Glu298Asp polymorphism of the endothelial nitric oxide synthase gene in Turkish patients with ischemic stroke

The low plasma nitric oxide concentrations and reduced vascular reactivity are considered major proatherogenic mechanisms in cardiovascular diseases. The present study aimed to assess the allelic frequency and the genotypic distribution of the Glu298Asp gene polymorphism at exon 7 of endothelial nitric oxide synthase (eNOS) gene in Turkish ischemic stroke patients compared to appropriate healthy controls, and to correlate the genetic findings with stroke subtypes. The study population included 146 (75 males, 71 females) patients with ischemic stroke which were categorized according to the Trial of ORG 10172 in Acute Stroke Treatment (TOAST) and 133 (34 males, 99 females) healthy subjects. The eNOS polymorphism was identified with a PCR followed by RFLP with the restriction enzyme BanII. Genotypes were defined as GG, GT, and TT according to the presence of the G and T alleles. In this case-control study, we did not find any significant difference in either the genotypic distribution or allelic frequency of Glu298Asp gene polymorphism between the patients and the controls. In addition, there was also no significant difference for the genotype distribution and the allelic frequency among the stroke subtypes. The results suggested the lack of the association between the Glu298Asp gene polymorphism and ischemic stroke or subtypes of ischemic stroke in the Turkish population.     

Association of the Glu298Asp polymorphism in the endothelial nitric oxide synthase gene with essential hypertension resistant to conventional therapy

Endothelial nitric oxide synthase (eNOS) produces nitric oxide (NO) which, after diffusing into vascular smooth muscle cells, activates guanylate cyclase leading to vasodilatation. A polymorphism (894G to T) in exon 7 of the eNOS gene causes the conversion of Glu to Asp in position 298. The recently described crystal structure of the heme domain of eNOS protein shows that Glu298 is fully solvent accessible and distant from regions integral to enzyme function. Searching for phenotypic expression of eNOS gene variants, we genotyped a group of patients with essential hypertension (H, n = 119) for the Glu298Asp polymorphism and compared them with age- and sex-matched healthy normals (N, n = 85). To specify phenotypic expression further, the hypertensive patients were subdivided into one group that responded well to regular antihypertensive therapy (CH, n = 45) and one group that was resistant to the therapy (RH, n = 74). Patients with BP higher than 140/90 mmHg when on adequate lifestyle modification and triple-combination therapy (including diuretics) were considered resistant. In RH and H groups, a significantly higher frequency of T alleles (P = 0.022 and P = 0.046, respectively) was found compared to normotonics (N). In well-controlled hypertonics, the same tendency was found, but did not reach statistical significance. The Glu298Asp polymorphism may contribute to the complex pathogenesis of essential hypertension and may be a factor in the resistance of these patients to conventional antihypertensive therapy. The presence of this allele may thus be predictive of the patients' therapeutic response.     

The missense Glu298Asp variant of the endothelial nitric oxide synthase gene is strongly associated with placental abruption

We recently identified a missense variant (Glu298Asp) that lies within exon 7 of the endothelial nitric oxide synthase (eNOS) gene, and that is associated with severe preeclampsia (proteinuric hypertension that develops as a consequence of pregnancy). Maternal hypertension is the most consistently identified factor predisposing to placental abruption. Our objective, therefore, was to analyze the association between the Glu298Asp eNOS gene variant and placental abruption. The study participants included 35 patients with histories of placental abruption and 170 control subjects. Screening for the Glu298Asp eNOS gene variant was carried out by analysis of polymerase chain reaction/restriction fragment length polymorphism. The analyses revealed that the frequency of the Glu298Asp variant (Glu298Asp homozygotes and heterozygotes) was significantly (P<0.001) higher in the placental abruption group (n=14; 40%) than in the control group (n=24; 14%). We conclude that the presence of the Glu298Asp eNOS gene variant could be a marker of increased risk of developing placental abruption.     

