Blockade of geranylgeranylation by rosuvastatin upregulates eNOS expression in human venous endothelial cells

Endothelial dysfunction is associated with a reduction in nitric oxide (NO) bioavailability. Positive effects of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) on the improvement of endothelial dysfunction have been shown. We investigated the effects of rosuvastatin and isoprenoid metabolites on endothelial NO synthase (eNOS) mRNA and protein expression in human umbilical venous endothelial cells after exposure to 10(-8)-10(-5) mol/l rosuvastatin for 8 and 12 h. Cell viability was not significantly altered after exposure to the statin for 12h. In a concentration-dependent manner, rosuvastatin upregulated eNOS mRNA and protein expression. The effects on eNOS expression mediated through rosuvastatin could be reversed by treatment with mevalonate indicating inhibition of HMG-CoA reductase as the underlying mechanism. Treatment with geranylgeranylpyrophosphate, but not farnesylpyrophosphate, reversed the increase of eNOS expression induced by rosuvastatin. Rosuvastatin may have beneficial effects on endothelial dysfunction associated with cardiovascular diseases beyond its effects on lowering cholesterol.     

The effect of high glucose on NO and O2- through endothelial GTPCH1 and NADPH oxidase

Although endothelial dysfunction deteriorates diabetic angiopathy, the mechanisms are obscure. We revealed that high glucose augmented eNOS through stimulation of eNOS mRNA in cultured BAECs. NO was decreased and O2- was increased simultaneously. NOS inhibitor, inhibited O2- release, so did NADPH oxidase inhibitor. The effects were synergistic. Both intracellular BH4 level and GTPCH1 activity were decreased by high glucose, in line with decrease of GTPCH1 mRNA. HMG-CoA reductase inhibitor, atorvastatin increased GTPCH1 mRNA and activity, and BH4 level. Conclusively, high glucose leads to eNOS dysfunction by inhibiting BH4 synthesis and atorvastatin stimulate BH4 synthesis directly, and it may work as atherogenic process.     

Involvement of Ca2+/calmodulin-dependent protein kinase II in endothelial NO production and endothelium-dependent relaxation

Nitric oxide (NO) is synthesized from l-arginine by the Ca(2+)/calmodulin-sensitive endothelial NO synthase (NOS) isoform (eNOS). The present study assesses the role of Ca(2+)/calmodulin-dependent protein kinase II (CaMK II) in endothelium-dependent relaxation and NO synthesis. The effects of three CaMK II inhibitors were investigated in endothelium-intact aortic rings of normotensive rats. NO synthesis was assessed by a NO sensor and chemiluminescence in culture medium of cultured porcine aortic endothelial cells stimulated with the Ca(2+) ionophore A23187 and thapsigargin. Rat aortic endothelial NOS activity was measured by the conversion of l-[(3)H]arginine to l-[(3)H]citrulline. Three CaMK II inhibitors, polypeptide 281-302, KN-93, and lavendustin C, attenuated the endothelium-dependent relaxation of endothelium-intact rat aortic rings in response to acetylcholine, A23187, and thapsigargin. None of the CaMK II inhibitors affected the relaxation induced by NO donors. In a porcine aortic endothelial cell line, KN-93 decreased NO synthesis and caused a rightward shift of the concentration-response curves to A23187 and thapsigargin. In rat aortic endothelial cells, KN-93 significantly decreased bradykinin-induced eNOS activity. These results suggest that CaMK II was involved in NO synthesis as a result of Ca(2+)-dependent activation of eNOS.     

