内皮型一氧化氮合酶脱偶联的研究进展

血管内皮功能障碍(endothelial dysfunction)是多种心脑血管疾病的共同病理机制,其突出表现为内皮依赖性血管舒张功能障碍,主要由NO减少及氧自由基增加所致。最新研究发现,内皮型一氧化氮合酶脱偶联(eNOS uncoup ling)是导致NO水平下降和氧自由基水平升高的重要机制,是高血压、糖尿病、动脉粥样硬化等疾病中内皮功能障碍的重要原因。通过纠正eNOS脱偶联可有效改善内皮功能,有望为保护血管内皮功能提供有效途径。     

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.     

阿托伐他汀对2型糖尿病患者外周血内皮祖细胞功能的影响及其机制的初步探讨

目的观察阿托伐他汀对2型糖尿病患者(T2DM)患者外周血内皮祖细胞功能的影响并初步探讨其机制。方法将40例糖尿病患者随机分为他汀治疗组和对照组,前者给予阿托伐他汀40 mg/d口服,治疗前及治疗后2、4周抽取外周血,采用密度梯度离心法分离培养内皮祖细胞,应用噻唑蓝(MTT)法检测内皮祖细胞增殖情况,Transwell小室检测迁移,Matrigel管腔形成实验检测管腔形成能力。另取20例T2DM患者血培养内皮祖细胞,加入阿托伐他汀、一氧化氮合酶(NOS)/NO途径抑制剂L-NAME后观察对其功能的影响。结果阿托伐他汀治疗后2周、4周外周血内皮祖细胞数量明显上升,功能明显改善(P<0.01)。体外实验他汀+L-NAME组的血内皮祖细胞数目较他汀组明显下降(P<0.01)。结论阿托伐他汀对糖尿病患者内皮祖细胞的功能具有一定保护作用,这种作用能被L-NAME阻断。阿托伐他汀的作用机制可能与激活NOS/NO信号通路有关。     

普伐他汀对人内皮祖细胞一氧化氮合成的影响

目的观察普伐他汀对人内皮祖细胞（EPCs）一氧化氮（NO）合成的影响。方法密度梯度离心法获取外周血单个核细胞,培养7d后,收集贴壁细胞并分别加入普伐他汀,10μmol/L及100μmol/L干预48h,免疫组化、荧光显微镜和流式细胞仪鉴定EPC,用RT-PCR方法测定对细胞内皮型一氧化氮合酶（eNOS）mRNA表达的影响,并用硝酸还原酶法测定培养液中一氧化氮（NO）的水平。结果普伐他汀组的人内皮祖细胞eNOS mRNA的表达、NO的合成明显增加。结论普伐他汀可增加人内皮祖细胞eNOS mRNA的表达和NO的合成     

Exendin-4对Ⅰ型糖尿病小鼠内皮祖细胞功能及AKT/eNOS信号通路的影响

目的探讨Exendin-4对Ⅰ型糖尿病小鼠内皮祖细胞(endothelial progenitor cells,EPCs)增殖、迁移、黏附、衰老的影响及对AKT/eNOS信号通路的影响。方法采用密度梯度离心法分离并培养6月龄Ⅰ型糖尿病小鼠EPCs,并用不同浓度的Exendin-4(1、5、10、25μmol·L^-1)处理EPCs,观察EPCs体外克隆形成及增殖能力。同时观察其对糖尿病小鼠EPCs迁移、黏附及衰老的影响。Western blot检测Exendin-4处理对EPCs蛋白激酶B/内皮型一氧化氮合酶(AKT/eNOS)信号通路的影响。结果Exendin-4处理可增加Ⅰ型糖尿病小鼠EPCs克隆形成及增殖能力,尤以10μmol·L^-1时作用效果最佳。Exendin-4处理可增加Ⅰ型糖尿病小鼠EPCs的体外迁移、黏附功能,并抑制细胞衰老。Western blot结果显示,Exendin-4可增强AKT及eNOS磷酸化水平,而GLP-1R抑制剂Exendin-9-39和AKT抑制剂MK-2206则可以阻断这种效应。结论Exendin-4可改善Ⅰ型糖尿病小鼠EPCs增殖、迁移、黏附能力并抑制细胞衰老,其机制可能与调控AKT/eNOS信号通路有关。     