Relationship between hemorheology and Glu(298)Asp polymorphism of endothelial nitric oxide synthase gene in patients with coronary artery disease

This study aimed to investigate the relationship between endothelial nitric oxide synthase Glu(298)Asp gene polymorphism and hemorheological parameters. Red blood cell (RBC) deformability, aggregation were measured using an ectacytometry, whole blood, plasma viscosities were determined by a viscometer. Restriction fragment length polymorphism was used to detect polymorphism. Plasma nitrite, nitrate concentrations were determined by Griess method. The genotype distribution of the control group was as follows: 50 (67.5%) GG, 21 (28.4%) GT, 3 (4.1%) TT. A 48 (57.8%) of the patients with CAD had GG, 28 (33.7%) GT, 7 (8.5%) of them TT genotype. RBC aggregation index of CAD patients with G allele was higher and t½ lower compared to controls carrying the same allele. The amplitude of RBC aggregation of healthy subjects with T allele, who are under increased cardiovascular risk was lower compared to control subjects with G allele. The results of this study indicate that, alterations in RBC aggregation seem to be a consequence of CAD, more than being a preexisting cause. Additionally, some compensatory mechanisms by causing decrements in RBC aggregation, may help regulation of circulation in healthy individuals with high cardiovascular risk.     

A missense Glu298Asp variant in the endothelial nitric oxide synthase gene is associated with coronary spasm in the Japanese

Coronary spasm plays an important role in the pathogenesis of not only variant angina but also ischemic heart disease in general. However, the precise mechanism(s) by which coronary spasm occurs remains to be elucidated. Coronary spasm may arise from interactions between environmental and genetic factors. Endothelial derived nitric oxide (NO) has been implicated in the control of vascular tone. We have recently shown that both basal and acetylcholine (ACh)-induced NO activities are impaired in the coronary arteries of patients with coronary spasm. The purpose of this study has been to elucidate the possible variants that occur in the coding region of the endothelial nitric oxide synthase (eNOS) gene and that may be associated with coronary spasm. After initial screening in the entire 26 coding regions of the eNOS gene, we found a missense Glu298Asp variant in exon 7 in patients with coronary spasm. We subsequently performed a larger scale study involving 113 patients with coronary spasm and 100 control subjects, who were all diagnosed by intracoronary injection of ACh. The analysis revealed a significant difference in the distribution of the variant between the coronary spasm group (21.2%) and control group (9.0%; P=0.014 for dominant effect). Thus, we have found the missense Glu298Asp variant in the eNOS gene by the analysis of its entire 26 coding regions. The variant is significantly associated with coronary spasm. Received: 2 February 1998 / Accepted: 9 April 1998     

Functional characterization of Glu298Asp mutant human endothelial nitric oxide synthase purified from a yeast expression system.

The Glu298Asp polymorphism of human endothelial nitric oxide synthase (eNOS) has been reported to be associated with several cardiovascular diseases, including hypertension and myocardial infarction. Therefore, we investigated the effect of the Glu298Asp (E298D) mutation on the function of purified recombinant eNOS expressed in the yeast Pichia pastoris. Wild type (WT) and mutant exhibited comparable affinities for L-arginine (K(m) values 4.4+/-0.6 and 5.2+/-0.8 microM, respectively) and V(max) values (142+/-36 and 159+/-29 nmol of L-citrulline/mg min, respectively). The E298D mutation affected neither electron transfer through the reductase domain (measured as cytochrome c reduction) nor reductive O(2) activation (measured either as NADPH oxidation or as H(2)O(2) formation in the absence of L-arginine and tetrahydrobiopterin (BH4)). The mutant was activated by BH4 with an EC(50) of 0.24+/-0.04 microM, a value comparable to that obtained with WT eNOS (0.22+/-0.02 microM). Activation of the enzyme by Ca(2+) was not affected (EC(50)=0.50+/-0.04 and 0.49+/-0.02 microM for WT and E298D eNOS, respectively). Calmodulin (CaM) affinity, studied by radioligand binding using 125I-labeled CaM, revealed virtually identical K(D) (3.2+/-0.5 and 4.0+/-0.3nM) and B(max) (1.4+/-0.2 and 1.2+/-0.3 pmol/pmol subunit) values for WT and E298D eNOS, respectively. Furthermore, E298D eNOS did not differ from the WT enzyme with respect to heme and flavin content or the ability to form SDS-resistant dimers. To summarize, we obtained no evidence for altered enzyme function of the eNOS mutant that could explain endothelial dysfunction associated with the E298D polymorphism.     

