HUVECs from newborns with a strong family history of diabetes show diminished ROS synthesis in the presence of high glucose concentrations

BACKGROUND: A family history of type 2 diabetes mellitus (DM) increases the probability to develop DM and endothelial dysfunction. The probable mechanism involves augmented reactive oxygen species (ROS) synthesis. The aim of this study was to evaluate the synthesis of ROS in human umbilical vein endothelial cells (HUVECs) obtained from healthy newborns with (experimental) and without (control) a strong family history of type 2 DM, exposed to different glucose concentrations. METHODS: HUVECs were exposed to various glucose concentrations for 24 and 48 h periods, before cell proliferation, mitochondrial activity, and mitochondrial membrane potential were determined. Intracellular ROS synthesis in the presence or absence of the mitochondrial uncoupler CCCP, cytochalasin B, or diphenyleneiodonium (DPI) was also evaluated. RESULTS: As opposed to control HUVECs, we found that experimental HUVECs exposed to 30 mmol/L glucose showed a 50% decrease in cell proliferation, a 90% reduction in mitochondrial activity, and a statistically significant inhibition of ROS synthesis in the presence of CCCP or cytochalasin B; DPI had no effect. CONCLUSIONS: Our results suggest that mitochondria and NAD(P)H-oxidase from HUVECs obtained from healthy newborns with a family history of DM have an innate deficient response to high glucose concentrations.     

HUVECs from newborns with a strong family history of diabetes show increased apoptosis by flow cytometry with annexin V

A family history of type 2 diabetes mellitus (DM) increases the probability to develop DM and endothelial dysfunction. Human umbilical vein endothelial cells (HUVECs) isolated from healthy newborns with familiar background of diverse diseases show early alterations such as less resistance to shear stress. The aim of this study was to evaluate the apoptosis by flow cytometry in HUVECs obtained from healthy newborns with (experimental) and without (control) a strong family history of DM, exposed to different glucose concentrations.MethodsHUVECs were incubated in M-199 culture media (containing a 5 mmol/L physiological glucose concentration) or supraphysiological glucose concentrations (15 or 30 mmol/L), for 48 h. Apoptosis was quantified by flow cytometry with annexin V and a polycaspase assay kit (FLICA, Immunochemistry Technologies LLC), and cell death was measurement by propidium iodide (PI, St Cruz Biotechnology Inc) positive stain.ResultsExperimental HUVECs showed higher levels of apoptosis in the presence of increasing glucose concentration (p < 0.01), whereas control HUVECs showed low levels of apoptosis.ConclusionsOur results suggest that HUVECs, isolated from healthy newborns with a strong family history of DM, have an abnormal predisposition to apoptosis.     

高糖诱导的大鼠肾小球系膜细胞中eNOS/NO的变化及调节机制研究

目的:探讨高糖条件下大鼠肾小球系膜细胞(GMCs)中内皮一氧化氮合酶一氧化氮(eNOS/NO)的变化及可能的调节机制.方法:将大鼠GMCs常规培养在含5.5 mmol/L葡萄糖的RPMIl640培养液中,用胰蛋白酶-乙二胺四乙酸(EDTA)混合消化酶传代.用RT、实时-PCR和Western blot测定GMCs中eN...     

