高糖作用下脐静脉内皮细胞中血管内皮生长因子基因表达与蛋白激酶C通路的关系

目的观察高糖作用人脐静脉内皮细胞时血管内皮生长因子mRNA和蛋白的表达与蛋白激酶C通路被激活和抑制的关系。方法提取并体外培养人脐静脉内皮细胞,分别加入不同浓度葡萄糖(5.5、11、22及44mmol/L)、蛋白激酶C激活剂和阻断剂并分别作用不同时间,应用逆转录聚合酶链反应检测血管内皮生长因子mR-NA水平,免疫细胞化学检测血管内皮生长因子及蛋白激酶C蛋白表达。结果经高糖处理的人脐静脉内皮细胞中血管内皮生长因子mRNA的转录水平明显高于对照组(P<0.05或P<0.01),但脐静脉内皮细胞经22 mmol/L葡萄糖作用72 h或佛波酯作用6 h,血管内皮生长因子mRNA水平不再升高,而呈下降趋势(P<0.05)。经22 mmol/L葡萄糖作用72 h后,血管内皮生长因子蛋白表达也逐渐下降。蛋白激酶C在22 mmol/L葡萄糖处理2 h后出现膜转位。而蛋白激酶C抑制剂GF109203X可阻断上述现象。结论高糖能引起脐静脉内皮细胞血管内皮生长因子mR-NA和蛋白表达增高。高糖引起脐静脉内皮细胞血管内皮生长因子基因表达增高,其机制与高糖激活蛋白激酶C通路有关。     

TL1A在高糖诱导的人脐静脉内皮细胞凋亡中的作用及机制探讨

目的 研究肿瘤坏死因子配体相关分子1A（TL1A）在高糖诱导的人脐静脉内皮细胞凋亡中的作用,并探讨机制.方法 采用RT-PCR、Western blot法检测不同浓度（5.6、11.2、22.4、33.6 mmol/L）葡萄糖与人脐静脉内皮细胞共同孵育48 h以及22.4 mmol/L葡萄糖作用于内皮细胞12、24、48、96 h后,TL1A mRNA及蛋白质在人脐静脉内皮细胞中的表达情况.在22.4 mmol/L葡萄糖培养的人脐静脉内皮细胞中加入不同浓度的TL1A单克隆抗体（1、3、9 μg/mL）,48 h后进行Annexin V/PI双染色流式细胞凋亡计数,以纯化重组人TL1A作为内源性TL1A的对照.结果 在人脐静脉内皮细胞中,随着葡萄糖浓度升高及作用时间延长,TL1A mRNA和蛋白水增高（P＜0.05）.流式细胞计数显示,高糖培养人脐静脉内皮细胞经TL1A单克降抗体处理后凋亡明显减少（P＜0.05）.结论 高浓度葡萄糖诱导体外培养的人脐静脉内皮细胞TL1A的表达呈浓度、时间依赖性增高;TL1A表达增多可能是高糖诱导人脐静脉内皮细胞凋亡的重要因素.     

内皮型一氧化氮合酶基因启动子区DNA甲基化

目的 探讨内皮型一氧化氮合酶基因启动子区DNA甲基化在同型半胱氨酸致内皮细胞损伤中的作用机制.方法 原代培养人脐静脉内皮细胞,加入0、50、100、200、500 μmol/L同型半胱氨酸和100 μmol/L同型半胱氨酸+维生素B12+叶酸的培养液中孵育72 h.MTT法检测人脐静脉内皮细胞的增殖活性;实时定量PCR测定内皮型一氧化氮合酶mRNA表达;化学比色法测定内皮型一氧化氮合酶活性;硝酸还原酶法检测一氧化氮生成量.巢式降落式甲基化特异性PCR法检测内皮型一氧化氮合酶基因启动子区DNA甲基化改变;同位素法检测DNA甲基化转移酶的活性.结果 人脐静脉内皮细胞与不同浓度同型半胱氨酸孵育72 h后,人脐静脉内皮细胞的增殖活性降低,内皮型一氧化氮合酶mRNA表达、内皮型一氧化氮合酶活性和一氧化氮的含量明显下降.内皮型一氧化氮合酶基因启动子序列DNA甲基化程度随同型半胱氨酸浓度的升高而增加,且DNA甲基化转移酶活性升高,而叶酸和维生素B12有一定拮抗作用,与对照组比较差异有显著性(P＜0.05和P＜0.01).结论 同型半胱氨酸可导致内皮型一氧化氮合酶基因启动子区序列高甲基化修饰,并出现相应的内皮型一氧化氮合酶基因表达下调,这可能是同型半胱氨酸致内皮细胞损伤的重要机制之一.     

