Statins blunt thrombin-induced down-regulation of endothelial nitric oxide synthase expression in human endothelial cells

Thrombin plays a pivotal role in the pathophysiology of acute coronary syndromes by mediating thrombus formation and endothelium-dependent vasomotor dysfunction. In human endothelial cells, prolonged incubation with thrombin down-regulates endothelial nitric oxide synthase (eNOS) expression via activation of Rho. Statins are effective in patients with acute coronary syndromes. These beneficial effects are attributed to their pleiotropic effects and also to an improved lipid profile. We hypothesized that statins may prevent the down-regulation of eNOS induced by thrombin in human endothelial cells. Human umbilical vein endothelial cells were used. Expression and activity of eNOS protein were evaluated by Western blotting and L-citrulline assay, respectively. Rho A membrane translocation was evaluated by Wesern blotting after fractionation. Stimulation of human umbilical vein endothelial cells with thrombin (4 U/mL, 24 h) significantly decreased eNOS expression. The addition of simvastatin significantly prevented thrombin-induced down-regulation of eNOS expression in a concentration-dependent manner (100 nmol/L to 10 micromol/L). Cerivastatin (10 micromol/L) also reversed the down-regulation of eNOS by thrombin. Both simvastatin and cerivastatin-blocked thrombin-induced decrease in NOS activity. Stimulation with thrombin (4 U/mL, 10 min) significantly increased the membrane translocation of Rho A. Simvastatin (10 micromol/L) and cerivastatin (10 micromol/L) significantly decreased thrombin-induced membrane translocation of Rho A. Therefore, statins blunt thrombin-induced down-regulation of eNOS expression in human endothelial cells. This finding provides a novel mechanism of the pleiotropic effects of statins, which may be beneficial for patients with acute coronary syndromes.     

Regulation of endothelial nitric oxide synthase and endothelin-1 expression by fluvastatin in human vascular endothelial cells

We investigated the effects of fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, on endothelial vasoactive substances using human umbilical vein endothelial cells (HUVECs). Incubation of HUVECs with fluvastatin for 12 h increased endothelial nitric oxide synthase (eNOS) mRNA expression in a concentration-dependent manner (peak, 276 +/- 38%, mean +/- S.D., of the control, at 1.0 microM fluvastatin, P<0.01). In addition, fluvastatin increased eNOS protein production (245 +/- 51% of the control level, P<0.05) as well as nitrite production (165 +/- 35% of the control level, P<0.01). In contrast, incubation of HUVECs with 1.0 microM fluvastatin for 12 h significantly reduced the production of endothelin-1 (ET-1) and preproET-1 mRNA expression in HUVECs (28 +/- 1% and 39 +/- 1% of the control level, respectively, P<0.01). Our results suggest that fluvastatin might be involved in improvement of endothelial function and prevention of the progression of atherosclerosis.     

Polychlorinated biphenyls alter the expression of endothelial nitric oxide synthase mRNA in human umbilical vein endothelial cells

Polychlorinated biphenyls (PCBs) are a group of persistent pollutants that are detected in maternal serum and umbilical cord, suggesting that fetal exposure also needs to be considered. The effects of dioxin-like PCB congeners 3,3',4,4'-tetrachlorobiphenyl (PCB77) and 3,3',4,4',5-pentachlorobiphenyl (PCB126) and a non-dioxin-like compound 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153) on the expression of endothelial nitric oxide synthase (eNOS), known to maintain blood flow to the fetus, in human umbilical vein endothelial cells (HUVECs) were investigated. The mRNA levels of eNOS, aryl hydrocarbon receptor (AhR) and cytochrome P450 (CYP) 1A1 in cells treated with 5 microM PCBs for 24 hours were analysed by real-time RT-PCR. Cells were also treated with alpha-naphthoflavone (alpha NF), an AhR antagonist or ICI 182780, an estrogen receptor (ER) antagonist, one hour prior to PCB exposure, to observe the effects of these receptors on eNOS modulation. Each PCB increased the eNOS mRNA level by 4.5-fold that was markedly inhibited by alphaNF. ERs were also suspected of altering eNOS levels because ICI 182780 treatment resulted in a decrease in the eNOS level. These results suggest that the eNOS mRNA expression increases due to the action of PCBs related to both AhR and ERs in HUVECs, and that maternal PCB exposure could influence fetal circulation.     

