C-reactive protein decreases endothelial nitric oxide synthase activity via uncoupling

C-reactive protein (CRP), a cardiovascular risk marker, induces endothelial dysfunction. We have previously shown that CRP decreases endothelial nitric oxide synthase (eNOS) expression and bioactivity in human aortic endothelial cells (HAECs). In this study, we examined the mechanisms by which CRP decreases eNOS activity in HAECs. To this end, we explored different strategies such as availability of tetrahydrobiopterin (BH4)-a critical cofactor for eNOS, superoxide (O₂⁻) production resulting in uncoupling of eNOS and phosphorylation/dephosphorylation of eNOS. CRP treatment significantly decreased levels of BH4 thereby promoting eNOS uncoupling. Pretreatment with sepiapterin, a BH4 precursor, prevented CRP-mediated effects on BH₄ levels, superoxide production as well as eNOS activity. The gene expression and enzymatic activity of GTPCH1, the first enzyme in the de novo biosynthesis of BH₄, were significantly inhibited by CRP. Importantly, GTPCH1 is known to be regulated by cAMP-mediated pathway. In the present study, CRP-mediated inhibition of GTPCH1 activity was reversed by pretreatment with cAMP analogues. Furthermore, CRP-induced O₂⁻ production was reversed by pharmacologic inhibition and siRNAs to p47 phox and p22 phox. Additionally, CRP treatment significantly decreased the eNOS dimer: monomer ratio confirming CRP-mediated eNOS uncoupling. The pretreatment of cells with NO synthase inhibitor (N-nitro-l-arginine methyl ester [l-NAME]) also prevented CRP-mediated O₂⁻ production further strengthening CRP-mediated eNOS uncoupling. Additionally, CRP decreased eNOS phosphorylation at Ser1177 as well as increased phosphorylation at Thr495. CRP appears to mediate these effects through the Fcγ receptors, CD32 and CD64. To conclude, CRP uncouples eNOS resulting in increased superoxide production, decreased NO production and altered eNOS phosphorylation.     

Dexamethasone, tetrahydrobiopterin and uncoupling of endothelial nitric oxide synthase

Objective To find out whether dexamethasone induces an uncoupling of the endothelial nitric oxide synthase (eNOS). Methods & Results A major cause of eNOS uncoupling is a deficiency of its cofactor tetrahydrobiopterin (BH₄). Treatment of human EA.hy 926 endothelial cells with dexamethasone decreased mRNA and protein expression of both BH₄-synthesizing enzymes: GTP cyclohydrolase I and dihydrofolate reductase. Consistently, a concentration- and time-dependent reduction of BH₄, dihydrobiopterin (BH₂) as well as BH₄: BH₂ ratio was observed in dexamethasone-treated cells. Surprisingly, no evidence for eNOS uncoupling was found. We then analyzed the expression and phosphorylation of the eNOS enzyme. Dexamethasone treatment led to a down-regulation of eNOS protein and a reduction of eNOS phosphorylation at serine 1177. A reduction of eNOS expression may lead to a relatively normal BH₄: eNOS molar ratio in dexamethasone-treated cells. Because the B H₄-eNOS stoichiometry rather than the absolute B H₄ amount is the key determinant of eNOS functionality (i.e., coupled or uncoupled), the down-regulation of eNOS may represent an explanation for the absence of eNOS uncoupling. Phosphorylation of eNOS at serine 1177 is needed for both the NO-producing activity of the coupled eNOS and the superoxide-producing activity of the uncoupled eNOS. Thus, a reduction of serine 1177 phosphorylation may render a potentially uncoupled eNOS hardly detectable. Conclusions Although dexamethasone reduces BH₄ levels in endothelial cells, eNOS uncoupling is not evident. The reduction of NO production in dexamethasone-treated endothelial cells is mainly attributable to reduced eNOS expression and decreased eNOS phosphorylation at serine 1177.     