渗透压对血管内皮细胞中NO合成酶活性的影响及其机制研究

目的：研究非等渗压浓度对血管内皮细胞NO合成酶活性的影响,并探索其发生机制。方法：使血管内皮细胞暴露于低渗（205mOsm)或高渗透压（410mOsm)培养液,用Griess法测定NO合成酶（NOS）活性,以Northern blot ting观测细胞iNOS和eNOS基因表达的变化。结果： 非等渗压浓度可使血管内皮细胞中NOS活性显著升高。细胞NOS活性变化具有明显的时间效应规律,低渗透压浓度效应产生的效应早于高渗透压浓度,且低渗透压浓度的影响较高渗透压浓度更为明显。Dexamethasone对这种非等渗透压诱导的NOS活性没有明显作用,给予cycloheximide,不影响非等渗压诱导的这种差异。Nothern blot分析表明：非等渗压浓度不诱导iNOS基因表达,而使eNOSmRNA表达增加。结论：非等渗透压浓度诱导血管内皮细胞NOS活性升高,eNOS基因表达增强是其主要机制之一。     

Dual effects of histone deacetylase inhibition by trichostatin A on endothelial nitric oxide synthase expression in endothelial cells

Inhibition of histone deacetylases by trichostatin A (TSA) has pleiotropic effects on gene expression. We demonstrated that at low dose (0.1 microg) TSA increased the eNOS mRNA levels, which was followed by a time- and dose-dependent down-regulation. Cycloheximide, a protein synthesis inhibitor, completely abolished TSA-induced decrease in eNOS expression, indicating that new protein synthesis is required for the inhibiting effect. Mevastatin--an inhibitor HMG-CoA reductase and geranylgeranylation reaction dose-dependently antagonized TSA-induced reduction. This mevastatin-mediated antagonism was completely abolished by geranylgeranylpyrophosphate, suggesting that geranylgeranyl modification is needed to activate the eNOS mRNA destabilizing factor--a mechanism responsible for statin-mediated eNOS upregulation.     

Differential effects of different statins on endothelin-1 gene expression and endothelial NOS phosphorylation in porcine aortic endothelial cells

It has been reported that 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase inhibitors (statins) produce a variety of cardiovascular protective effects independent of their ability to lower total and low-density lipoprotein cholesterol. Recent studies have also reported that statins produce pleiotropic effects through improved endothelial function, enhanced fibrinolysis, and antithrombotic actions. In the present study, we examined the effects of pitavastatin, pravastatin, atorvastatin, and cerivastatin on endothelin (ET)-1 production in cultured porcine aortic endothelial cells (PAECs). Treatment with cerivastatin but not pitavastatin, pravastatin, or atorvastatin decreased basal and TNF-alpha-stimulated ET-1 release from PAECs in a dose-dependent manner (1-10 microM). Northern blot analysis showed that cerivastatin markedly suppressed prepro ET-1 mRNA expression in both conditions. In addition, these inhibitory effects of cerivastatin on ET-1 release and prepro ET-1 mRNA expression were completely abolished by simultaneous treatment with 200 microM mevalonate. Furthermore, cerivastatin did not have any effects on endothelial nitric oxide synthase (eNOS) protein levels, but induced eNOS phosphorylation at Ser1177. From these findings, it is most likely that cerivastatin suppresses ET-1 production, possibly through an increase in eNOS activity and the subsequent nitric oxide production in PAECs. These findings also suggest that cerivastatin may have beneficial effects on ET-1-related diseases.     

NO and NOS isoforms in the development of apoptosis in renal ischemia/reperfusion

Nitric oxide (NO) and the expression of endothelial (eNOS) and inducible (iNOS) isoforms of nitric oxide synthase (NOS) are recognized as important mediators of physiological and pathological processes of renal ischemia/reperfusion (I/R) injury, but little is known about their role in apoptosis. The ability of the eNOS/NO system to regulate the iNOS/NO system and thus promote apoptosis was assessed during experimental renal I/R. Renal caspase-3 activity and the number of TUNEL-positive cells increased with I/R, but decreased when NOS/NO systems were blocked with L-NIO (eNOS), 1400W (iNOS), and N-nitro-l-arginine methyl ester (L-NAME; a nonselective NOS inhibitor). I/R increased renal eNOS and iNOS expression as well as NO production. The NO increase was eNOS- and iNOS-dependent. Blockage of NOS/NO systems with L-NIO or L-NAME also resulted in a lower renal expression of iNOS and iNOS mRNA; in contrast, eNOS expression was not affected by iNOS-specific blockage. In conclusion, two pathways define the role of NOS/NO systems in the development of apoptosis during experimental renal I/R: a direct route, through eNOS overexpression and NO production, and an indirect route, through expression/activation of the iNOS/NO system, induced by eNOS.     