Preliminary computational modeling of nitric oxide synthase 3 interactions with caveolin-1： influence of exon 7 Glu298Asp polymorphism

The significance of endothelial nitric oxide synthase 3 （NOS3） activity has been recognized for many years, however it was only recently that the complicated regulation of this constitutively expressed enzyme in endothelial cells was identified. A critical component of the NOS3 regulatory cyde in endothelial cells is its intracellnlar localization to caveolae. The caveolar coordination of NOS3, more specifically its interaction with caveolin-1 （Cav-1）, plays a major role in normal endothelial NOS3 activity and vascular bioavailability of nitric oxide. We have recently shown that the presence of NOS3 exon 7 Glu298Asp polymorphism caused diminished shear-dependent NOS activation, was less extensively associated with caveolae, and had a decreased degree of interaction with Cav-1. Here, we carried out preliminary investigations to identify possible mechanisms of the genotype-dependent endothelial cell responses we observed in our previous investigations. Through this approach we tested the hypothesis that computer simulations could provide insights regarding the contribution of this single nucleotide polymorphism to regulation of the NOS3 isoform. We observed that in the Glu/Asp and Asp/Asp mutant genotypes, the amount of NOS3 associated with Cav-1 was significantly lower. Additionally, we have shown, using a theoretical computational model, that mutation of an amino acid at position 298 might affect the protein-protein interactions and localization of the NOS3 protein. These alterations might also affect the protein function and explain the enhanced disease risk associated with the presence of Glu298Asp polymorphism in the NOS3 protein.     

NO Level and Endothelial NO Synthase Gene Polymorphism （Glu298Asp） in the Patients with Coronary Artery Disease from the Turkish Population

A healthy endothelium plays a core role in cardiovascu-lar control [1]. In the endothelial cell, nitric oxide (NO) issynthesized by the endothelial nitric oxide synthase (eNOS)encoded by a 26-exon gene (NOS 3) located on chromo-some 7 [2]. Besides its regulatory functions on vasomotortone and blood flow, endothelial NO is known to inhibitthe platelet activation and modulate migration and growthof the vascular smooth muscle [3]. Indirect evidence sug-gests that alterations of the NO pathwa…     

Association between Glu298Asp/677C-T single nucleotide polymorphism in the eNOS/MTHRF gene and blood stasis syndrome of ischemic stroke

Blood stasis syndrome of ischemic stroke (BSS-IS) is a common clinical phenotype that may be affected by certain mutagenic environmental factors or chemotherapeutic drugs; however, the role of susceptibility genes remains unclear. Previous studies have shown that ischemic stroke (IS) was closely associated with the Glu298Asp polymorphism in the eNOS gene and the 677C-T (Ala→Val) polymorphism in methylenetetrahydrofolate reductase (MTHRF) gene. Therefore, these two single nucleotide polymorphisms (SNPs) were selected to detect their associations with BSS-IS in this study. A SNP chip was employed to screen the SNP variation between both groups, and the results were verified using denaturing high-performance liquid chromatography (DHPLC) and restriction fragment length polymorphism (RFLP). The results confirmed that the TT genotype of Glu298Asp in the eNOS gene may be one of the risk factors associated with BSS-IS, while the genotype of 677C-T (Ala→Val) in the MTHRF gene may not be relevant to BSS-IS.     

The polymorphism for endothelial nitric oxide synthase gene,the level of nitric oxide and the risk for pre-eclampsia: A meta-analysis

Endothelial NO, which is synthesized by endothelial nitric oxide synthase (eNOS), has been reported to be related with the occurrence of pre-eclampsia (PE). However, the polymorphisms of eNOS (− 786 T > C, 4b/a and G894T), the level of nitric oxide and the risk of PE remain unclear. Thus we performed this meta-analysis to determine the associations between them in order to predict the risk for PE and interference with PE development in the early period of antenatal care. All studies investigating the associations between PE risk and polymorphisms of eNOS, or PE risk and serum concentration of NO were reviewed. Finally, 29 studies were included, involving 11 for − 786 T > C, 11for 4b/a, and 22 for G894T polymorphisms and PE risk. In the overall analysis, − 786 T > C polymorphism was found to be related with increased PE risk in the dominant model (OR = 1.17, 95% CI = 1.02-1.35). a allele for 4b/a suffers the high risk of PE (OR = 1.46, 95% CI = 1.01–2.10). In the subgroup analysis, significantly increased risk was detected among Europeans for − 786 T > C polymorphism (OR = 1.40, 95%CI = 1.14–1.73).However, no significant association was detected for G894T polymorphism in the overall and subgroup analysis. The comprehensive evaluation of 9 available studies indicated that serum NO level was significantly decreased in case group (SMD = − 0.96 umol/mL, 95%CI = − 1.80, − 0.12 umol/mL).Hence, we concluded that eNOS gene − 786 T > C and 4b/a except for G894T polymorphisms were contributed significantly to PE risk, especially for Europeans, and a low NO concentration in serum increased the risk for PE.     