体外研究胰岛素及磷脂酰肌醇3-激酶途径对一氧化氮生成的影响

目的探讨胰岛素及磷脂酰肌醇3-激酶(PI3-K)途径对NO生成的影响。方法检测胰岛素、葡萄糖以及PI3-K活性不可逆的抑制剂(Wortmannin)对培养的人脐静脉内皮细胞(HUVECs)PI3-K表达以及NO、超氧阴离子(O_2~-)产生和内皮型一氧化氮合酶(eNOS)活性的影响。实验分为对照组、10 mU/L胰岛素组、100 mU/L胰岛素组、甘露醇组、5 mmol/L葡萄糖+10 mU/L胰岛素组(5 mmol/L G1组)、25 mmol/L葡萄糖+100 mU/L胰岛素组(25 mmol/L G2组)、50 nmol/L Wortmannin组(50 nmol/L W组)、50 nmol/L Wortmannin+10 mU/L胰岛素组(50 nmol/L W1组)和50 nmol/L Wortmannin+100 mU/L胰岛素组(50 nmol/L W2组)。结果与对照组比较,不同浓度胰岛素组eNOS活性及NO水平显著升高(P<0.01);25 mmol/L G2组、50 nmol/L W组、50 nmol/LW1组和50 nmol/L W2组eNOS活性及NO水平均显著降低,O_2~-生成明显增加(P<0.01);与对照组比较,不同浓度胰岛组、50 nmol/L W组、50 nmol/L W1组和50 nmol/L W2组PI3-K蛋白表达显著升高(P<0.05,P<0.01)。结论 PI3-K信号途径对于促进NO产生、维持血管内皮细胞的正常功能具有重要作用,在高糖、高胰岛素状态下该条途径受损并由此引发内皮功能障碍。     

前列腺素EI对人脐静脉内皮细胞中NO和eNOS的影响

目的探讨前列腺素EI(prostaglandin EI,PGEI)对内皮细胞一氧化氮(NO)表达和内皮型一氧化氮合酶(eNOS)活性的影响。方法以人脐静脉内皮细胞(HUVEC)为实验对象,检测不同浓度PGEI作用不同时间后,细胞培养上清液和细胞中NO水平的变化,以及细胞eNOS活性的改变。结果(1)随着PGEI浓度的升高,eNOS的活性和NO的含量均逐渐增加(P<0.05);(2)短时间PGEI的干预对eNOS和NO的影响均不明显,24h后细胞中eNOS活性明显升高(P<0.05),NO的含量自12h起随时间延长而增加(P<0.05);(3)用不同PGEI浓度预处理,使TNF-α对eNOS活动的抑制作用减弱。结论PGEI可能通过诱导eNOS的表达,促进NO的释放,且可以重新激活被TNF-α抑制的eNOS活性。     

Insulin and lysophosphatidylcholine synergistically stimulate NO-dependent cGMP production in human endothelial cells.

AIMS: Nitric oxide (NO) is an important regulator of cardiovascular homeostasis. Lysophosphatidylcholine (lyso-PC), a major constituent of oxidized low density lipoproteins (oxLDL), has been reported to impair nitric oxide-dependent vasodilatation. This study investigated the possible mechanism of the lyso-PC effect on insulin-stimulated NO-dependent of cyclic guanosine 3',5'-monophosphate (cGMP) generation in human endothelial cells. METHODS: The intracellular concentration of cGMP in cultured human umbilical vein endothelial cells (HUVECs) was used to estimate NO production. The levels of endothelial nitric oxide synthase (eNOS) protein expression were assessed by Western blotting analyses. RESULTS: Both insulin, at physiological concentration, and lyso-PC stimulated rapid and prolonged intracellular of cGMP production, and together induced a marked synergistic response (for short-term stimulation: 1185 +/- 285.9% over control level (100%) compared with insulin and lyso-PC alone (384.8 +/- 67.4% and 357 +/- 205%, respectively; P < 0.001), for long-term stimulation: 3495 +/- 1377%, compared with insulin and lyso-PC alone (663 +/- 131% and 487 +/- 250%, P = 0.002)). Stimulated levels of cGMP accumulation were completely abrogated by NOS inhibitor, indicating NO involvement in the effects of insulin and lyso-PC. Stimulated NO synthesis was not associated with altered eNOS protein expression. Cell subfractionation studies demonstrate that insulin and lyso-PC each alone induced translocation of eNOS from the membrane to the cytosolic compartment and together caused a synergistic translocation. CONCLUSIONS: The presented data suggest that insulin and lyso-PC synergistically upregulate endothelial NO production via eNOS translocation from the membrane fraction to the cytosol. This study raises the possibility that an interplay between various factors accompanying diabetes can lead to endothelial NO overproduction or desensitization of NO-dependent responses. Appropriate rather than necessarily high levels of nitric oxide is the determinant of vascular health.     