高糖对人脐静脉内皮细胞内皮抑素表达的影响

目的 研究高糖对人内皮细胞内皮抑素(ES)mRNA及蛋白表达水平的影响及其意义.方法 培养人脐静脉内皮细胞(HUVECs),分为正常糖(5.6mmol/L)对照组,高糖(11.2、22.4mmol/L)组.培养24、48、72、96h后,收集各组不同作用时间点细胞,用RT-PCR法检测ES mRNA表达水平,Western blot法检测ES蛋白的表达.结果 高糖对HUVECs中ES mRNA和蛋白表达水平的影响是双相的:短时间内起促进作用,具有时间依赖性;随作用时间延长转为抑制作用,22.4mmol/L糖培养HUVECs 96h时组ES mRNA和蛋白表达水平较对照组明显降低(P<0.05).结论 糖尿病(DM)慢性病程中,ES表达的降低可能与DM血管病变的发生有关.     

阿托伐他汀通过过氧化体增殖物激活型受体γ促进一氧化氮生成抑制炎性因子产生

目的 观察阿托伐他汀是否能通过激活人脐静脉内皮细胞的过氧化体增殖物激活型受体γ从而改善血管内皮功能.方法 将体外培养的人脐静脉内皮细胞的实验分为两部分,实验一分组:①对照组;②脂多糖组(1.0 mg/L);③阿托伐他汀1.0 mmol/L组;④脂多糖+阿托伐他汀1.0 mmol/L组;⑤脂多糖+阿托伐他汀5.0mmol/L组,经孵育24 h后,收集细胞和培养上清液,用RT-PCB方法测定不同浓度阿托伐他汀对人脐静脉内皮细胞的过氧化体增殖物激活型受体γ表达的影响,并用硝酸还原酶法测定不同浓度阿托伐他汀干预对脂多糖诱导后细胞培养上清液中的一氧化氮生成的影响,ELISA方法测定细胞培养上清液中人可溶性细胞间黏附分子含量的影响.实验二分组:①对照组;②阿托伐他汀5.0 mmol/L组;③0.2 mmol/L GW9662组;④脂多糖+阿托伐他汀5.0mmol/L组;⑤GW9662+脂多糖+阿托伐他汀5.0 mmol/L组,观察过氧化体增殖物激活型受体γ特异性阻断剂GW9662对阿托伐他汀与脂多糖共同作用后人脐静脉内皮细胞的过氧化体增殖物激活型受体γ表达及培养上清液中一氧化氮、人可溶性细胞问黏附分子1含量变化的影响.结果 不同浓度阿托伐他汀可上调人脐静脉内皮细胞的过氧化体增殖物激活型受体γ表达,且随着药物浓度的增加其上调受体表达的作用增强.不同浓度阿托伐他汀可干预脂多糖诱导的人脐静脉内皮细胞液中一氧化氮生成减少及人可溶性细胞间黏附分子1含量的增加,且随着药物浓度的增加上述作用增强.过氧化体增殖物激活型受体γ特异性阻断剂GW9662可部分阻断阿托伐他汀上述作用.结论 阿托伐他汀可能部分通过激活人脐静脉内皮细胞的过氧体增殖物激活型受体γ受体,促进一氧化氮生成,抑制炎性因子的产生,改善血管内皮功能.     