The Asp298 allele of endothelial nitric oxide synthase is a risk factor for myocardial infarction among patients with type 2 diabetes mellitus

Background

To establish an efficient prophylaxis of coronary artery disease reliable risk stratification is crucial, especially in the high risk population of patients suffering from diabetes mellitus. This prospective study determined the predictive value of coronary calcifications for future cardiovascular events in asymptomatic patients with diabetes mellitus.

Methods

We included 716 patients suffering from diabetes mellitus (430 men, 286 women, age 55.2 ± 15.2 years) in this study. On study entry all patients were asymptomatic and had no history of coronary artery disease. In addition, all patients showed no signs of coronary artery disease in ECG, stress ECG or echocardiography. Coronary calcifications were determined with the Imatron C 150 XP electron beam computed tomograph. For quantification of coronary calcifications we calculated the Agatston score. After a mean observation period of 8.1 ± 1.1 years patients were contacted and the event rate of cardiac death (CD) and myocardial infarction (MI) was determined.

Results

During the observation period 40 patients suffered from MI, 36 patients died from acute CD. The initial Agatston score in patients that suffered from MI or died from CD (475 ± 208) was significantly higher compared to those without cardiac events (236 ± 199, p < 0.01). An Agatston score above 400 was associated with a significantly higher annualised event rate for cardiovascular events (5.6% versus 0.7%, p < 0.01). No cardiac events were observed in patients with exclusion of coronary calcifications. Compared to the Framingham risk score and the UKPDS score the Agatston score showed a significantly higher diagnostic accuracy in the prediction of MI with an area under the ROC curve of 0.77 versus 0.68, and 0.71, respectively, p < 0.01.

Conclusion

By determination of coronary calcifications patients at risk for future MI and CD could be identified within an asymptomatic high risk group of patients suffering from diabetes mellitus. On the other hand future events could be excluded in patients without coronary calcifications.     

The role of vascular endothelial protein tyrosine phosphatase on nitric oxide synthase function in diabetes: from molecular biology to the clinic

Endothelial nitric oxide synthase (eNOS) and receptor-type vascular endothelial protein tyrosine phosphatase (VE-PTP) are one of the majors signaling pathways related to endothelial health in diabetes. Several reports have shown that the inhibition of VE-PTP can lead the nitric oxide production, although repeated studies showed that VE-PTP regulated the eNOS exclusive at Ser1177 in indirect-manner. A recent, exciting paper (Siragusa et al. in Cardiovasc Res, 2020. https://doi.org/10.1093/cvr/cvaa213), showing that VE-PTP regulates eNOS in a direct-manner, dephosphorylating eNOS at Tyr81 and indirect at Ser1177 and the effects of a VE-PTP inhibitor, AKB-9778, in the blood pressure from diabetic patients.     

Adult human vascular endothelial cells express the IL6 gene differentially in response to LPS or IL1

We investigated the regulation of IL6 biological activity, de novo synthesis, and mRNA levels in adult vascular endothelial cells (EC) by bacterial endotoxin or inflammatory cytokines. Cells incubated without stimulus released scant IL6 activity. IFN gamma, IL2, or PDGF did not augment IL6 release from EC. LPS, lipid A, and TNF increased IL6 release modestly (5 to 20-fold), while recombinant IL1s (rIL1s) stimulated this process 100 to 400-fold. Differential release of IL6 from EC treated with LPS or rIL1 continued for at least 144 hr. Exposure to LPS or rIL1 caused EC to synthesize IL6 de novo. EC secreted the newly synthesized IL6 into the supernatant, rather than retaining it within or bound to cells. EC accumulated IL6 mRNA after 3 hr of exposure to rIL1. However, we could only detect IL6 message in cells incubated with LPS under "superinduction" conditions with cycloheximide, consistent with lower levels of IL6 biological activity in response to LPS compared to IL1 stimulation. We propose that local production of IL6 by vascular EC, which comprise the barrier between tissues and the blood, may influence regional immune and inflammatory responses.     