非诺贝特通过上调三磷酸鸟苷环化水解酶Ⅰ促进eNOS复偶联

目的探讨非诺贝特发挥降脂外血管内皮保护作用的机制。方法体外培养人脐静脉内皮细胞（HU-VECs）,非诺贝特预处理HUVECs 2 h,再与脂多糖（LPS）共孵育24 h。采用Western blot检测三磷酸鸟苷环化水解酶Ⅰ（GTPCH-Ⅰ）的表达水平,高效液相色谱法检测四氢生物蝶呤（BH4）的表达水平,ELISA检测细胞上清一氧化氮（NO）浓度,利用Confocal方法检测细胞活性氧（ROS）产生水平。结果非诺贝特预处理后,较单纯LPS刺激组,细胞内BH4表达水平及细胞上清NO产生增多,伴有细胞内ROS产生减少（P均〈0.05）;此外,单独给予非诺贝特处理内皮细胞后,可见浓度依赖性上调细胞GTPCH-Ⅰ的表达,非诺贝特（10μmol/L）上调GTPCH-Ⅰ的表达在12 h达到高峰。结论非诺贝特通过上调内皮细胞GTPCH-Ⅰ的表达而增加BH4的水平,进而促进内皮型一氧化氮合酶的复偶联,改善血管内皮功能。     

利拉鲁肽通过抑制核因子κB p65磷酸化调控人脐静脉内皮细胞一氧化氮合酶的表达

目的 探讨利拉鲁肽在高糖环境下对人脐静脉内皮细胞（HUVECs）功能的影响及其可能机制.方法 高糖环境下（25 mmol/L葡萄糖）培养人脐静脉内皮细胞,分别给予10、100、1 000 μg/L利拉鲁肽进行干预,采用实时定量聚合酶链反应（RT-PCR）和Western blotting法分别检测内皮型一氧化氮合酶（eNOS）、诱导型一氧化氮合酶（iNOS）以及核因子κB p65 （NF-κB p65）的mRNA、蛋白表达水平;应用肿瘤坏死因子-α（TNF-α）干预利拉鲁肽处理后的HUVECs,观察eNOS、iNOS、NF-κB p65的mRNA、蛋白表达水平的变化.研究结果多组间采用单因素方差分析,两组间比较采用SLD分析.结果 与普通培养基（7 mmol/L葡萄糖）组相比,高糖环境下HUVECs的eNOS mRNA和蛋白表达均降低,iNOS mRNA和蛋白表达及磷酸化NF-κB p65蛋白表达均增高（t=2.79、5.75、4.32、4.85、7.12,均P＜0.05）.与0μg/L组相比,1 000μg/L利拉鲁肽干预组HUVECs的eNOS mRNA、eNOS蛋白表达均上调,iNOS mRNA、iNOS蛋白表达及NF-κB p65磷酸化蛋白表达均下调（t=5.12、9.34、6.70、5.50、8.94,均P＜0.05）.与单独使用利拉鲁肽组相比,TNF-α联合利拉鲁肽组HUVECs的eNOSmRNA和蛋白表达均下调,iNOS mRNA和蛋白表达及磷酸化NF-κB p65蛋白表达均上调（t=3.33～7.87,均P＜0.05）.结论 利拉鲁肽可通过抑制NF-κB p65磷酸化在转录和翻译水平上增加eNOS表达、降低iNOS的表达,从而改善内皮细胞功能,预防糖尿病动脉粥样硬化.     

同型半胱氨酸对人脐静脉内皮细胞eNOS表达及NO含量的影响

目的 通过观察同型半胱氨酸(Hcy)对人脐静脉内皮细胞(HUVECs)一氧化氮合酶(eNOS)表达及NO含量的影响,探讨Hcy诱导动脉粥样硬化形成的病理机制.方法 将体外培养的人脐静脉内皮细胞分为5组,分别加入Hcy0、2.5、5、10及15 mmol/L,培养24h.通过MTT比色法测定细胞活力,采用RT-PCR法检测eNOS mRNA的表达,通过硝酸还原酶法测NO含量.结果 与对照组比较,Hcy(5 ～ 15 mmol/L)剂量处理组细胞活力明显下降(P＜0.05,P＜0.01),eNOS mRNA的表达显著降低(P＜0.01);NO的含量明显下降(P＜0.01),eNOS mRNA的表达变化及NO的含量变化呈现明显的剂量依赖性(P＜0.01).结论 Hcy可通过抑制eNOS mRNA的表达,使eNOS合成减少,活性降低,进而减少HUVECs内NO的生成,这可能是Hcy诱导动脉粥样硬化形成的病理机制之一.     