The role of nitric oxide on rosuvastatin-mediated S-nitrosylation and translational proteomes in human umbilical vein endothelial cells

ABSTRACT: BACKGROUND: The pleiotropic effects of 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins), which are independent from their cholesterol-lowering action, have been widely recognized in various biological systems. Statins can affect endothelial homeostasis, which is partly modulated by the production of nitric oxide (NO). However, it is unclear how statin/NO-mediated posttranslational S-nitrosylation of endothelial proteins and changes in translational profiles may benefit endothelial integrity. Therefore, it is important to understand the statin/NO-mediated S-nitrosylation in endothelial cells. RESULTS: Rosuvastatin treatment of human umbilical vein endothelial cells (ECs) enhanced the enzymatic activity of endothelial nitric oxide synthase (eNOS) and the expression of 78 S-nitrosoproteins. Among these S-nitrosoproteins, we identified 17 proteins, including protein disulfide bond isomerase, phospholipase C, transaldolase and heat shock proteins. Furthermore, a hydrophobic Cys66 was determined as the S-nitrosylation site of the mitochondrial HSP70. In addition to the statin-modulated posttranslational S-nitrosylation, changes in the NO-mediated translational proteome were also observed. Seventeen major proteins were significantly upregulated after rosuvastatin treatment. However, 12 of these proteins were downregulated after pretreating ECs with an eNOS inhibitor (L-NAME), which indicated that their expression was modulated by NO. CONCLUSIONS: ECs treated with rosuvastatin increase eNOS activation. The increased NO production is involved in modulating S-nitrosylation and translation of proteins. We provide further evidence of the pleiotropic effect of rosuvastatin on endothelial physiology.     

Palmitate increases nitric oxide synthase activity that is involved in palmitate-induced cell death in cardiomyocytes.

The objective of this study was to test the hypothesis that nitric oxide synthase (NOS) is subjected to regulatory control by palmitate, and that nitric oxide (NO) is operative in palmitate-induced cell death. Palmitate induced a significant ( p<0.05 ) concentration-dependent increase in NOS activity measured by the conversion of [(3)H]arginine to [3H]citrulline in embryonic chick cardiomyocytes. Cellular eNOS and iNOS, determined by immunocytochemistry, were increased by palmitate. Western blotting also showed that palmitate, 500 microM for 4h, significantly increased the amount of cellular of eNOS and iNOS by 36.2+/-6.5% ( p<0.001 ) and 38.4+/-14.4% ( p<0.05 ), respectively. The NOS inhibitor L-NAME significantly ( p<0.05 ) accentuated palmitate-induced cell death These data suggest that palmitate has a bifunctional effect on cell viability--in addition to loss of cell viability, palmitate stimulates NOS activity by inducing an increase in cellular eNOS and iNOS with the resultant NO production serving to protect cardiomyocytes from palmitate-induced cell death.     

Differential expression and localization of nitric oxide synthases in cirrhotic livers of bile duct-ligated rats.