Ethanol consumption increases the expression of endothelial nitric oxide synthase, inducible nitric oxide synthase and metalloproteinases in the rat kidney

Objectives The effects of longterm ethanol consumption on the levels of nitric oxide (NO) and the expression of endothelial NO synthase (eNOS), inducible NO synthase (iNOS) and metalloproteinase‐2 (MMP‐2) were studied in rat kidney. Methods Male Wistar rats were treated with 20% ethanol (v/v) for 6 weeks. Nitrite and nitrate generation was measured by chemiluminescence. Protein and mRNA levels of eNOS and iNOS were assessed by immunohistochemistry and quantitative real‐time polymerase chain reaction, respectively. MMP‐2 activity was determined by gelatin zymography. Histopathological changes in kidneys and indices of renal function (creatinine and urea) and tissue injury (mitochondrial respiration) were also investigated. Results Chronic ethanol consumption did not alter malondialdehyde levels in the kidney. Ethanol consumption induced a significant increase in renal nitrite and nitrate levels. Treatment with ethanol increased mRNA expression of both eNOS and iNOS. Immunohistochemical assays showed increased immunostaining for eNOS and iNOS after treatment with ethanol. Kidneys from ethanol‐treated rats showed increased activity of MMP‐2. Histopathological investigation of kidneys from ethanol‐treated animals revealed tubular necrosis. Indices of renal function and tissue injury were not altered in ethanol‐treated rats. Conclusions Ethanol consumption increased renal metalloproteinase expression/activity, which was accompanied by histopathological changes in the kidney and elevated NO generation. Since iNOS‐derived NO and MMPs contribute to progressive renal injury, the increased levels of NO and MMPs observed in ethanol‐treated rats might contribute to progressive renal damage.     

Feedback inhibition of nitric oxide synthase activity by nitric oxide.

1. A murine macrophage cell line, J774, expressed nitric oxide (NO) synthase activity in response to interferon-gamma (IFN-gamma, 10 u ml-1) plus lipopolysaccharide (LPS, 10 ng ml-1). The enzyme activity was first detectable 6 h after incubation, peaked at 12 h and became undetectable after 48 h. 2. The decline in the NO synthase activity was not due to inhibition by stable substances secreted by the cells into the culture supernatant. 3. The decline in the NO synthase activity was significantly slowed down in cells cultured in a low L-arginine medium or with added haemoglobin, suggesting that NO may be involved in a feedback inhibitory mechanism. 4. The addition of NO generators, S-nitroso-acetyl-penicillamine (SNAP) or S-nitroso-glutathione (GSNO) markedly inhibited the NO synthase activity in a dose-dependent manner. The effect of NO on the enzyme was not due to the inhibition of de novo protein synthesis. 5. SNAP directly inhibited the inducible NO synthase extracted from activated J774 cells, as well as the constitutive NO synthase extracted from the rat brain. 6. The enzyme activity of J774 cells was not restored after the removal of SNAP by gel filtration, suggesting that NO inhibits NO synthase irreversibly.     

高糖抑制牛主动脉内皮细胞诱导型和结构型一氧化氮合酶的表达

目的:研究高糖对新生小牛主动脉内皮细胞(BAEC)一氧化氮合酶(NOS)表达的影响。方法:BAEC培养并传代于含正常葡萄糖(5.5mmol·L~(-1),NGBAEC),高糖(25 mmol·L~(-1),HG-BAEC)或高渗(葡萄糖5.5 甘露醇19.5 mmol·L~(-1),Mann-BAEC)的无酚红M1640培养基.Griess反应检测脂多糖(LPS)诱导的一氧化氮(NO)产生.Westem blot法检测结构型NOS(ecNOS)及诱导型NOS(iNOS)表达。结果:LPS(0.25-2 mg·L~(-1))剂量依赖性刺激BAEC产生NO,并在LPS 1 mg·L~(-1)达峰值。高糖显著抑制LPS诱导的NO产生(亚硝酸μmol·L~(-1):HG-BAEC 43±8,vs NG-BAEC 71±11,Mann-BAEC 70±9,n=4,P<0.01)。同样,与NG-和Mann-BAEC相比,HG-BAEC iNOS表达下降39.9％和39.3％,ecNOS表达下降28％和24％,而NG-与Mann-BAEC之间,LPS诱导的NO产量和iNOS和ecNOS的表达无差别。结论:高糖抑制BAEC NO的释放,与NOS的低表达有关。     

Glucagon-like peptide-1 activates endothelial nitric oxide synthase in human umbilical vein endothelial cells

Aim:

To investigate the effects of glucagon-like peptide-1 (GLP-1) on endothelial NO synthase (eNOS) in human umbilical vein endothelial cells (HUVECs), and elucidate whether GLP-1 receptor (GLP-1R) and GLP-1(9–36) are involved in these effects.

Methods:

HUVECs were used. The activity of eNOS was measured with NOS assay kit. Phosphorylated and total eNOS proteins were detected using Western blot analysis. The level of eNOS mRNA was quantified with real-time RT-PCR.