七氟醚预处理对高糖诱导的人脐静脉内皮细胞损伤的保护作用

目的研究七氟醚对高糖诱导的人脐静脉内皮细胞(HUVECS)损伤的保护作用及可能机制。方法原代培养人脐静脉内皮细胞并鉴定;将人脐静脉内皮细胞生长至70%～80%融合时分为4组:正常葡萄糖浓度组(5.5 mmol/L葡萄糖)、高葡萄糖组(25mmol/L葡萄糖)、甘露醇组(5.5 mmol/L葡萄糖+19.5 mmol/L甘露醇)、高葡萄糖(25 mmol/L葡萄糖)+七氟醚组。各组细胞培养第1天、第4天、第7天评估细胞功能改变:采用MTT法观察细胞增殖的改变;流式细胞术PI/Annexin双染法检测细胞凋亡水平;Griess检测一氧化氮(NO);RT-PCR测定细胞中内皮型一氧化氮合酶(eNOS)mRNA的表达;Western Blot检测Bcl-2蛋白表达水平。结果各组细胞增殖水平比较差异无统计学意义。与正常糖浓度组比较,高葡萄糖组细胞凋亡增加,Bcl-2表达下调(P <0.05),Bax表达上调(P <0.05),eNOS表达下降(P <0.05);与高葡萄糖组比较,高葡萄糖+七氟醚组水平细胞存活数量增多(P <0.05),SOD活性增强(P <0.05)。结论七氟醚对高糖诱导的人脐静脉内皮细胞有抑制凋亡和抗氧化的保护作用。     

葡萄糖对血管内皮细胞小凹蛋白1和血管内皮生长因子表达的影响

为了探讨高浓度葡萄糖损伤血管内皮细胞及其对小凹蛋白-1和血管内皮生长因子表达的影响。将人脐静脉内皮细胞株ECV304．分别培养在对照组和含5.5mmol／L、11.1mmol／L、22.0mmol／L、33.0mmol／L葡萄糖的培养基中。经葡萄糖培养24h后，噻唑蓝法测定细胞增殖活性，硝酸还原酶法测定培养上清液中一氧化氮浓度，免疫组织化学和免疫印迹方法检测细胞中小凹蛋白-1和血管内皮生长因子的表达。结果发现，随着葡萄糖浓度的增加，内皮细胞增殖活性呈浓度依赖性抑制(r=-0.776，P=0.000)；一氧化氮浓度呈浓度依赖性增加(r=0.698，P=0.000)；小凹蛋白-1和血管内皮生长因子为棕黄色颗粒，主要分布于胞浆中；血管内皮生长因子的表达呈浓度依赖性增加(r=0.645，P=0.009)；小凹蛋白-1的表达也呈浓度依赖性增加(r=0.808，P=0.000)。提示高糖可诱导血管内皮细胞的血管内皮生长因子和小凹蛋白-1的表达，此变化可能与糖尿病患者高糖致血管病变有关。     