Increased vascular nitric oxide (NO) production has been implicated in the pathogenesis of the hyperdynamic circulation in liver cirrhosis. This study investigated the expression of three isoforms of NO synthase (NOS) in rat cirrhotic livers. Cirrhosis was induced by chronic bile duct ligation (BDL). NOS enzyme activity was assessed by L-citrulline generation. Competitive RT-PCR was performed to detect the mRNA levels of NOS. In situ hybridization was done to localize NOS mRNA. Protein expression of NOS was evaluated by Western blotting and immunohistochemistry. The L-citrulline assay showed that constitutive NOS (cNOS) enzymatic activity was decreased, while inducible NOS (iNOS) activity was increased in BDL livers. Both endothelial NOS (eNOS) and neuronal NOS (nNOS) mRNA were detected in BDL and sham rats, but with enhanced expression in BDL rats. eNOS protein was redistributed with less expression in sinusoidal endothelial cells, but the total levels in liver were not changed. nNOS was induced in hepatocytes of BDL rats, in contrast to only a weak signal observed around some blood vessels in sham livers. Intense mRNA and protein expression of iNOS was induced in livers of BDL rats and was localized in hepatocytes, with no or a negligible amount in control livers. In conclusion, iNOS was induced in cirrhotic liver with its activity increased. In contrast, cNOS activity was impaired, regardless of unchanged eNOS protein levels and enhanced nNOS expression. These results suggest that all three types of NOS have a role in cirrhosis, but their expression and regulation are different.     

Induction of endothelial nitric-oxide synthase in rat brain astrocytes by systemic lipopolysaccharide treatment

In the brain, three isoforms of nitric oxide (NO) synthase (NOS), namely neuronal NOS (nNOS, NOS1), inducible NOS (iNOS, NOS2), and endothelial NOS (eNOS, NOS3), have been implicated in biological roles such as neurotransmission, neurotoxicity, immune function, and blood vessel regulation, each isoform exhibiting in part overlapping roles. Previous studies showed that iNOS is induced in the brain by systemic treatment with lipopolysaccharide (LPS), a Gram-negative bacteria-derived stimulant of the innate immune system. Here we found that eNOS mRNA is induced in the rat brain by intraperitoneal injection of LPS of a smaller amount than that required for induction of iNOS mRNA. The induction of eNOS mRNA was followed by an increase in eNOS protein. Immunohistochemical analysis revealed that eNOS is located in astrocytes of both gray and white matters as well as in blood vessels. Induction of eNOS in response to a low dose of LPS, together with its localization in major components of the blood-brain barrier, suggests that brain eNOS is involved in early pathophysiologic response against systemic infection before iNOS is induced with progression of the infection.     

Simvastatin attenuates expression of cytokine-inducible nitric-oxide synthase in embryonic cardiac myoblasts

Cardiac stem cells or myoblasts are vulnerable to inflammatory stimulation in hearts with infarction or ischemic injury. Widely used for the prevention and treatment of atherosclerotic heart disease, the cholesterol-lowering drugs statins may exert anti-inflammatory effects. In this study, we examined the impact of inhibition of hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase with simvastatin on the expression of inducible nitric-oxide synthase (iNOS) in embryonic cardiac myoblasts stimulated with the proinflammatory cytokines, interleukin-1 or tumor necrosis factor. Treatment with simvastatin significantly reduced the levels of iNOS mRNA and protein in cytokine-treated rat H9c2 cardiac embryonic myoblasts. Addition of the HMG-CoA reductase product, L-mevalonate, and the by-product of cholesterol synthesis, geranylgeranyl pyrophosphate, could reverse the statin inhibitory effect on iNOS expression. Simvastatin treatment lowered the Rho GTPase activities, whereas the Rho-associated kinase inhibitor Y27632 partially blocked the statin inhibitory effect on nitrite production in the cytokine-treated H9c2 cells. Treatment with simvastatin led to inactivation of NF-kappaB by elevation of the NF-kappaB inhibitor IkappaB and reduction of the NF-kappaB nuclear contents in the cytokine-stimulated H9c2 cells. Hence, treatment with simvastatin can attenuate iNOS expression and NO synthesis in cytokine-stimulated embryonic cardiac myoblasts. The statin inhibitory effect may occur through isoprenoid-mediated intracellular signal transduction, which involves several key signal proteins, such as Rho kinase and IkappaB/NF-kappaB. These data suggest that statin therapy may protect the cardiac myocyte progenitors against the cytotoxicity of cytokine-induced high output of NO production in infarcted or ischemic hearts with inflammation.     