Results:

Incubation of HUVECs with GLP-1 (50–5000 pmol/L) for 30 min significantly increased the activity of eNOS. Incubation of HUVECs with GLP-1 (500–5000 pmol/L) for 5 or 10 min increased eNOS phosphorylated at ser-1177. Incubation with GLP-1 (5000 pmol/L) for 48 h elevated the level of eNOS protein, did not affect the level of eNOS mRNA. GLP-1R agonists exenatide and GLP-1(9–36) at the concentration of 5000 pmol/L increased the activity, phosphorylation and protein level of eNOS. GLP-1R antagonist exendin(9–39) or DPP-4 inhibitor sitagliptin, which abolished GLP-1(9–36) formation, at the concentration of 5000 pmol/L partially blocked the effects of GLP-1 on eNOS.

Conclusion:

GLP-1 upregulated the activity and protein expression of eNOS in HUVECs through the GLP-1R-dependent and GLP-1(9–36)-related pathways. GLP-1 may prevent or delay the formation of atherosclerosis in diabetes mellitus by improving the function of eNOS.     

Mechanisms underlying nebivolol-induced endothelial nitric oxide synthase activation in human umbilical vein endothelial cells

1. Nebivolol (NEB) has been shown to be a selective blocker of beta1-adrenoceptors with additional vasodilating properties that are mediated, at least in part, by an endothelial-dependent liberation of nitric oxide (NO). In the present study, we investigated the underlying mechanisms of NEB-induced vasodilation. 2. Immunohistochemical staining of endothelial nitric oxide synthase (eNOS) was performed in the absence and presence of NEB in human umbilical vein endothelial cells (HUVEC). In addition, we measured the release of nitric oxide (NO) using diaminofluorescein. Metoprolol (MET) was used for comparison. 3. Nebivolol, but not MET (each at 10 micromol/L), caused a time-dependent increase in NO release from HUVEC, as demonstrated by an increase in DAF fluorescence at 0 versus 10 min (+234 +/- 7 and 55 +/- 22% basal, respectively). Blockade of beta3-adrenoceptors by SR 59230A (1 micromol/L) partially reduced the NEB-induced increase in DAF fluorescence. Complete inhibition of NEB-induced NO liberation was achieved by the simultaneous blockade of beta3-adrenoceptors and oestrogen receptors (with 1 micromol/L ICI 182,780). 4. Application of NEB significantly increased eNOS translocation and serine 1177 phosphorylation of eNOS. However, NEB did not alter eNOS-phosphorylation at threonine 495 and at serine 114. 5. In conclusion, the endothelium-dependent NO liberation induced by NEB is due to stimulation of beta3-adrenoceptors and oestrogen receptors and coincides with eNOS translocation and a phosphorylation at eNOS-serine 1177. These characteristics of NEB may be beneficial not only when treating patients suffering from cardiovascular disease, but may also prevent further deterioration of endothelial dysfunction.     

Down-regulation of endogenous nitric oxide synthase inhibitors on endothelial SK3 expression

ObjectivesTo investigate role of endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) in down-regulation of the expression of endothelial SK3 in atherosclerosis.MethodsApolipoprotein E deficient (apo E^−/−) mice aged 11 ∼ 12 weeks were treated with ADMA (5 mg/kg per day, subcutaneous injection) for 4 weeks. Cultured human umbilical venous endothelial cells (HUVECs) were treated with different concentrations of lysophosphatidylcholine (LPC) or ADMA for 48 h. Plasma levels of ADMA were determined by high performance liquid chromatogram (HPLC); protein and mRNA levels of SK3 in the aortas of mice and cultured cells were detected by immunofluorescence, western blot and RT-PCR, respectively.ResultsConcomitantly with the elevated plasma levels of ADMA, the expressions of both SK3 protein and mRNA in aortas of apo E^−/− mice were significantly reduced in comparison to those of the wild-type mice. Moreover, 4-week treatment of ADMA made levels of SK3 expression even lower. In cultured HUVECs, either LPC or ADMA notably decreased the expressions of both SK3 protein and mRNA in a concentration dependent manner.ConclusionsEndogenous ADMA may be an important factor for down-regulation of the expression of endothelial SK3 in atherosclerotic animals.     