高同型半胱氨酸血症诱导eNOS脱偶联损害冠状动脉内皮功能

目的探讨高同型半胱氨酸血症（HHcy）患者冠状动脉内皮功能是否被损伤,以及这种损伤是否通过内皮型一氧化氮合酶（eNOS）脱偶联实现的。方法 71例参与者被分成健康对照组（n=50）和HHcy组（n=21）,利用多普勒超声心动测定腺苷诱导下冠状动脉左前降支的舒张功能的改变,冠脉血流速度储备（CFVR）由最大血流速度与基线水平的比值计算得出。采用ELISA以及高效液相色谱法测定血浆一氧化氮（NO）、四氢生物蝶呤（BH4）的水平。结果与健康对照组相比,HHcy组患者血浆NO、BH4的水平降低（P〈0.05）;HHcy组CFVR低于健康对照组（P〈0.05）;血浆Hcy水平与NO及CFVR呈负相关（P〈0.05）。结论 HHcy可能通过降低BH4生物利用度,诱导eNOS脱偶联,而导致冠状动脉内皮功能损伤,进而促进不良冠脉事件的发生。     

Endothelial nitric oxide synthase dysfunction in diabetic mice: importance of tetrahydrobiopterin in eNOS dimerisation

Aims/hypothesis Impaired nitric oxide (NO) bioactivity and increased superoxide (SO) production are characteristics of vascular endothelial dysfunction in diabetes. The underlying mechanisms remain unknown. In this regard, we investigated the role of tetrahydrobiopterin (BH4) bioavailability in regulating endothelial nitric oxide synthase (eNOS) activity, dimerisation and SO production in streptozotocin-induced diabetic mice.Methods Mouse aortas were used for assays of the following: (1) aortic function by isometric tension; (2) NO by electronic paramagnetic resonance; (3) SO by lucigenin-enhanced chemiluminescence and dihydroethidine fluorescence; (4) total biopterin and BH4 by high-performance liquid chromatography; and (5) eNOS protein expression and dimerisation by immunoblotting.Results In diabetic mouse aortas, relaxations to acetylcholine and NO levels were significantly decreased, but SO production was increased, in association with reductions in total biopterins and BH4. Although total eNOS levels were increased in diabetes, the protein mainly existed in monomeric form. Conversely, specifically augmented BH4 in diabetic endothelium preserved eNOS dimerisation, but the expression remained unchanged.Conclusions/interpretation Our results demonstrate that BH4 plays an important role in regulating eNOS activity and its functional protein structure, suggesting that increasing endothelial BH4 and/or protecting it from oxidation may be a rational therapeutic strategy to restore eNOS function in diabetes.     

内皮细胞型NO合酶基因第7外显子894G→T点突变与糖尿病肾病相关性研究

目的 探讨内皮细胞型NO合酶（eNOS)基因第7外显子894G→T点突变与中国北方汉族人2型糖尿病（T2DM）合并肾病（DN）之间的关系。方法 运用聚合酶链式反应限制性片段长度多态性技术（PCR－RFLP），结合DNA测序技术，检测了228例中国北方汉族人的eNOS基因第7外显子894G→T错义突变位点的基因型，其中T2DM患者143例（DN79例），健康成人85例，并对各组间的等位基因频率与基因型频率进行了比较。结果 ①T2DM组的T等位基因及TG基因型频率与正常对照（N）组无显著性差异（P＞0．05）。②DN＋组T等位基因及TG基因型频率显著高于糖尿病非肾病患者（P＜0．05）。③SBP、HbA1c、TC、TG和eNOS基因第7外显子894G→T点突变均与糖尿病肾病有关（P＜0．05）。结论 eNOS基因第7外显子894G→T点突变的T等位基因可能是中国人2型糖尿病易患肾病的独立危险因素。     

High nitric oxide synthase activity in endothelial cells in ulcerative colitis

Endothelial nitric oxide (NO) synthase, a unique NO synthase (NOS) isoform that is expressed constitutively by the vascular endothelium both in vivo and in vitro, is believed to be essential to systemic and/or local vascular integrity. NOS expression by endothelial cells may indicate vascular activation. We successfully established a simple method for the culture of microvascular endothelial cells from a small amount of tissue and investigated ulcerative colitis (UC), in which condition vascular factors have not been studied extensively. We cultured endothelial cells from the mesenteries of surgical patients with UC and assayed NOS activity by reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry. Strong NOS activity was demonstrated in the cells from all UC patients (5/5), whereas no activity was detected in the cells from human umbilical veins and the mesenteries of colon cancer patients (0/10 and 0/5, respectively). This strong NOS activity was not diminished by incubation with a high concentration of glucocorticoid, suggesting that it was constitutive. These results indicate a close relationship of vascular activation (high NOS activity) with the pathogenesis of UC.     