肿瘤坏死因子相关的凋亡诱导配体对2型糖尿病大鼠内皮依赖性血管舒张功能的影响

目的 观察TNF相关的凋亡诱导配体（TRAIL）对T2DM大鼠主动脉内皮依赖性血管舒张功能的影响及其可能的机制. 方法 选取4周龄雄性SD大鼠,分为正常对照组（NC,n=10）与模型组（n=40）,随机将模型组分为T2DM亚组（n=10）与TRAIL亚组（TRAIL,n=10）.TRAIL干预6周后,检测各组FPG及FIns水平,计算ISI.检测NC组与TRAIL亚组血清TRAIL水平.观察大鼠离体主动脉内皮依赖性血管舒张反应,并检测主动脉一氧化氮（NO）含量、内皮型一氧化氮合酶活性（eNOS）.结果 与NC组比较,T2DM亚组FPG、FIns水平升高（P＜0.01）,ISI降低（P＜0.01）,血清TRAIL水平降低（P＜0.01）;血管内皮功能失调,乙酰胆碱（Ach）引起的血管最大舒张率降低（P＜0.01）,血管中NO含量及eNOS阳性表达降低（P＜0.01或P＜0.01）.TRAIL亚组血管Ach的舒张反应改善（P＜0.01）;血管中NO含量增加且eNOS阳性表达上调（P＜0.05或P＜0.01）;FPG、FIns降低,ISI提高（P＜0.01）. 结论 TRAIL改善T2DM大鼠内皮依赖性血管舒张功能的同时,可促进具有血管保护作用的NO生成.     

氟伐他汀对人脐静脉内皮细胞游离钙离子水平及内皮型一氧化氮合酶活性的影响

目的:观察氟伐他汀对人脐静脉内皮细胞(HUVECs)游离钙离子水平及内皮型一氧化氮合酶(eNOS)活性的影响及可能机制。方法:体外培养HUVECs,随机分为5组:空白对照组,氟伐他汀(10-8,10-7,10-6,10-5mol/L)组。采用硝酸还原酶法测定细胞上清液中NO含量,液体闪烁计数仪测定L-[3H]-精氨酸和L-[3H]-瓜氨酸的含量,用激光共聚焦扫描显像系统检测内皮细胞内游离钙离子浓度([Ca2 ]i)水平的变化。结果:与空白对照组比较,10-8,10-7,10-6,10-5mol/L氟伐他汀孵育细胞12h后可显著升高HUVECs细胞内eNOS活性,促进NO释放,同时伴有[Ca2 ]i升高,且呈浓度依赖性。另外,10-5mol/L氟伐他汀在0～12h时间段呈时间依赖性增高eNOS活性,作用12h使eNOS活性达到最高(P<0.01)。结论:氟伐他汀呈浓度依赖性升高HUVECseNOS活性和促进NO释放,该作用与其增加内皮细胞内[Ca2 ]i有关。     

High glucose mediates pro-oxidant and antioxidant enzyme activities in coronary endothelial cells