阿托伐他汀通过调节Bcl-2/Bax蛋白表达抑制高糖诱导的人脐静脉内皮细胞凋亡

目的研究阿托伐他汀对高浓度葡萄糖诱导的人脐静脉内皮细胞凋亡的影响以及其分子机制。方法将培养的人脐静脉内皮细胞与不同浓度的葡萄糖(5.6 mmol/L、17.6 mmol/L、33.3 mmol/L)及阿托伐他汀(0.1μmol/L、1μmol/L、10μmol/L)作用24 h后用吖啶橙/溴化已啶荧光染色观察凋亡细胞形态,四甲基偶氮唑蓝比色法测定人脐静脉内皮细胞增殖率,流式细胞仪和W estern Blotting分别检测细胞早期凋亡率及Bcl-2/Bax蛋白表达。结果随着葡萄糖浓度的增加,人脐静脉内皮细胞增殖率逐渐降低(P0.05),细胞早期凋亡率逐渐升高(P0.05)。人脐静脉内皮细胞Bcl-2蛋白表达逐渐减弱,Bax蛋白表达逐渐增强。用不同浓度的阿托伐他汀干预后,人脐静脉内皮细胞的增殖率逐渐升高(P0.05),而凋亡率逐渐降低(P0.05);人脐静脉内皮细胞Bcl-2蛋白表达逐渐增强,Bax蛋白表达逐渐减弱。其中,与高糖组比较,10μmol/L阿托伐他汀干预组能提高Bcl-2蛋白表达(P0.05),抑制Bax蛋白表达(P0.05)。结论阿托伐他汀可能通过调节Bcl-2/Bax蛋白表达抑制高浓度葡萄糖诱导的人脐静脉内皮细胞凋亡。     

糖基化终产物对人脐静脉内皮细胞一氧化氮合酶活性和表达的影响及二甲双胍的保护作用

目的 探讨糖基化终产物及二甲双胍对人脐静脉内皮细胞一氧化氮合酶活性和表达的影响.方法 用胶原酶法分离人脐静脉内皮细胞并加以培养.将内皮细胞与不同浓度的糖基化终产物和二甲双胍分别孵育3、6、12、24 h,CCK-8法测定人脐静脉内皮细胞增殖活性.硝酸还原酶法测定一氧化氮含量,分光光度法测定一氧化氮合酶活性,蛋白免疫印迹法检测内皮型一氧化氮合酶蛋白表达水平.结果 糖基化终产物抑制人脐静脉内皮细胞增殖,二甲双胍促进人脐静脉内皮细胞增殖.糖基化终产物抑制人脐静脉内皮细胞的一氧化氮生成和一氧化氮合酶活性(P<0.01),呈剂量、时间依赖关系.二甲双胍(与对照组相比)或与糖基化终产物共同干预(与糖基化终产物组相比)均增加人脐静脉内皮细胞一氧化氮生成和一氧化氮合酶活性(P<0.01).糖基化终产物与人脐静脉内皮细胞共同孵育24 h后,内皮型一氧化氮合酶表达水平明显下降;二甲双胍上调内皮型一氧化氮合酶的表达;与糖基化终产物组相比,糖基化终产物与二甲双胍共同干预组内皮型一氧化氮合酶表达上调(P<0.01).结论 二甲双胍能够改善糖基化终产物导致的人脐静脉内皮细胞损伤.     

球状脂联素抑制波动性高血糖诱导人脐静脉内皮细胞凋亡与脂联素受体1有关

目的 探讨球状脂联素在抑制波动性高血糖诱导人脐静脉内皮细胞凋亡中的机制.方法 不同条件下体外培养人脐静脉内皮细胞5天.实验分为对照组(葡萄糖5.5 mmol/L)、高糖组(葡萄糖25 mmol/L)、葡萄糖交替组(葡萄糖5.5/25 mmol/L,每8 h更换培养液一次)、高渗组(甘露醇25 mmol/L)和高渗交替组(甘露醇5.5/25 mmol/L,每8 h更换培养液一次).葡萄糖交替组中部分细胞用不同浓度(0、0.5、1.0和3 mg/L)球状脂联素干预,用脂联素受体1特异性小干扰RNA作用内皮细胞.采用流式细胞仪检测细胞凋亡及RT-PCR检测脂联素受体1和受体2 的mRNA表达.结果 与对照组比较,高糖组和葡萄糖交替组细胞凋亡明显增加,脂联素受体1 mRNA的表达显著降低(P<0.01).与高糖组比较,葡萄糖交替组细胞凋亡显著增加,脂联素受体1 mRNA的表达显著降低(P<0.01).不同浓度(0、0.5、1.0和3 mg/L)球状脂联素作用内皮细胞,发现3 mg/L球状脂联素能显著抑制内皮细胞凋亡(P<0.01),并能显著拮抗波动性高血糖诱导的脂联素受体1 mRNA表达的下降(P<0.01).不同组间内皮细胞脂联素受体2 mRNA表达差异无显著性.siRNA作用内皮细胞后球状脂联素这种抑制凋亡作用显著减弱(P<0.01).结论 与持续性高血糖条件比较,波动性高血糖显著降低人脐静脉内皮细胞脂联素受体1 mRNA的表达,而对脂联素受体2 mRNA的表达无影响.球状脂联素可能通过脂联素受体1拮抗波动性高血糖诱导的人脐静脉内皮细胞凋亡.     