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。     

洛伐他汀保护内皮祖细胞的机制研究

目的探讨洛伐他汀(lovastatin)保护内皮祖细胞(en-dothelial progenitor cells,EPCs)的机制。方法EPCs与洛伐他汀或者血凝素样氧化型低密度脂蛋白受体(lectin-like oxi-dized low density lipoprotein receptor,LOX-1)的特异性阻断抗体(LOX-1 mAb)预处理24 h后,再与氧化型低密度脂蛋白(oxidized low density lipoprotein,oxLDL)孵育48 h。然后,检测EPCs迁移、粘附和管状结构形成能力。为探讨洛伐他汀的作用机制,检测EPCs生成一氧化氮(nitric oxide,NO)的量,内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)和LOX-1蛋白及mRNA表达。结果oxLDL抑制EPCs迁移、粘附及管状结构形成能力,降低NO产生、eNOS蛋白及mRNA表达,增加LOX-1蛋白及mRNA表达。洛伐他汀和LOX-1 mAb恢复EPCs功能,逆转oxLDL对NO、eNOS及LOX-1的调节。结论洛伐他汀通过调节eNOS和LOX-1而保护EPCs免受oxLDL的损害。     

激活Sonic hedgehog通路改善1型糖尿病小鼠内皮祖细胞功能

目的研究激活Sonic hedgehog通路对1型糖尿病小鼠内皮祖细胞(EPCs)生物学功能的影响。方法用链脲佐菌素(STZ)诱导建立1型糖尿病小鼠模型;采用密度梯度离心法分离并培养糖尿病小鼠骨髓EPCs;体外给予Sonic hedgehog(Shh)信号通路配体蛋白Shh和受体激动剂SAG,通过MTT法、改良Boyden小室、Matrigel和β-半乳糖苷酶分别检测各组EPCs的增殖、迁移、小管形成和衰老的功能性指标。结果 1型糖尿病小鼠EPCs与正常对照组相比功能明显下降,体外给予Shh蛋白和受体激动剂SAG,可促进糖尿病EPCs增殖,减少衰老,改善迁移和小管形成能力。结论体外激活Sonic hedgehog通路可以改善1型糖尿病小鼠内皮祖细胞受损的功能。     

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.     

糖尿病患者血管内皮祖细胞研究进展

血管内皮祖细胞(EPC)来源于骨髓,是具有修复内皮和新生血管功能的干细胞。糖尿病患者外周血EPC数量和功能均出现下降,EPC已成为糖尿病及其并发症治疗的一个新靶点。本文综述EPC在糖尿病病理生理中的作用和药物干预机理的研究进展。     

Ameliorative effect of berberine on endothelial dysfunction in diabetic rats induced by high-fat diet and streptozotocin

Berberine can improve insulin resistance, lower blood glucose, and regulate lipid metabolism disorders which cause endothelial dysfunction, leading to vascular complications of type 2 diabetes mellitus. The aim of the present study was to investigate the effects of berberine on endothelial dysfunction of aortas in type 2 diabetes mellitus rats and its mechanism. Wistar rats were randomly divided into four groups: diabetic rats, control rats, diabetic rats treated with berberine (100 mg/kg), and control rats treated with berberine. The serum fasting blood glucose, insulin, total cholesterol, triglyceride and nitric oxide (NO) levels were tested. Acetylcholine-induced endothelium-dependent relaxation and sodium nitroprusside induced endothelium-independent relaxation were measured in aortas for estimating endothelial function. The expression of endothelial nitric oxide synthase (eNOS) mRNA was measured by RT-PCR, and the protein expressions of eNOS and NADPH oxidase (NOX4) were analyzed by western blot. The results showed that berberine significantly decreased fasting blood glucose, and triglyceride levels in diabetic rats. Berberine also improved endothelium-dependent vasorelaxation impaired in aorta. The expressions of eNOS mRNA and protein were significantly increased, while NOX4 protein expression was decreased in aortas from diabetic rats with berberine treatment. Moreover, serum NO levels were elevated after berberine treatment. In conclusion, berberine restores diabetic endothelial dysfunction through enhanced NO bioavailability by up-regulating eNOS expression and down-regulating expression of NADPH oxidase.     