AIM: Excess levels of free radicals such as nitric oxide (NO) and superoxide anion (O(2)(-)) are associated with the pathogenesis of endothelial cell dysfunction in diabetes mellitus. This study was designed to investigate the underlying causes of oxidative stress in coronary microvascular endothelial cells (CMECs) exposed to hyperglycaemia. METHODS: CMECs were cultured under normal (5.5 mmol/l) or high glucose (22 mmol/l) concentrations for 7 days. The activity and expression (protein level) of endothelial NO synthase (eNOS), inducible NOS (iNOS), NAD(p)H oxidase and antioxidant enzymes, namely, superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) were investigated by specific activity assays and Western analyses, respectively, while the effects of hyperglycaemia on nitrite and O(2)(-) generation were investigated by Griess reaction and cytochrome C reduction assay, respectively. RESULTS: Hyperglycaemia did not alter eNOS or iNOS protein expressions and overall nitrite generation, an index of NO production. However, it significantly reduced the levels of intracellular antioxidant glutathione by 50% (p < 0.05) and increased the protein expressions and activities of p22-phox, a membrane-bound component of pro-oxidant NAD(p)H oxidase and antioxidant enzymes (p < 0.05). Free radical scavengers, namely, Tiron and mercaptopropionylglycine (MPG) (0.1-1 micromol/l) reduced hyperglycaemia-induced antioxidant enzyme activity and increased glutathione and nitrite generation to the levels observed in CMEC cultured in normoglycaemic medium (p < 0.01). The differences in enzyme activity and expressions were independent of the increased osmolarity generated by high glucose levels as investigated by using equimolar concentrations of mannitol in parallel experiments. CONCLUSIONS: These results suggest that hyperglycaemia-induced oxidative stress may arise in CMEC as a result of enhanced pro-oxidant enzyme activity and diminished generation of antioxidant glutathione. By increasing the antioxidant enzyme capacity, CMEC may protect themselves against free radical-induced cell damage in diabetic conditions.     

非诺贝特促进人内皮细胞内皮一氧化氮合酶复偶联的作用研究

目的探讨非诺贝特能否对脂多糖（LPS）诱导的血管内皮一氧化氮合酶（eNOS）脱偶联发挥保护作用。方法体外培养人脐静脉内皮细胞（HUVECs）,用非诺贝特预处理HUVECs 2 h,再与LPS共孵育24 h,采用高效液相色谱法检测细胞四氢生物蝶呤（BH4）的表达水平,ELISA检测细胞eNOS表达水平和细胞上清一氧化氮（NO）浓度,利用Confocal方法检测细胞内活性氧（ROS）产生水平。结果与对照组比较,单纯LPS刺激组内皮细胞BH4表达水平降低,伴有eNOS表达下调和NO水平降低,而内皮细胞内ROS产生增加（P均〈0.05）。与单纯LPS刺激组比较,非诺贝特预处理组内皮细胞BH4表达水平升高,同时伴有eNOS表达上调和NO水平增加,而内皮细胞内ROS产生降低（P均〈0.05）。结论非诺贝特通过上调BH4水平,对LPS诱导的血管内皮细胞eNOS脱偶联有逆转作用,这可能是其发挥血管内皮保护作用的机制之一。     

Nitric oxide stimulates glucose transport through insulin-independent GLUT4 translocation in 3T3-L1 adipocytes

OBJECTIVE: It is well known that nitric oxide synthase (NOS) is expressed and that it modulates glucose transport in skeletal muscles. Recent studies have shown that adipose tIssues also express inducible and endothelial nitric oxide synthase (eNOS). In the present study, we investigated whether nitric oxide (NO) induces glucose uptake in adipocytes, and the signaling pathway involved in the NO-stimulated glucose uptake in 3T3-L1 adipocytes. METHODS: First, we determined the expression of eNOS in 3T3-L1 adipocytes, and then these cells were treated with the NO donor sodium nitroprusside (SNP) and/or insulin, and glucose uptake and phosphorylation of insulin receptor substrate (IRS)-1 and Akt were evaluated. Moreover, we examined the effects of a NO scavenger, a guanylate cyclase inhibitor or dexamethasone on SNP-stimulated glucose uptake and GLUT4 translocation. RESULTS: SNP at a concentration of 50 mmol/l increased 2-deoxyglucose uptake (1.8-fold) without phosphorylation of IRS-1 and Akt. Treatment with the NO scavenger or guanylate cyclase inhibitor decreased SNP-stimulated glucose uptake to the basal level. Dexamethasone reduced both insulin- and SNP-stimulated glucose uptake with impairment of GLUT4 translocation. CONCLUSION: NO is capable of stimulating glucose transport through GLUT4 translocation in 3T3-L1 adipocytes, via a mechanism different from the insulin signaling pathway.     

