol-lowering drugs, statins, have been shown to improve endothelial function in patients with hypercholesterolemia and atherosclerosis. Promising results were also obtained in some, but not all, vascular diseases after treatment with antioxidant vitamins (C and E) and after administration of eNOS substrate, L-arginine, or its cofactor, tetrahydrobiopterin (BH(4)). Novel strategies, which may produce beneficial changes in the vascular endothelium, include the use of natural extracts from plant foods rich in phytochemicals.     

The effects of modulating eNOS activity and coupling in ischemia/reperfusion (I/R)

The in vivo role of endothelial nitric oxide synthase (eNOS) uncoupling mediating oxidative stress in ischemia/reperfusion (I/R) injury has not been well established. In vitro, eNOS coupling refers to the reduction of molecular oxygen to L-arginine oxidation and generation of L-citrulline and nitric oxide NO synthesis in the presence of an essential cofactor, tetrahydrobiopterin (BH(4)). Whereas uncoupled eNOS refers to that the electron transfer becomes uncoupled to L-arginine oxidation and superoxide is generated when the dihydrobiopterin (BH(2)) to BH(4) ratio is increased. Superoxide is subsequently converted to hydrogen peroxide (H(2)O(2)). We tested the hypothesis that promoting eNOS coupling or attenuating uncoupling after I/R would decrease H(2)O(2)/increase NO release in blood and restore postreperfused cardiac function. We combined BH(4) or BH(2) with eNOS activity enhancer, protein kinase C epsilon (PKC ε) activator, or eNOS activity reducer, PKC ε inhibitor, in isolated rat hearts (ex vivo) and femoral arteries/veins (in vivo) subjected to I(20 min)/R(45 min). When given during reperfusion, PKC ε activator combined with BH(4), not BH(2), significantly restored postreperfused cardiac function and decreased leukocyte infiltration (p?相似文献   

Inhibition of nitric oxide synthase uncoupling by sepiapterin improves left ventricular function in streptozotocin-induced diabetic mice

1. Uncoupling of nitric oxide synthase (NOS) has been implicated in the pathogenesis of left ventricular (LV) dysfunction in diabetes mellitus. In the present study, we investigated the role of NOS uncoupling in oxidative/nitrosative stress and LV dysfunction in the diabetic mouse heart. 2. Diabetes was induced in wild-type (WT), endothelial (e) NOS knockout (eNOS(-/-)), inducible (i) NOS knockout (iNOS(-/-)) and neuronal (n) NOS knockout (nNOS(-/-)) mice by streptozotocin (STZ) treatment. 3. In the diabetic heart, iNOS, but not eNOS or nNOS, expression was increased. Levels of malondialdehyde (MDA), 4-hydroxy-noneal (HNE) and nitrotyrosine (NT), as markers of oxidative/nitrosative stress, were increased in the diabetic mouse heart, but the increase in oxidative/nitrosative stress was significantly repressed in the iNOS(-/-) diabetic mouse heart. Levels of nitrite and nitrate (NO(x)), as an index of nitric oxide, bioavailability were significantly decreased in the iNOS(-/-) diabetic mouse heart. 4. Oral administration of sepiapterin (10 mg/kg per day), a precursor of tetrahydrobiopterin (BH(4)), significantly increased BH(4) and the BH(4)/BH(2) ratio in diabetic mouse heart. Similarly, sepiapterin inhibited the formation of HNE, MDA and NT in diabetic hearts from all three genotypes, but the increase in NO(x) following sepiapterin treatment was significantly attenuated in the iNOS(-/-) diabetic mouse heart. Percentage fractional shortening (FS), evaluated by echocardiography, decreased significantly in all genotypes of diabetic mice. Sepiapterin significantly increased percentage FS in diabetic mice, except in iNOS(-/-) mice. 5. These results suggest that sepiapterin inhibits uncoupling of NOS and improves LV function presumably by increasing iNOS-derived nitric oxide in the diabetic heart.     