黄芪提取物对大鼠骨髓源性内皮祖细胞的作用

摘要： 目的 探讨黄芪提取物对大鼠骨髓源性内皮祖细胞 （EPCs） 黏附、 迁移、 活力、 血管形成能力及内皮型一氧 化氮合成酶 （eNOS） 表达的影响。方法 体外培养、 分离和鉴定 EPCs, 设 10-4、 10-3、 10-2 g/L 黄芪提取物组和对照组。 倒置显微镜下观察并比较各组 EPCs 黏附、 迁移、 血管形成能力的差异; MTT 法检测 EPCs 的活力变化; RT-PCR 法检 测 EPCs 中 eNOS mRNA 的表达;Western blot 检测 EPCs 中 eNOS 蛋白的表达。结果 与对照组相比, 不同浓度黄芪 提取物组促 EPCs 黏附、 迁移、 血管形成能力均显著增强, 且呈浓度依赖性(F 值分别为 15.256、 13.633、 97.549, 均 P < 0.05); EPCs 的活力显著增加, 呈时间 （F 时间=9.755） 和浓度依赖性 （F 组间=10.018）; 且 EPCs 中 eNOS mRNA 和蛋白的表 达水平均明显升高, 呈浓度依赖性(F 值分别为 56.356、 77.125, 均 P < 0.05)。结论 黄芪提取物具有调控 EPCs 促血 管新生的作用, 该作用可能与上调eNOS 的表达水平密切相关。     

Akt/eNOS pathway activation in endothelium-dependent relaxation is preserved in aortas from female, but not from male, type 2 diabetic mice

Cardiovascular problems are major causes of morbidity and mortality, the main problems being coronary artery disease and atherosclerosis, in type 2 diabetes mellitus. However, female gender is a protective factor in the development of, for example, atherosclerosis and hypertension. Our aim was to investigate possible gender differences in the activation of Akt/eNOS signaling in aortas from a mouse type 2 diabetic model. Nonfasting plasma glucose was significantly above control in the diabetic mice (both males and females). Plasma insulin was not different between the age-matched controls and the diabetic mice (of either gender). In diabetic males (vs male controls and/or diabetic females): (a) systemic blood pressure was elevated, (b) the clonidine- and insulin-induced Akt-dependent aortic relaxations were impaired, but the ACh-induced Akt-independent and SNP-induced endothelium-independent aortic relaxations were not, (c) Akt and eNOS expression levels were lower, (d) both Akt phosphorylation at Ser(473) and eNOS phosphorylation at Ser(1177) in the aorta were lower under clonidine- or insulin-stimulation, but not under ACh-stimulation. These results suggest that in mice: (i) endothelial functions mediated via the Akt/eNOS pathway are abrogated in type 2 diabetes only in males and (ii) in females (vs males), eNOS expression is elevated and the endothelium resists dysfunction.     

Endothelial nitric oxide synthase inhibition does not alter endothelial progenitor cell colony forming capacity or migratory activity

Endothelial nitric oxide synthase (eNOS) activity has been shown to play a pivotal role in the mobilization of endothelial progenitor cells (EPCs) into the circulation from bone marrow. Indeed, in eNOS-deficient mice, exercise-induced EPC mobilization is severely diminished. We determined ex vivo whether circulating EPC colony-forming capacity and migratory activity are influenced by eNOS activity. Peripheral-blood mononuclear cells were isolated from 20 healthy adults and preplated for 2 days, and nonadherent cells were further cultured for 7 days in the presence and absence of N-nitro-L-arginine methyl ester (L-NAME, 300 microM, an eNOS antagonist) to determine EPC colony-forming units. Migratory activity of EPCs, cultured with and without L-NAME (300 microM) was determined by utilizing a modified Boyden chamber. The number of EPC colony-forming units was not significantly different when cultured in the absence or presence of L-NAME (21+/-5 vs 18+/-5). Moreover, eNOS inhibition did not alter EPC migratory activity; mean fluorescence was similar in samples cultured with (983+/-126 RFUs) and without (962+/-105 RFUs) L-NAME. These in vitro results suggest that, in contrast to EPC mobilization from the bone marrow, eNOS does not exert a modulatory influence on the functional capacity of circulating EPCs to either form colonies or migrate.     

Lycopene ameliorates erectile dysfunction in streptozotocin-induced diabetic rats

Diabetes mellitus (DM) is characterized by oxidative stress, which is one of the major pathophysiological mechanisms underlying diabetic erectile dysfunction (ED). Lycopene is one of the most potent antioxidants among the natural carotenoids. The present study was aimed to investigate whether lycopene could lower oxidative stress and attenuate ED in diabetic rats. Lycopene (10, 30, 60 mg/kg/d) was administered via intragastric intubation for 8 weeks to streptozotocin (STZ) (50 mg/kg, i.v.) induced diabetic rats. The results showed that chronic lycopene treatment significantly and dose dependently restored ED in diabetic rats by lowering blood glucose, reducing oxidative stress and up-regulating eNOS expression. These results indicated that lycopene treatment is potentially a new strategy for treating diabetic ED.     