甲基亚砷酸对内皮型一氧化氮合酶活性的影响

目的研究甲基亚砷酸（MMA^Ⅲ）对内皮型一氧化氮合酶（eNOS）活性的影响，并初步探讨MMA^Ⅲ抑制eNOS活性的作用机制。方法从体外培养的牛大动脉血管内皮细胞中制备eNOS提取液，单独暴露于MMA^Ⅲ（暴露剂量分别为1、2．5、5、10、15μmol／L）或其他砷化合物（亚砷酸钠、砷酸钠、甲基砷酸或二甲基砷酸，暴露剂量均为10μmol／L）；或者联合暴露于MMA^Ⅲ（10μmol／L）和二硫苏糖醇（DTT，100μmol／L）。37℃暴露5min后测定L-[^3H]精氨酸产生的L-[^3H]瓜氨酸量来反映eNOS活性。结果eNOS活性随着MMA^Ⅲ暴露剂量增加而逐渐降低，MMA^Ⅲ的2．5、5、10、15μmol／L剂量组的eNOS活性分别是对照组的44．4％、23．4％、11．7％和6．6％，均显著低于对照组（P〈0．01）；MMA^Ⅲ对eNOS活性的50％抑制剂量（IC50）为2．1μmol／L；其他砷化合物10μmol／L暴露对eNOS活性均无明显影响（P〉0．05）；DTT（100μmol／L）能够部分恢复MMA^Ⅲ暴露引起的eNOS活性降低。结论MMA^Ⅲ对eNOS活性具有显著抑制作用；MMA^Ⅲ抑制eNOS活性很可能与MMA^Ⅲ和eNOS分子的二巯基结构的相互作用有关。     

小凹蛋白与内皮型一氧化氮合成酶在大鼠肝硬化组织中的表达

目的探讨小凹蛋白Caveolin-1、内皮型一氧化氮合成酶eNOS在大鼠肝硬化组织中的异常表达及其意义。方法构建二甲基亚硝胺（DMN）致肝纤维化大鼠模型，在造模4周后观察肝纤维化程度。免疫组织化学染色检测30例大鼠肝硬化肝组织和30例正常大鼠肝组织中Caveolin-1和eNOS的细胞定位；Western Blot检测Caveolin-1和eNOS的蛋白表达水平变化。结果Caveolin-1和eNOS均主要分布于肝窦内皮细胞中，Caveolin-1在肝硬化组表达阳性率为90％，对照组为37％，两组比较差异有统计学意义（P〈0．05）；eNOS在肝硬化组表达阳性率为30％，对照组为66％，两组比较差异有统计学意义（P〈0．05）。Westem Blot检测Caveolin-1在肝硬化组织中较正常肝组织中表达明显增强；eNOS在肝硬化组织中呈低水平表达，较正常肝组织中表达明显减少。结论肝硬化肝窦内皮细胞中Caveolin-1的异常表达促进eNOS-Caveolin-1复合物的生成，结合形式的eNOS活性降低，导致NO合成减少，肝内血管阻力持续增加，从而导致了门静脉高压症的形成。     

Subcellular distribution of nitric oxide synthase isoforms in the rat duodenum

AIM:To study the cell-type specific subcellular distribution of the three isoforms of nitric oxide synthase(NOS) in the rat duodenum.METHODS:Postembedding immunoelectronmicroscopy was performed,in which primary antibodies for neuronal NOS(nNOS),endothelial NOS(eNOS),and inducible NOS(iNOS),were visualized with protein A-gold-conjugated secondary antibodies.Stained ultrathin sections were examined and photographed with a Philips CM10 electron microscope equipped with a MEGAVIEW II camera.The specificity of t...     

基因芯片技术分析内皮型一氧化氮合酶基因多态性与冠心病的关系

目的探讨内皮型一氧化氮合酶（eNOS）Glu298Asp基冈多态性与冠心病的关系。方法以89例冠心病患者为病例组（男性62例,女性27例）,均行冠状动脉造影确诊,对照组78例（男性44例,女性34例）均行冠状动脉造影排除冠心病,运用基因芯片技术方法检测eNOS Glu298 Asp基因的多态性,对比分析冠心病组及对照组eNOS Glu298 Asp基因型及等位基因的差异。结果eNOS Glu298 Asp基因型分布冠心病组与对照组比较差异无统计学意义（P=0．495）,D等位基因频率冠心病组（10．11％）与对照组（10．27％）相比差异无统计学意义（P=0．965）。结论eNOS Glu298 Asp基因变异与冠心病的发病可能无相关性。     