Globular adiponectin upregulates nitric oxide production in vascular endothelial cells

Aims/hypothesis Adiponectin, also called ACRP30, is a novel adipose tissue-specific protein that has been shown to improve insulin sensitivity and to exert anti-atherogenic effects. It is known that knockout mice lacking endothelial NO synthase (eNOS) develop hypertension, insulin resistance, hyperlipidaemia, and show augmented ischaemia-reperfusion damage. Thus, we examined whether globular adiponectin activates eNOS to produce NO.Methods To analyze NO production in bovine aortic endothelial cells (BAE), NOx (nitrite and nitrate) was measured in the medium with an automated NO detector/high-performance liquid chromatography system. eNOS activation was assessed by phosphorylation of the enzyme and its activity was evaluated by citrulline synthesis in human umbilical vein endothelial cells (HUVEC). eNOS mRNA and protein expressions in HUVEC were evaluated by Realtime PCR and Western blot analysis.Results Gobular adiponectin increased NO production in BAE. It also caused eNOS phosphorylation and potentiated eNOS activity in HUVEC. In addition, globular adiponectin up-regulated the eNOS gene to increase protein expression in HUVEC.Conclusion/interpretation Globular adiponectin increases NO production through two mechanisms, namely, by activation of eNOS enzyme activity and via an increase in eNOS expression. Activation and up-regulation of eNOS could explain some of the observed vasoprotective properties of globular adiponectin, as well as its beneficial effects on the cardiovascular system.Abbreviations NO nitric oxide - eNOS endothelial NO synthase - BAE bovine aortic endothelial cells - HUVEC human umbilical vein endothelial cells - ACRP30 adipocyte complement-related protein of 30 kDa - GAPDH glyceraldehyde-3-phosphate dehydrogenase     

The impact of estrogen and L-arginine on serum NO and eNOS expression in aorta of aged rats

Background Cardiovascular diseases (CVD) are less prevalent in postmenopausal women received estrogen replacement therapy (ERT) than those who did not receive ERT.Previous study has shown that the increase of nitric oxide (NO) synthesis is one of the cardioprotective effects of estrogen.This study investigated the effects of estrogen and L-arginine (L-Arg) on serum NO concentrations and the possible regulatory role in endothelial nitric oxide synthase (eNOS) expression in aortas of aged rats.Methods Fifty aged female wistar rats (18-20 months) were randomly divided into five groups (n=10):Sham group (sham operated,0.9 % NaCl 10 μg every three day for 4 months),OVX group (ovariectomized,0.9 % NaCl 10 μg i.m every three day for 4 months),OVE group (ovariectomized + 17β-estradiol 10 μg i.m every three day for 4 months),OVE + L-Arg group (ovariectomized + 17β-estradiol 10ug i.m every three day + 2.25 % L-Arg contained in drinking water every day for 4 months) and L-Arg group (ovariectomized + 2.25 % L-Arg contained in drinking water every day for 4 months).NO concentration and the expression of eNOS mRNA in aorta were measured after 4 months.Results Serum NO synthesis did not alter after ovariectomized (P=0.362),but were increased in OVE group,L-Arg group and OVE + L-Arg group compared with OVX group (P < 0.05,P < 0.05,P < 0.01,respectively).NO concentration also increased in OVE + L-Arg group when compared with OVE group (P < 0.05) or L-Arg group (P < 0.05).There was no significant difference in eNOS mRNA expression in aortas of aged rats between sham,OVX,OVE,OVE + L-Arg and L-Arg group (F=0.550,P=0.700).Conclusions Estrogen treatment and L-Arg supplementation increase serum NO synthesis,but do not upregulate eNOS mRNA expression in aortas of aged rats.     