κ-阿片受体激活后通过caveolin-eNOS途径抑制由软脂酸钠诱导的内皮损伤

目的 探讨κ-阿片受体在对抗软脂酸钠（sodium palmitate,SP）诱导的人脐静脉内皮细胞（HUVECs）损伤中的作用及其作用机制。 方法 体外培养HUVECs并分为6组即正常对照组、κ-阿片受体激动剂U50,488H组、SP组、SP+U50,488H组、SP+U50,488H+κ-阿片受体阻断剂nor-BNI组、SP+U50,488H+eNOS抑制剂L-NAME组。检测各组细胞生存率、测定各组caveolin-1、eNOS的蛋白表达水平,以及NO的产生及细胞凋亡情况。 结果 与正常对照组相比,SP组细胞的生存率明显降低（P<0.01）,caveolin-1的蛋白表达显著增加（P<0.01）,eNOS的活性明显受到抑制（P<0.05）,NO生成量大幅下降（P<0.01）,细胞凋亡显著增加（P<0.01）;而κ-阿片受体激动剂U50,488H可以显著抑制SP的上述作用,使细胞生存率升高（P<0.01）,caveolin-1的蛋白表达均显著降低（P<0.05或P<0.01）,eNOS的蛋白表达显著增加（P<0.05）,NO生成量显著增多（P<0.01）,细胞凋亡明显减少（P<0.01）。U50,488H的作用可被κ-阿片受体阻断剂nor-BNI或NO合酶抑制剂L-NAME所阻断（P<0.05或P<0.01）。 结论 激活κ-阿片受体能够抑制软脂酸钠诱导的内皮损伤,其机制可能与下调caveolin-1表达,增强eNOS活性有关。     

Sesamin induces nitric oxide and decreases endothelin-1 production in HUVECs: possible implications for its antihypertensive effect

OBJECTIVE: Sesamin has been proved to be antihypertensive. Nitric oxide (NO) is the most important vascular relaxing factor that is regulated in endothelium. Endothelin-1 (ET-1) is characterized as a potent vasoconstrictor and is also regulated in endothelium. Alterations in the endothelial production of NO and ET-1 are known to correlate with hypertension. This study investigated the effect of sesamin on NO and ET-1 in the human umbilical vein endothelial cells (HUVECs). DESIGN: The concentrations of NO and ET-1 in the medium of HUVECs treated by sesamin were measured. The mRNA and protein expressions of nitric oxide synthase (NOS), endothelin converting enzyme-1 (ECE-1), and endothelin-1 (ET-1) were also investigated. Other than the mRNA and protein expression, NOS activity and cyclic GMP (cGMP) were detected. METHODS: The NO concentration was detected by colorimetric assay. The cGMP and ET-1 were analyzed by EIA. The eNOS, ECE-1, and ET-1 mRNA expressions were assayed by Northern blot. The eNOS and ECE-1 protein expressions were analyzed by Western blot. The NOS activity was assayed by detecting the level of [H]-1-citrullin transformed from [H]-1-arginine. RESULTS: Sesamin not only increased the NO concentration in the medium of HUVECs in a dose-dependent manner after 24 h, but also induced eNOS mRNA and protein expressions. NOS activity in the HUVECs was also induced by sesamin. The content of cGMP was induced by sesamin through NO signaling. On the other hand, the ET-1 concentration in the medium of HUVECs treated by sesamin was suppressed in a dose-dependent manner after 24 h. The ECE-1 protein and mRNA expressions were also inhibited by sesamin. However, the mRNA expression of prepro ET-1 was not influenced by sesamin. CONCLUSION: From the above results, it is suggested that sesamin may improve hypertension by its ability to induce NO and inhibit ET-1 production from endothelial cells. The increase of NO by sesamin is through the induction of eNOS gene expression. The decrease of ET-1 by sesamin is through the inhibition of ECE gene expression, but is not through the inhibition of prepro ET-1 gene expression.     