Nitric oxide dynamics and endothelial dysfunction in type II model of genetic diabetes

Although diabetes is a major risk factor for vascular diseases, e.g., hypertension and atherosclerosis, mechanisms that underlie the "risky" aspects of diabetes remain obscure. The current study is intended to examine the notion that diabetic endothelial dysfunction stems from a heightened state of oxidative stress induced by an imbalance between vascular production and scavenging of reactive oxygen/nitrogen species. Goto-Kakizaki (GK) rats were used as a genetic animal model for non-obese type II diabetes. Nitric oxide (NO) bioavailability and O2- generation in aortic tissues of GK rats were assessed using the Griess reaction and a lucigenin-chemiluminescence-based technique, respectively. Organ chamber-based isometric tension studies revealed that aortas from GK rats had impaired relaxation responses to acetylcholine whereas a rightward shift in the dose-response curve was noticed in the endothelium-independent vasorelaxation exerted by the NO donor sodium nitroprusside. An enhancement in superoxide (O2-) production and a diminuation in NO bioavailability were evident in aortic tissues of GK diabetic rats. Immunoblotting and high-performance liquid chromatography (HPLC)-based techniques revealed, respectively, that the above inverse relationship between O2- and NO was associated with a marked increase in the protein expression of nitric oxide synthase (eNOS) and a decrease in the level of its cofactor tetrahydrobiopterin (BH4) in diabetic aortas. Endothelial denudation by rubbing or the addition of pharmacological inhibitors of eNOS (e.g. N(omega)-nitro-L-arginine methyl ester (L-NAME)), and NAD(P)H oxidase (e.g. diphenyleneiodonium, apocynin) strikingly reduced the diabetes-induced enhancement in vascular O2- production. Aortic contents of key markers of oxidative stress (isoprostane F2alpha III, protein-bound carbonyls, nitrosylated protein) in connection with the protein expression of superoxide generating enzyme NAD(P)H oxidase (e.g. p47phox, pg91phox), a major source of reactive oxygen species in vascular tissue, were elevated as a function of diabetes. In contrast, the process involves in the vascular inactivation of reactive oxygen species exemplified by the activity of CuZnSOD was reduced in this diseased state. Our studies suggest that diabetes produces a cascade of events involving production of reactive oxygen species from the NADPH oxidase leading to oxidation of BH4 and uncoupling of NOS. This promotes the oxidative inactivation of NO with subsequent formation of peroxynitrite. An alteration in the balance of these bioactive radicals in concert with a defect in the antioxidant defense counteracting mechanism may favor a heightened state of oxidative stress. This phenomenon could play a potentially important role in the pathogenesis of diabetic endothelial dysfunction.     

Endothelial NO synthase as a source of NO and superoxide

Endothelial nitric oxide (NO) synthase (eNOS) is responsible for most of the vascular NO produced. A functional eNOS transfers electrons from nicotinamide adenine dinucleotide phosphate (NADPH) via flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) in the carboxy-terminal reductase domain to the heme in the amino-terminal oxygenase domain where the substrate L-arginine is oxidized to L-citrulline and NO. This normal flow of electrons requires dimerization of the enzyme, the presence of the substrate L-arginine, and presence of the cofactor (6R)-5,6,7,8-tetrahydro-L-biopterin (BH₄), one of the most potent naturally occurring reducing agents. Cardiovascular risk factors, such as hypertension, hypercholesterolemia, diabetes mellitus, or chronic smoking, stimulate the production of reactive oxygen species (ROS) in the vascular wall. NADPH oxidases represent major sources of this ROS and have been found upregulated in animal models of hypertension, diabetes, and sedentary lifestyle. Superoxide avidly interacts with vascular NO to form peroxynitrite (ONOO⁻). BH₄ is highly sensitive to oxidation, e.g., by ONOO⁻, and reduced levels of BH₄ promote eNOS uncoupling. In fact, in many cases, supplementation with BH₄ is capable of correcting eNOS dysfunction. Alternatively, an oxidation of the zinc-thiolate complex of eNOS by ONOO⁻ has been proposed as a mechanism for eNOS uncoupling. Under uncoupled conditions, superoxide is generated from the oxygenase domain of eNOS. eNOS uncoupling and its change from a protective enzyme to a contributor to oxidative stress has been observed in several in vitro models and in animals with cardiovascular pathophysiology such as spontaneously hypertensive rats (SHR), angiotensin-II-induced hypertension, or diabetes. Taken together, several mechanisms seem to underlie endothelial dysfunction, but an uncoupled eNOS markedly contributes to this phenomenon.