Activation of liver X receptor enhances the proliferation and migration of endothelial progenitor cells and promotes vascular repair through PI3K/Akt/eNOS signaling pathway activation

Vascular endothelial injury is a major cause of many cardiovascular diseases. The proliferation and migration of endothelial progenitor cells (EPCs) play a pivotal role in endothelial regeneration and repair after vascular injury. Recently, liver X receptor (LXR) activation has been suggested as a potential target for novel therapeutic interventions in the treatment of cardiovascular disease. However, the effects of LXR activation on endothelial regeneration and repair, as well as EPC function, have not been investigated. In the present study, we demonstrate that LXRs, including LXRα and LXRβ, are expressed and functional in rat bone marrow-derived EPCs. Treatment with an LXR agonist, TO901317 (TO) or GW3965 (GW), significantly increased the proliferation and migration of EPCs, as well as Akt and eNOS phosphorylation in EPCs. Moreover, LXR agonist treatment enhanced the expression and secretion of vascular endothelial growth factor in EPCs. LXR agonists accelerated re-endothelialization in injured mouse carotid arteries in vivo. These data confirm that LXR activation may improve EPC function and endothelial regeneration and repair after vascular injury by activating the PI3K/Akt/eNOS pathway. We conclude that LXRs may be attractive targets for drug development in the treatment of cardiovascular diseases associated with vascular injury.     

Effects of resveratrol on endothelial progenitor cells and their contributions to reendothelialization in intima-injured rats

The aim of this study was to investigate the effects of resveratrol on endothelial progenitor cell (EPC) activities in vitro and on the mobilization of circulating EPCs, and reendothelialization in balloon-injured aorta of rats. After being isolated, cultured, and characterized, human EPCs were stimulated with resveratrol. We found that a low concentration of resveratrol (1 microM) led to significant enhanced activities of proliferation, migration, and adhesion, as well as promoting endothelial nitric acid synthetase (eNOS) expression in EPCs, whereas a high concentration (60 microM) inhibited the aforementioned functions and eNOS expression. In a rat model of injured aorta, a low dosage of resveratrol (10 mg/kg) increased the amount of EPCs in rat circulation as compared with placebo, whereas the result of a high dosage (50 mg/kg) did not reach statistical difference. In addition, 10 mg/kg of resveratrol both accelerated reendothelialization and inhibited neointimal formation; however, 50 mg/kg only reduced neointimal formation, which was not as effective as the previous one. eNOS expression in injured arteries was potently enhanced in the 10 mg/kg group, but not in the 50 mg/kg group.These findings suggest that a low dosage of resveratrol could markedly raise the proliferative, migrative, and adhesive activities of EPCs and upgrade eNOS expression in vitro as well as increase EPC mobilization, enhance eNOS expression, and accelerate the repair of injured artery; however, a high dosage cannot.     

Effects of treatment for diabetes mellitus on circulating vascular progenitor cells

The circulating endothelial progenitor cells (EPCs) have an important role in angiogenesis, and the smooth muscle progenitor cells (SMPCs) participate in atherosclerosis. However, little is known about the effects of treatment of diabetes mellitus (DM) on EPCs and SMPCs. Therefore, we investigated the relations between the number of circulating vasucular progenitor cells before and after the treatment for DM. Ten previously untreated DM patients were enrolled in this study. Blood samples were collected before and after treatment. The peripheral mononuclear cells were purified and cultured to differentiate them into EPCs and SMPCs. After two weeks, the number of EPCs was determined by Dil-labeled acetylated low density lipoprotein and lectin binding. The number of SMPCs was evaluated by immunocytochemical staining of alpha-smooth muscle actin. Before treatment, the number of EPCs and SMPCs was significantly related to hemoglobin A1c and blood sugar. Serial examination revealed that improvement of glycemic control significantly increased the number of both EPCs and SMPCs. DM reduces the number of circulating EPCs and SMPCs according to its severity, and treatment of DM significantly increases the number of EPCs and SMPCs, which may be involved in angiogenesis and atherosclerosis in diabetes.