Endothelial nuclear factor-kappaB translocation and vascular cell adhesion molecule-1 induction by complement: inhibition with anti-human C5 therapy or cGMP analogues

We have previously shown that reoxygenation of hypoxic human umbilical vein endothelial cells (HUVECs) leads to the activation and deposition of complement. In the present study, we investigated whether the terminal complement complex (C5b-9) influences HUVEC nuclear factor-kappaB (NF-kappaB) translocation and vascular cell adhesion molecule-1 (VCAM-1) protein expression after hypoxia/reoxygenation by decreasing endothelial cGMP. Additionally, we investigated the action of anti-human C5 therapy on endothelial cGMP, NF-kappaB translocation, and VCAM-1 protein expression. Reoxygenation (0.5 to 3 hours, 21% O(2)) of hypoxic (12 hours, 1% O(2)) HUVECs in human serum (HS) significantly increased C5b-9 deposition, VCAM-1 expression, and NF-kappaB translocation compared with hypoxic/reoxygenated HUVECs treated with the recombinant human C5 inhibitor h5G1.1-scFv. Acetylcholine (ACh)-induced cGMP synthesis was significantly higher in normoxic HUVECs compared with hypoxic HUVECs reoxygenated in HS but did not differ from hypoxic HUVECs reoxygenated in buffer or HS treated with h5G1.1-scFv. Treatment of hypoxic/reoxygenated HUVECs with h5G1.1-scFv or cGMP analogues significantly attenuated NF-kappaB translocation and VCAM-1 protein expression. Treatment with NO analogues, but not a cAMP analogue, cGMP antagonists, or an NO antagonist, also significantly attenuated VCAM-1 expression. We conclude that (1) C5b-9 deposition, NF-kappaB translocation, and VCAM-1 protein expression are increased in hypoxic HUVECs reoxygenated in HS; (2) reoxygenation of hypoxic HUVECs in HS, but not buffer alone, attenuates ACh-induced cGMP synthesis; and (3) treatment of hypoxic/reoxygenated HUVECs with h5G1.1-scFv attenuates C5b-9 deposition, NF-kappaB translocation, and VCAM-1 expression while preserving ACh-induced cGMP synthesis. C5b-9-induced VCAM-1 expression may thus involve an NO/cGMP-regulated NF-kappaB translocation mechanism.     

脂联素抑制炎症小体NLRP3表达减轻高糖高脂诱导内皮细胞损伤

目的 研究脂联素（APN）是否通过抑制炎症小体NLRP3表达减轻高糖高脂所致的内皮细胞损伤。方法 将培养的人脐静脉内皮细胞（human umbilical vein endothelial cells,HUVECs）分为6组:对照组、高糖高脂组、对照+ NLRP3 siRNA组,高糖高脂组+NLRP3 siRNA组,对照+APN组,高糖高脂+APN组。培养48 h后检测细胞存活率、凋亡率、炎症小体NLRP3表达水平。结果 与对照组相比,高糖高脂组细胞存活率显著下降,细胞凋亡显著升高,炎症小体NLRP3表达水平显著升高（均P<0.05）;炎症小体NLRP3 siRNA可有效的抑制炎症小体NLRP3的表达,改善高糖高脂引起的内皮细胞损伤（均P<0.05）;APN能抑制高糖高脂引起的炎症小体NLRP3表达增多,进而减轻高糖高脂引起的内皮损伤（均P<0.05）。结论 炎症小体NLRP3表达增多是高糖高脂诱导人脐静脉内皮细胞凋亡的内在机制,而脂联素可以通过抑制炎症小体NLRP3的过度表达发挥内皮保护作用。伤,降低心肌炎症反应。     