Arginine vasopressin increases iNOS-NO system activity in cardiac fibroblasts through NF-kappaB activation and its relation with myocardial fibrosis

Previous studies have shown that arginine vasopressin (AVP) promotes myocardial fibrosis (MF), whereas nitric oxide (NO) inhibits MF. Cardiac fibroblasts (CFs) are the main target cells of MF. However, the modulatory effect of AVP on NO production in CFs and the role of this effect in MF are still unknown. In the present study, CFs obtained from Sprague-Dawley rats were stimulated with or without AVP and pyrrolidine dithiocarbamate (PDTC), a specific inhibitor of nuclear factor kappa-B (NF-kappaB). NO production and NOS activity were detected with absorption spectrometry, inducible nitric oxide synthase (iNOS) protein with Western blot analysis, iNOS mRNA with real-time PCR, CF collagen synthesis with [(3)H]proline incorporation, and NF-kappaB activation with immunofluorescence staining and Western blot analysis. The results showed that AVP increased NO production in a dose- and time-dependent manner, with maximal effects at 10(-7) mol/l after 24-h stimulation. AVP also increased NOS activity, protein and mRNA levels of iNOS in a coincident manner. Furthermore, AVP also increased CF collagen synthesis in a dose- and time-dependent manner. In addition, it was found that NF-kappaB was activated by AVP, and that PDTC could inhibit NO production, NOS activity, protein and mRNA levels of iNOS stimulated by AVP in a dose-dependent manner. The inhibitory effects of PDTC on NF-kappaB translocation were coincident with the effects of PDTC on iNOS-NO system activity. It is suggested that AVP increases NO production via the regulation of iNOS gene expression, and the upregulation of iNOS gene expression stimulated by AVP is mediated through NF-kappaB activation. NO production induced by AVP may counteract the profibrotic effects of AVP, thus the development of MF perhaps depends on the balance between profibrotic AVP and antifibrotic NO effects on MF.     

组胺对肺动脉内皮细胞一氧化氮合酶基因表达的影响

本实验研究了组胺对原代培养的肺动脉内皮细胞一氧化氮合酶(nitric oxidCsynthase,NOS)基因表达的影响及分子机制。采用RT-PCR和免疫印迹技术分别检测mRNA和蛋白质的表达水平,用荧光素酶报告基因实验检测eNOS基因转录起始点上游长1.6-kb的启动子活性,用硝酸还原酶法检测NO的产量。结果发现,组胺增强eNOS表达,呈浓度和时间依赖性,10μmol/L组胺处理肺动脉内皮细胞24h可使eNOS mRNA和蛋白质的表达达到高峰,eNOS mRNA水平为正常对照组的160.8±12.2%(P<0.05),蛋白质水平为正常对照组的136.2±11.2%(P<0.05)。特异性CaMK Ⅱ抑制剂KN-93可抑制组胺的这一效应,表明组胺可通过激活CaMK Ⅱ增强肺动脉内皮细胞eNOS基因的表达。报告基因实验表明,10μmol/L组胺处理24h后肺动脉内皮细胞eNOS基因启动子的活性增强,为正常对照组的148.2±33.7%(P<0.05)。组胺可使肺动脉内皮细胞产生NO增加。这些结果表明组胺在转录水平增强肺动脉内皮细胞eNOS基因的表达,并使细胞产生NO增加,这可能是组胺调节肺血管张力的机制之一。CaMK Ⅱ可能是组胺增强肺动脉内皮细胞eNOS基因表达的途径之一。     

Resistance to endotoxic shock in endothelial nitric-oxide synthase (eNOS) knock-out mice: a pro-inflammatory role for eNOS-derived no in vivo