     

Cell type-specific recycling of tetrahydrobiopterin by dihydrofolate reductase explains differential effects of 7,8-dihydrobiopterin on endothelial nitric oxide synthase uncoupling

(6R)-5,6,7,8-Tetrahydro-l-biopterin (BH4) availability regulates nitric oxide and superoxide formation by endothelial nitric oxide synthase (eNOS). At low BH4 or low BH4 to 7,8-dihydrobiopterin (BH2) ratios the enzyme becomes uncoupled and generates superoxide at the expense of NO. We studied the effects of exogenously added BH2 on intracellular BH4/BH2 ratios and eNOS activity in different types of endothelial cells. Incubation of porcine aortic endothelial cells with BH2 increased BH4/BH2 ratios from 8.4 (controls) and 0.5 (BH4-depleted cells) up to ∼20, demonstrating efficient reduction of BH2. Uncoupled eNOS activity observed in BH4-depleted cells was prevented by preincubation with BH2. Recycling of BH4 was much less efficient in human endothelial cells isolated from umbilical veins or derived from dermal microvessels (HMEC-1 cells), which exhibited eNOS uncoupling and low BH4/BH2 ratios under basal conditions and responded to exogenous BH2 with only moderate increases in BH4/BH2 ratios. The kinetics of dihydrofolate reductase-catalyzed BH4 recycling in endothelial cytosols showed that the apparent BH2 affinity of the enzyme was 50- to 300-fold higher in porcine than in human cell preparations. Thus, the differential regulation of eNOS uncoupling in different types of endothelial cells may be explained by striking differences in the apparent BH2 affinity of dihydrofolate reductase.     

Targeting vascular redox biology through antioxidant gene delivery: a historical view and current perspectives

Oxidative stress, resulting from a deregulated equilibrium between superoxide and nitric oxide (NO) production, contributes to the progression of different vascular diseases such as atherosclerosis, hypertension, ischemia/reperfusion injury and restenosis. Despite disappointing results of various oral antioxidant treatment trials, promising findings have been reported using gene delivery of enzymes to improve NO bioavailability and decrease oxidative stress in animal models for vascular diseases. NO production can be increased by overexpression of endothelial NO synthase (eNOS) in the vascular wall. However, the complex regulation of NOS needs to be carefully considered in the context of gene therapy along with the availability of its cofactor tetrahydrobiopterin and eNOS uncoupling. Furthermore, preclinical studies demonstrated that gene delivery of antioxidative vascular wall-specific enzymes, such as heme oxygenase-1, superoxide dismutase, catalase and glutathione peroxidase, has the potential to attenuate oxidative stress and inhibit atherosclerosis. Another option is to transfect vascular disease patients with secreted antioxidants such as high density lipoprotein-associated enzymes or soluble scavenger receptors. The advantage of the latter is that gene delivery of these enzymes and receptors does not need to be endothelium specific. Nonetheless, techniques to deliver genes specifically to the vascular wall are under development and hold interesting perspectives for the treatment of vascular diseases in the future. The patents relevant to gene delivery are also discussed in this review article.     