葡萄糖对人血管内皮细胞多元醇通路的激活作用及其机理

观察葡萄糖对人血管内皮细胞多元醇通路的影响 ,并探讨其作用机理。体外培养人脐静脉内皮细胞 ,加不同浓度葡萄糖或作用不同时间 ,采用高效液相色谱仪、硝酸还原酶法、生物化学检测及逆转录聚合酶链反应等方法测定内皮细胞山梨醇、一氧化氮、醛糖还原酶活性及醛糖还原酶基因mRNA。结果发现 ,经高浓度葡萄糖处理的人脐静脉内皮细胞山梨醇浓度明显高于对照组 (P <0 .0 5) ,一氧化氮浓度明显低于对照组 (P <0 .0 5)。醛糖还原酶基因mRNA水平及其活性均呈浓度及时间依赖性 ,但是内皮细胞经 2 2mmol L葡萄糖作用 48h或 44mmol L葡萄糖作用 2 4h后 ,醛糖还原酶基因mRNA水平及其活性均不再升高 ,而呈下降趋势 (P <0 .0 5)。结果提示 ,高浓度葡萄糖能引起内皮细胞功能改变 ,其机制可能是高浓度葡萄糖能增强醛糖还原酶基因的转录并提高其活性 ,从而活化多元醇通路。     

17Beta-estradiol modulates endothelin-1 expression and release in human endothelial cells

OBJECTIVE: In this study the role of 17beta-estradiol (E2) in the regulation of endothelin-1 (ET-1) mRNA expression and secretion was investigated in cultured human umbilical vein endothelial cells (HUVECs). METHODS: Endothelial cells were either deprived of or treated with 17beta-estradiol (10(-9), 10(-7) M) for 48 h. After the incubation, the effect of E2 on ET-1 gene expression was evaluated by Northern blot analysis. ET-1 release into the media was measured by radioimmunoassay after 6 h of incubation under basal conditions and upon stimulation with thrombin (4 U/ml). In addition, the cyclic guanosine 5'-monophosphate (cGMP) content of cells was assayed by immunoassay. In order to exclude the role of nitric oxide (NO) in E2-induced effects on endothelin-1 gene expression and secretion, nitric oxide synthase (NOS) inhibitor, N-nitro L-arginine methyl ester (1 mM) (L-NAME) was added to the media of some cultures. RESULTS: Incubation of HUVECs with 10(-9) and 10(-7) M E2 for 48 h resulted in a 30 and 47% inhibition of ET-1 mRNA expression, respectively. Incubation with E2 also decreased the basal and thrombin-stimulated ET-1 release while increasing the cGMP content of cells significantly. NOS inhibitor L-NAME increased the release of ET-1 from E2-incubated cells but did not alter the ET-1 release from hormone-deprived cells. However, ET-1 secretion of E2-treated cells were significantly less than the deprived ones. Northern blot analyses also demonstrated that inhibition of NOS only partly attenuated the effect of E2 on ET-1 gene expression. In the presence of L-NAME, treatment with 10(-7) M E2 caused a 12% decrease in ET-1 gene expression. CONCLUSION: The results demonstrate that E2 may play both direct and indirect role in regulation of ET-1 gene expression and production in human endothelial cells. E2-induced increase in NO but decrease in ET-1 production may partly explain the mechanism of the protective effects of the hormone on the cardiovascular system.     