Aging enhances the sensitivity of endothelial cells toward apoptotic stimuli: important role of nitric oxide

Advanced aging leads to impaired endothelial NO synthesis and enhanced endothelial cell apoptosis; therefore, we investigated the sensitivity of aged endothelial cells toward apoptotic stimuli and determined the role of NO. Human umbilical vein endothelial cells (HUVECs) were cultured until 14th passage. In aged cells, oxLDL and tumor necrosis factor-alpha-induced apoptosis and caspase-3-like activity were significantly enhanced more than 3-fold compared with young cells (passage 3). Because NO contributes to protection against endothelial cell death via S-nitrosylation of caspases, we determined endothelial NO synthase (eNOS) protein expression and the content of S-nitrosylated proteins. Aged HUVECs showed significantly reduced eNOS expression (35+/-10%) and a decrease in the overall S-NO content (33+/-3%), suggesting that eNOS downregulation may be involved in age-dependent increase of apoptosis sensitivity. Indeed, eNOS knockout endothelial cells showed a significantly enhanced apoptosis induction. Exogenous NO donors abolished increased apoptosis and caspase-3-like activity. In contrast, the application of shear stress, which exerts a profound apoptosis inhibitory effect via upregulation of NO synthesis in young cells, failed to inhibit apoptosis in aged cells. Moreover, no upregulation of eNOS protein expression and S-NO content in response to shear stress was detected in aged cells. Overexpression of wild-type eNOS completely restored the antiapoptotic effect of shear stress, whereas only a partial inhibitory effect was detected under steady conditions. Strikingly, transfection of constitutively active phosphomimetic eNOS (S1177D) further abrogated apoptosis in aged HUVECs. Thus, aging of endothelial cells is associated with decreased NO synthesis and concomitantly increased sensitivity of apoptosis, which may contribute to functional impairment of the endothelial monolayer.     

抵抗素对内皮细胞NO生成的影响及Akt信号机制

目的探讨抵抗素对内皮细胞NO生成的影响及其可能的信号机制。方法分离、培养人脐静脉内皮细胞（HUVECs）,以不同浓度抵抗素（15、50、100ng／ml）干预。荧光显微镜检测各组细胞中N0的生成,RT-PCR检测eNOS mRNA表达水平,Western blot检测Akt和eNOS磷酸化水平。结果15、50、100ng／ml抵抗素干预HUVECs 24h后,胰岛素刺激的内皮NO生成显著降低（三组分别为4．01±0．69、3．764±0．71、3．73±0．45,vs对照组P均〈0．05）,同时伴有内皮Akt和eNOS磷酸化水平的降低（us对照组P均〈0．05）,而eNOS mRNA表达无显著改变。结论抵抗素可通过P13K／Akt途径影响HuVECs eNOS磷酸化水平,进而调节内皮细胞NO生成,但该影响并非Akt依赖性。     

Decreased nitric oxide synthesis in human endothelial cells cultured on type I collagen

Endothelial dysfunction, considered as a defective vascular dilatation after certain stimuli, is characteristic of different pathological conditions, such as hypertension, atherosclerosis, or diabetes. A decreased synthesis or an increased degradation of nitric oxide (NO) has been postulated as the mechanism responsible for this alteration. The present experiments were designed to test the hypothesis that the presence of an abnormal extracellular matrix in vessel walls could be responsible for the decreased NO synthesis observed in these pathological conditions. Experiments were performed in cultured human umbilical vein endothelial cells (HUVECs) grown on type IV (Col. IV) or type I (Col. I) collagen. Cells seeded on Col. I showed decreased nitrite synthesis, nitric oxide synthase activity, eNOS protein content, and eNOS mRNA expression when compared with cells grown on Col. IV. Moreover, cells grown on Col. I failed to respond to glucose oxidase activation of the eNOS system. In both cases, the changes in the eNOS mRNA expression seemed to depend on the modulation of eNOS promoter activity. The downregulation of eNOS induced by Col. I was blocked by D6Y, a peptide that interferes with the Col. I-dependent signals through integrins, as well as by specific anti-integrin antibodies. Moreover, a decreased activation of integrin-linked kinase (ILK) may explain the effects observed in Col. I-cultured cells because the activity of this kinase was decreased in these cells and ILK modulation prevented the Col. I-induced changes in HUVECs. Taken together, these findings may contribute to explaining the basis of endothelial dysfunction in some vascular diseases.