Nitric oxide in coronary artery disease: effects of antioxidants

In this review article we examine the main mechanisms leading to decreased nitric oxide (NO) bioavailability, and we present the current strategies available to increase NO levels, mainly by using antioxidants in patients with coronary artery disease. Decreased NO bioavailability in the vasculature is a key feature of all the classic risk factors for atherosclerosis, and it can be the result of NO's decreased synthesis and increased oxidative deactivation. Increased NO synthesis can be achieved by improving the intracellular redox state in endothelial cells, stabilizing endothelial NO synthase (eNOS) dimers, and maintaining sufficient intracellular levels of eNOS substrate L-arginine. Antioxidant treatment may have a dual role by increasing NO synthesis and decreasing its oxidative deactivation. However, in patients with coronary artery disease, although intracoronary infusions of vitamins or chronic vitamin treatment improve endothelial function, their effect on clinical outcome is questioned. In conclusion, in coronary artery disease, NO bioavailability can be increased mainly by reversing the causes of endothelial dysfunction via treatment of classic risk factors, while the use of antioxidant vitamins is controversial and the ideal antioxidant strategy is still unknown.

     

Uncoupling of eNOS contributes to redox-sensitive leukocyte recruitment and microvascular leakage elicited by methylglyoxal

Elevated levels of the glycolysis metabolite methylglyoxal (MG) have been implicated in impaired leukocyte–endothelial interactions and vascular complications in diabetes, putative mechanisms of which remain elusive. Uncoupling of endothelial nitric oxide synthase (eNOS) was shown to be involved in endothelial dysfunction in diabetes. Whether MG contributes to these effects has not been elucidated. By using intravital microscopy in vivo, we demonstrate that MG-triggered reduction in leukocyte rolling velocity and increases in rolling flux, adhesion, emigration and microvascular permeability were significantly abated by scavenging reactive oxygen species (ROS). In murine cremaster muscle, MG treatment reduced tetrahydrobiopterin (BH4)/total biopterin ratio, increased arginase expression and stimulated ROS and superoxide production. The latter was significantly blunted by ROS scavengers Tempol (300 μM) or MnTBAP (300 μM), by BH4 supplementation (100 μM) or by NOS inhibitor N^G-nitro-l-arginine methyl ester (l-NAME; 20 μM). In these tissues and cultured murine and human primary endothelial cells, MG increased eNOS monomerization and decreased BH4/total biopterin ratio, effects that were significantly mitigated by supplementation of BH4 or its precursor sepiapterin but not by l-NAME or tetrahydroneopterin, indicative of MG-triggered eNOS uncoupling. MG treatment further decreased the expression of guanosine triphosphate cyclohydrolase I in murine primary endothelial cells. MG-induced leukocyte recruitment was significantly attenuated by supplementation of BH4 or sepiapterin or suppression of superoxide by l-NAME confirming the role of eNOS uncoupling in MG-elicited leukocyte recruitment. Together, our study uncovers eNOS uncoupling as a pivotal mechanism in MG-induced oxidative stress, microvascular hyperpermeability and leukocyte recruitment in vivo.     

The soy isoflavone genistein induces a late but sustained activation of the endothelial nitric oxide-synthase system in vitro

Cardiovascular diseases are known as the major causes of death or disability in western countries. Decreased bioavailability of endothelial derived nitric oxide (NO) is recognized as an important promoter in cardiovascular disease. In vivo studies suggest that phytoestrogens, especially isoflavones from soy, enhance endothelium-dependent vasoreactivity. We hypothesized that isoflavones may affect the expression of endothelial-type nitric oxide synthase (eNOS) and thereby NO formation in vitro. Human EA.hy926 endothelial cells were treated with the soybean isoflavones biochanin A and formononetin and with their metabolites genistein and daidzein. eNOS promoter activity was examined by a luciferase reporter gene assay (20 h). Active eNOS was detected by quantifying conversion of L-arginine to L-citrulline and by measuring NO released from endothelial cells using the fluorescent probe DAF-2 (20-96 h).eNOS promoter activity increased in response to isoflavone treatment (20 h). NO and L-citrulline production by EA.hy926 cells rose up to 1.7-fold of control levels after stimulation with genistein for 48-96 h. From these results, we conclude that the suggested positive effects of soy isoflavones on vascular reactivity may be indeed mediated via a long-term effect on the eNOS system.