Liposome-mediated gene transfer of endothelial nitric oxide synthase to cirrhotic rat liver decreases intrahepatic vascular resistance

Background and Aims:  Nitric oxide (NO) production by endothelial nitric oxide synthase (eNOS) in sinusoidal endothelial cells is reduced in the injured liver and leads to intrahepatic portal hypertension. The present study evaluates the effects of liposome-mediated gene transfer of eNOS on the intrahepatic vascular resistance and portal venous pressure (PVP) in cirrhotic rats.
Methods:  Hepatic cirrhosis was induced in male Sprague–Dawley rats by intraperitoneal injection of carbon tetrachloride (CCl₄), whereas the control normal rats were given the same dose of peanut oil. Plasmid eukaryotic expression vector (liposome-pcDNA3/eNOS) was injected into the portal vein of CCl₄ cirrhotic rats, whereas cirrhotic controls received the same dose of naked plasmid (liposome-pcDNA3) or Tris buffer, and control normal rats received the same dose of Tris buffer. Five days after gene transfer, the levels of eNOS mRNA and protein, NO production, PVP and the changes of hepatic intrahepatic vascular resistance were investigated.
Results:  Five days after eNOS gene transfer, the levels of eNOS mRNA, eNOS protein and NO production in cirrhotic rats increased remarkably, while hepatic vascular resistance and PVP decreased significantly in cirrhotic rats.
Conclusion:  Liposome-mediated eNOS gene transfer via intraportal injection is feasible and the increase of intrahepatic eNOS leads to a marked decrease in introhepatic vascular resistance and PVP. These data indicate that intrahepatic eNOS plays an important role in the pathogenesis of portal hypertension and gene transfer of eNOS is a potential and novel therapy for portal hypertension.     

内皮型一氧化氮合酶基因多态性与急性心肌梗死的相关性探讨

目的　探讨内皮型一氧化氮合酶 (eNOS)基因多态性与急性心肌梗死 (AMI)的相关性。方法　依据eNOS基因外显子 7G894T位点设计引物 ,通过巢式聚合酶链反应 (PCR)扩增目的片段 ,限制性内切酶消化目的片段 ,琼脂糖凝胶电泳 ,紫外透射分析仪检测 ,计数 10 7例AMI病人及 81例健康者基因型及突变基因频率 ,通过χ2 检验有无统计学意义。结果　eNOS基因外显子 7的 894位点有 3种基因型 :GG、GT、TT。AMI组 10 7例中2 5例发生G894T突变 ,纯合子TT 9例 ,杂合子GT 16例。对照组 81例中 13例发生G894T突变 ,均为杂合子。两组等位基因纯合子突变具有非常显著统计学意义 ,x2 =5 4 2 9,P <0 0 5 ,两组等位基因总突变率 (纯合子 +杂合子 )无明显统计学意义 ,x2 =1 5 2 9,P >0 0 5。结论　eNOS基因 894位点TT型突变与AMI发病密切相关 ,是AMI发病的危险因子     

Uncoupled endothelial nitric oxide synthase and oxidative stress in a rat model of pregnancy-induced hypertension

BACKGROUND: Preeclampsia is a human pregnancy-associated syndrome associated with hypertension, proteinuria, and endothelial dysfunction. We tested whether increased reactive oxygen species (superoxide and peroxynitrite) production and decreased bioavailability of the endothelial nitric oxide (NO) synthase (eNOS) cofactor tetrahydrobiopterin (BH4) contributes to maternal endothelial dysfunction in rats with pregnancy-induced hypertension and several characteristics of preeclampsia. METHODS: Nonpregnant (DS) and pregnant (PDS) rats were treated with deoxycorticosterone acetate and 0.9% saline for approximately 3 weeks and nonpregnant (Con) and pregnant (P) rats received tap water. Blood pressure, urinary protein levels, mesenteric vascular reactivity, aortic protein expression, and aortic reactive oxygen species levels were compared between the four groups. RESULTS: The PDS rats had significantly decreased mesenteric endothelium-dependent relaxation responses and aortic NO production compared to Con, DS, and P rats despite increased aortic eNOS expression. Aortic superoxide and peroxynitrite levels were increased in PDS rats compared with Con, DS, and P rats. Scavenging of reactive oxygen species or increasing tetrahydrobiopterin levels normalized mesenteric endothelium-dependent relaxation responses, aortic NO production, and aortic superoxide and peroxynitrite levels in PDS rats. CONCLUSIONS: These data suggest that increased superoxide production by NADPH oxidase, peroxynitrite degradation of BH4, and uncoupled eNOS contribute to endothelial dysfunction in a rat model of pregnancy-induced hypertension.     