Down-regulation of endogenous nitric oxide synthase inhibitors on endothelial SK3 expression

OBJECTIVES: To investigate role of endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) in down-regulation of the expression of endothelial SK3 in atherosclerosis. METHODS: Apolipoprotein E deficient (apo E(-/-)) mice aged 11 approximately 12 weeks were treated with ADMA (5 mg/kg per day, subcutaneous injection) for 4 weeks. Cultured human umbilical venous endothelial cells (HUVECs) were treated with different concentrations of lysophosphatidylcholine (LPC) or ADMA for 48 h. Plasma levels of ADMA were determined by high performance liquid chromatogram (HPLC); protein and mRNA levels of SK3 in the aortas of mice and cultured cells were detected by immunofluorescence, western blot and RT-PCR, respectively. RESULTS: Concomitantly with the elevated plasma levels of ADMA, the expressions of both SK3 protein and mRNA in aortas of apo E(-/-) mice were significantly reduced in comparison to those of the wild-type mice. Moreover, 4-week treatment of ADMA made levels of SK3 expression even lower. In cultured HUVECs, either LPC or ADMA notably decreased the expressions of both SK3 protein and mRNA in a concentration dependent manner. CONCLUSIONS: Endogenous ADMA may be an important factor for down-regulation of the expression of endothelial SK3 in atherosclerotic animals.     

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。     

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.     

Inducible nitric oxide synthase inhibition by mycophenolic acid

The focus of this review is the influence of an immunosuppressive xenobiotic drug mycophenolic acid on the induction of nitric oxide production in various cell types. The potential therapeutic significance of the cell-specific fine-tuning of nitric oxide release by mycophenolic acid, as well as the mechanisms behind the drug action are discussed.     

Effects of simvastatin on endothelial function after chronic inhibition of nitric oxide synthase by L-NAME

Blood pressure, plasma NO(2) and NO(3) level, heart weight index, antioxidant enzyme activity, and vascular reactivity in rat intact aortic rings were assessed to investigate the effects of 8-week treatment with the hydroxy-methyl-glutaryl coenzyme A reductase inhibitor simvastatin (1 mg/kg per day) on endothelial dysfunction induced by chronic Nomega-nitro-l-arginine methyl ester (l-NAME 70 mg/kg per day). Results were compared with those obtained in rats receiving l-NAME, simvastatin or control animals. Coadministration of simvastatin did not restore l-NAME-increased blood pressure but normalized heart weight index (P < 0.05), endothelium-dependent relaxation to acetylcholine (P < 0.001), and plasma NO(2) and NO(3) concentration (P < 0.001) without affecting relaxation to sodium nitroprusside. Endothelium-dependent relaxation in these animals was abolished by acute incubation with l-NAME, unaffected by thromboxane synthetase inhibitor and TXA(2)/PGH(2) receptor antagonist, ridogrel, and decreased by indomethacin. Simvastatin treatment also increased plasma NO(2)+NO(3) without affecting endothelial function, heart weight index, and blood pressure of control rats. The presence of superoxide dismutase (SOD) and catalase improved endothelial relaxation only in l-NAME-treated rats, but O(2)- generated by hypoxanthine and xanthine oxidase inhibited the relaxant effect in both l-NAME and simvastatin plus l-NAME-treated rats. SOD activity was increased in all groups receiving simvastatin. Long-term treatment with simvastatin restored l-NAME-induced endothelial dysfunction, probably by preventing nitric oxide decrease. Other effects of simvastatin, including release of compensating vasodilatory cyclo-oxygenase products and increased SOD activity, could also be involved.     

Induction of vasorelaxation through activation of nitric oxide synthase in endothelial cells by brazilin

The vasorelaxant activity of Caesalpinia sappan L., a traditional Chinese medicine, and its major component brazilin were investigated in isolated rat aorta and human umbilical vein endothelial cells. In isolated rat aorta, C. sappan L. extract and brazilin relaxed phenylephrine-induced vasocontraction and increased cyclic guanosine 3',5'-monophosphate (cGMP) content. Induction of vasorelaxation of brazilin was endothelium-dependent and could be markedly blocked by pretreatment with nitric oxide synthase (NOS) inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME); N(G)-monomethyl-L-arginine acetate (L-NMMA) and guanylyl cyclase inhibitor, methylene blue; 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and nitric oxide (NO) scavenger, hemoglobin. The increasing cGMP content induced by brazilin was also blocked by pretreatment with L-NAME, methylene blue, and the removal of extracellular Ca(2+). In human umbilical vein endothelial cells, brazilin dose-dependently induced an increase in NO formation and NOS activity, which were greatly attenuated by either the removal of extracellular Ca(2+) or the chelating of intracellular Ca(2+) chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM). Moreover, brazilin dose-dependently induced the influx of extracellular Ca(2+) in human umbilical vein endothelial cells. Collectively, these results suggest that brazilin induces vasorelaxation by the increasing intracellular Ca(2+) concentration in endothelial cells of blood vessels and hence activating Ca(2+)/calmodulin-dependent NO synthesis. The NO is released and then transferred into smooth muscle cells to activate guanylyl cyclase and increase cGMP content, resulting in vasorelaxation.