cGMP-mediated negative-feedback regulation of endothelial nitric oxide synthase expression by nitric oxide

Earlier studies have demonstrated that nitric oxide (NO) exerts a fast-acting inhibitory influence on endothelial NO synthase (eNOS) enzymatic activity in isolated vascular tissue preparations. The present study was designed to examine the possible effect of NO on eNOS protein expression in cultured endothelial cells and intact animals. Human coronary endothelial cells were incubated with S-nitroso-N-acetyl-penicillamine (SNAP, an NO donor), oxyhemoglobin (HGB, an NO trapping agent), SNAP plus HGB, or inactive vehicle (control). In other experiments, cells were treated with 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor), 1H-[1,2, 4]oxadiazolo-[4,3-2]quinoxalin-1-one (ODQ, a guanylate cyclase inhibitor), SNAP plus ODQ, 8-bromo-cGMP (8-Br-cGMP, a cell-permeable cGMP compound), 8-Br-cGMP plus HGB, or inactive vehicle in order to discern the effect of cGMP. The incubations were conducted for 24 hours, and total nitrate plus nitrite production and eNOS protein abundance (Western analysis) were measured. To determine the effect of NO on eNOS expression in vivo, rats were treated with either the NO donor isosorbide dinitrate or placebo by gastric gavage for 48 hours, and aortic eNOS protein expression was examined. The NO donor SNAP markedly depressed, whereas the NO scavenger HGB significantly raised, eNOS protein expression. The downregulatory action of SNAP was completely abrogated by HGB. Phosphodiesterase inhibitor and 8-Br-cGMP downregulated, whereas the guanylate cyclase inhibitor ODQ upregulated eNOS protein expression. The downregulatory action of SNAP was completely overcome by the guanylate cyclase inhibitor ODQ, and the upregulatory action of the NO scavenger HGB was abrogated by 8-Br-cGMP. Administration of NO donor resulted in a marked downregulation of aortic eNOS protein expression in intact animals, thus confirming the in vitro findings. NO serves as a negative-feedback regulator of eNOS expression via a cGMP-mediated process.     

Role of endothelial nitric oxide synthase in endothelial cell migration.

Endothelium-derived nitric oxide (NO) and its precursor L-arginine have been implied to promote angiogenesis, but little is known about the precise mechanism. The inhibition of endogenous NO formation by Nomega-nitro-L-arginine methyl ester (L-NAME) (1 mmol/L) but not its inactive enantiomer D-NAME (1 mmol/L) inhibited endothelial cell sprouting from the scratched edge of the cultured bovine aortic endothelial cell monolayer. Inhibition of endogenous NO release by L-NAME was confirmed by amperometric measurement using an NO-specific electrode. In the modified Boyden chamber, L-NAME (1 mmol/L) significantly inhibited endothelial cell migration, whereas L-NAME did not affect endothelial DNA synthesis as assessed by analysis of [3H]thymidine incorporation. We then examined alteration of endothelial cell adhesion molecule expression after the inhibition of NO by L-NAME in cultured human umbilical vein endothelial cells. In both normoxic and hypoxic conditions, L-NAME (1 mmol/L) inhibited surface expression of integrin alphavbeta3, which is an important integrin facilitating endothelial cell survival and angiogenesis. However, L-NAME did not affect the expression of platelet endothelial cell adhesion molecule-1, intercellular adhesion molecule-1, vascular endothelial adhesion molecule-1, gap junction protein connexin 43, and VE-cadherin, which have been reported to potentially affect angiogenesis. In summary, inhibition of endothelial NO synthase by L-NAME attenuated endothelial cell migration but not proliferation in vitro. Furthermore, endogenous endothelium-derived NO maintains the functional expression of integrin alphavbeta3, a mediator for endothelial migration, survival, and angiogenesis. Endothelium-derived NO, thus, may play an important role in mediating angiogenesis by supporting endothelial cell migration, at least partly, via an integrin-dependent mechanism.