诱导型一氧化氮合酶和解偶联蛋白-2对心肌缺血预适应的作用

目的 探讨诱导型一氧化氮合酶（inducible nitric oxide synthase,iNOS）和解偶联蛋白-2（uncoupling protein-2,UCP2）对大鼠心肌缺血预适应的保护机制。方法 结扎左冠状动脉复制大鼠心肌缺血再灌注模型。预适应组行3次缺血5min,再灌注10min的预处理,分别于预处理0,6,12,24和48h（分别为0,6,12,24和48h亚组）后行30min缺血及120min再灌注：对照组开胸后不结扎左冠状动脉,电镜观察心肌超微结构,据Rainio评分标准进行心肌超微结构损伤程度的半定量分析。采用Western Blot及比色法检测心肌UCP2活性及iNOS活性。结果 预适应各亚组UCP2活性均增高（P〈0．05）,0h亚组UCP2表达水平最高（P〈0．01）,24小时亚组和48小时亚组心肌iNOS活性显著升高（P〈0．05）。结论 UCP2和iNOS共同参与了大鼠心肌缺血预适应心肌保护作用。     

内皮型一氧化氮合酶基因G894T多态性与心肌梗死相关性的Meta分析

目的 采用Meta分析的方法评估内皮型一氧化氮合酶（eNOS）基因G894T多态性与心肌梗死（MI）相关性.方法 按照文献纳入标准制定检索策略后,采用计算机检索PubMed、中国期刊全文数据库和万方数据库中1995年至2012年12月30日间发表的探讨eNOS基因G894T多态性与MI相关性的病例-对照研究,由两名作者独立提取数据及评价方法学质量后,采用RevMan 5.1软件进行Meta分析.结果 最终纳入13个病例-对照研究,共计2264例MI患者,2774例健康对照人群.基于随机效应模型的Meta分析结果显示,eNOS G894T多态性能够显著增加MI的患病风险[T vs.G：OR=1.51,95%CI：1.21～1.89;TT vs.GG：OR=1.99,95%CI：1.21～3.26;GT vs.GG：OR=1.34,95%CI：1.07～1.68;（GT＋TT） vs.GG：OR=1.47,95%CI：1.14～1.88;TT vs.（GG＋GT）：OR=1.80,95%CI：1.13～2.86].亚组分析结果表明这一相关性存在于亚洲人群与急性心肌梗死（AMI）之间,而与非亚洲人群无相关性,与混合MI（AMI和陈旧性MI）间可能存在相关性,漏斗图提示有发表偏倚存在.结论 基于当前的证据,eNOS G894T多态性与MI存在相关性,特别是亚洲人群与AMI.但当前证据尚不能确定eNOS G894T多态性是否是MI的独立危险因素,以及eNOS G894T多态性与陈旧性MI、与非亚洲人群AMI的相关性.     

内皮型一氧化氮合酶基因多态性与冠心病的关系

内皮型一氧化氮合酶(eNOS)基因被列为冠心病(CHD)的一个候选易感基因。本文对与冠心病关系较为密切的三种eNOS基因多态性:4b/a、T786C和G894T的研究进展作一综述,对进一步认识冠心病的发病机制具有重要意义。     

内皮型一氧化氮合酶基因多态性与冠心病的研究进展

冠状动脉粥样硬化性心脏病（cronary atherosclerotic heart disease,CHD）简称冠心病,是指冠状动脉发生粥样硬化引起管腔狭窄或闭塞,导致心肌缺血缺氧或坏死引起的心脏病.吸烟、肥胖、高血压及代谢综合征等均已成为广为人知的几个冠心病重要危险因素.但也有患者没有这些危险因素存在,并且在一些个体中,常有明显的冠心病家族史,说明冠心病是环境与遗传危险因素动态相互作用所致的.目前已有诸多冠心病基因多态性研究,包括内皮型一氧化氮合酶（endothelial nitric oxide synthase,eNOS）.在近年的荟萃分析中显示,某些遗传变异可能为特定群体冠心病发病的危险因素之一.