The expression of inducible nitric-oxide synthase (iNOS) and subsequent "high-output" nitric oxide (NO) production underlies the systemic hypotension, inadequate tissue perfusion, and organ failure associated with septic shock. Therefore, modulators of iNOS expression and activity, both endogenous and exogenous, are important in determining the magnitude and time course of this condition. We have shown previously that NO from the constitutive endothelial NOS (eNOS) is necessary to obtain maximal iNOS expression and activity following exposure of murine macrophages to lipopolysaccharide (LPS). Thus, eNOS represents an important regulator of iNOS expression in vitro. Herein, we validate this hypothesis in vivo using a murine model of sepsis. A temporal reduction in iNOS expression and activity was observed in LPS-treated eNOS knock-out (KO) mice as compared with wild-type animals; this was reflected in a more stable hemodynamic profile in eNOS KO mice during endotoxaemia. Furthermore, in human umbilical vein endothelial cells, LPS leads to the activation of eNOS through phosphoinositide 3-kinase- and Akt/protein kinase B-dependent enzyme phosphorylation. These data indicate that the pathogenesis of sepsis is characterized by an initial eNOS activation, with the resultant NO acting as a co-stimulus for the expression of iNOS, and therefore highlight a novel pro-inflammatory role for eNOS.     

TGF-beta(1) modulates NOS expression and phosphorylation of Akt/PKB in rat myocytes exposed to hypoxia-reoxygenation

Myocardial hypoxia-reoxygenation (H-R) is associated with upregulation of inducible nitric oxide synthase (iNOS), decrease in endothelial NOS (eNOS), and increase in protein kinase B (PKB). Previous work also shows that transforming growth factor-beta(1) (TGF-beta(1)) can attenuate myocardial injury induced by H-R. We examined the modulation of NOS and PKB expression in response to H-R by TGF- beta(1). Myocytes from Sprague-Dawley rat hearts were cultured and exposed to hypoxia (95% N(2)-5% CO(2), PO(2) ~30 mmHg) for 24 h and reoxygenation (95% air-5% CO(2)) for 3 h. Myocytes were then examined for lactate dehydrogenase (LDH) release, iNOS activity (conversion of L-[(3)H]arginine to L-[(3)H]citrulline), iNOS and eNOS expression, and PKB phosphorylation. H-R alone resulted in myocyte injury, upregulation of iNOS activity and expression, decrease in eNOS expression, and increase in PKB phosphorylation (all P < 0.05 vs. cells cultured in normoxic conditions). Treatment of myocytes with TGF-beta(1) (1 ng/ml) resulted in a reduction in LDH release, attenuation of the alterations in NOS expression (both iNOS and eNOS), and PKB phosphorylation in response to H-R (all P < 0.05 vs. H-R alone). These observations suggest that TGF-beta(1) decreases H-R injury and attenuates alterations in NOS and PKB phosphorylation in myocytes exposed to H-R.     

Cell growth modulates nitric oxide synthase expression in fetal pulmonary artery endothelial cells

Nitric oxide (NO), produced by endothelial (e) nitric oxide synthase (NOS), is a critical mediator of vascular function and growth in the developing lung. Pulmonary eNOS expression is diminished in conditions associated with altered pulmonary vascular development, suggesting that eNOS may be modulated by changes in pulmonary artery endothelial cell (PAEC) growth. We determined the effects of cell growth on eNOS expression in cultured ovine fetal PAEC studied at varying levels of confluence. NOS enzymatic activity was sixfold greater in quiescent PAEC at 100% confluence compared with more rapidly replicating cells at 50% confluence. To determine if there is a reciprocal effect of NO on PAEC growth, studies of NOS inhibition or the provision of exogenous NO from spermine NONOate were performed. Neither intervention had a discernable effect on PAEC growth. The influence of cell growth on NOS activity was unique to pulmonary endothelium, because varying confluence did not alter NOS activity in fetal systemic endothelial cells. The effects of cell growth induced by serum stimulation were also evaluated, and NOS enzymatic activity was threefold greater in quiescent, serum-deprived cells compared with that in serum-stimulated cells. The increase in NOS activity observed at full confluence was accompanied by parallel increases in eNOS protein and mRNA expression. These findings indicate that eNOS gene expression in fetal PAEC is upregulated during cell quiescence and downregulated during rapid cell growth. Furthermore, the interaction between cell growth and NO in the PAEC is unidirectional.