A single nucleotide polymorphism in the promoter region of the human gene for osteoprotegerin is related to vascular morphology and function

Osteoprotegerin (OPG) is a secreted member of the tumor necrosis factor receptor family, and has previously been shown to regulate bone mass by inhibiting osteoclast differentiation and activation. Recent evidence indicates that OPG also plays a role in the vascular system, since ablation of the OPG gene in mice results in calcification of the aorta and renal arteries, and association has been found between serum levels of OPG and cardiovascular mortality. This study presents a novel single nucleotide polymorphism, a T/C transition located 129 bp upstream the TATA-box of the human OPG gene, detected by sequence analysis. The OPG genotype was determined by restriction fragment length polymorphism in a cohort consisting of 59 healthy subjects. The intima-media thickness (IMT) in the common carotid artery and maximal post-ischemic forearm blood flow (FBF) were investigated. Subjects with the CC genotype showed a significantly increased IMT (p<0.05) and a concommitantly reduced maximal FBF (p<0.01) as compared to those with the T allele. Thus, our results show that the polymorphism in the promoter region of OPG is associated with both vascular morphology and function in apparently healthy subjects.     

Mild increases in portal pressure upregulate vascular endothelial growth factor and endothelial nitric oxide synthase in the intestinal microcirculatory bed, leading to a hyperdynamic state

Increased nitric oxide (NO) is the main factor leading to the hyperdynamic circulation associated with advanced portal hypertension (PHT), but the initial mechanisms and the magnitude of increase in portal pressure required to trigger NO production are not known. We addressed these issues by studying systemic and splanchnic hemodynamics and endothelial NO synthase (eNOS) and VEGF expression in rats with different degrees of portal hypertension. Portal vein ligation (PVL) performed over needles of three different calibers (16-, 18-, and 20-gauge) yielded different degrees of PHT and portosystemic shunting. Compared with sham rats, all three groups of PVL rats exhibited features of hyperdynamic circulation. Rats with minimal portal hypertension (PVL with a 16-gauge needle) showed an early increase in VEGF and eNOS expression selectively at the jejunum. Immunofluorescence showed that VEGF expression was located in highly vascularized areas of the mucosa. Inhibition of VEGF signaling markedly attenuated the increase in eNOS expression. In conclusion, mild increases in portal pressure are enough to upregulate eNOS at the intestinal microcirculation, and this occurs, at least in part, through VEGF upregulation.     

Evidence for association of endothelial cell nitric oxide synthase gene polymorphism with earlier progression to end-stage renal disease in a cohort of Hellens from Greece and Cyprus

Nitric oxide (NO) is thought to be an important factor in the deterioration of renal function. A variable-number tandem 27-bp repeat in intron 4 of the endothelial cell nitric oxide synthase (NOS3) gene has been found to be associated with the plasma levels of NO metabolites. Two alleles are of varied frequencies in different populations (a and b). The shorter allele a has been associated in Japanese populations with the progression of renal disease. Here we investigated this hypothesis by studying the putative role of this polymorphism in a Hellenic population of patients with end-stage renal disease (ESRD). We analyzed the genotypes of 361 ESRD patients and 295 healthy Hellens from Greece and Cyprus. The frequencies of NOS3-4bb, NOS3-4ab, and NOS3-4aa were 0.69, 0.27, and 0.03, respectively, in the control group and 0.71, 0.24, and 0.04 in the group of patients. The data in the two populations were analyzed by the chi-square and Fisher's exact tests. The frequencies of these three genotypes of NOS3-4 polymorphism in the Hellenic population of Greece and Cyprus are similar to those observed in other Caucasian populations. Moreover, our results from three patient groups, autosomal dominant polycystic kidney disease (ADPKD), diabetes mellitus (DM), and non-DM, showed that the frequencies of aa and ab genotypes in the patient populations were not significantly different from those observed in the control group. This work indicates that NOS3-4 polymorphism does not show any association with the development of ESRD in this studied European population. However, examination of the data regarding progression to ESRD within 5 years or after more than 5 years following clinical diagnosis of ADPKD provided evidence of statistical difference (p = 0.048, before Bonferroni correction), with faster progression in the group of ADPKD patients who carried allele a.     

The 27-bp repeat polymorphism in intron 4 (27 bp-VNTR) of endothelial nitric oxide synthase (eNOS) gene is associated with albumin to creatinine ratio in Mexican Americans

Molecular and Cellular Biochemistry - The events leading to Schwannomas development are still largely unknown. Some studies have demonstrated that merlin acts as a tumor suppressor by blocking...