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1.  Association of endothelial nitric oxide synthase polymorphisms with coronary artery disease in Korean individuals with or without diabetes mellitus 
Polymorphisms of the endothelial nitric oxide synthase (eNOS) gene have been implicated in various diseases, but their roles as risk factors in type 2 diabetes mellitus (T2DM) with regard to coronary artery disease (CAD) are largely unknown. Therefore, we investigated the association of the genotypes and haplotypes of eNOS polymorphisms in CAD with T2DM. A case-control study was performed to evaluate the genotypes and haplotypes of the eNOS polymorphisms (-786T>C, 4a4b and 894G>T) in 192 CAD patients and 196 controls. The same population was also re-organized upon the status of T2DM. The genotypes of eNOS -786T>C, 4a4b and 894G>T polymorphisms were determined by polymerase chain reaction-restriction fragment length polymorphism. We found that eNOS -786TC+CC and 4a4b+4a4a genotypes were significantly prevalent in the diabetic controls and the diabetic CAD patients compared to the non-diabetic controls or non-diabetic CAD patients, respectively. The frequency of the -786C-4a-894G haplotype was significantly greater in the diabetic CAD patients (p=0.001) and diabetic controls (p=0.023) compared to the non-diabetic controls, whereas the haplotype of -786T-4b-894G was less prevalent in the diabetic CAD patients compared to the non-diabetic controls (p=0.018). Significant associations of the genotypes and the haplotypes were consistently observed in the T2DM group compared to non-DM group, regardless of CAD status. Our finding suggests that the eNOS -786T>C and 4a4b polymorphisms and the -786C-4a-894G haplotype are risk factors for T2DM, whereas the haplotype of -786T-4b-894G has a protective effect against the development of T2DM.
doi:10.3892/etm_00000111
PMCID: PMC3445891  PMID: 22993595
coronary artery disease; endothelial nitric oxide synthase; haplotype; polymorphism; type 2 diabetes mellitus
2.  Association of endothelial nitric oxide synthase promoter region (T-786C) gene polymorphism with acute coronary syndrome and coronary heart disease 
Background
Nitric oxide (NO) is an endothelium derived relaxing factor (EDRF) which has an important role for regulating the heart-vessel physiology. The objective of this study was to evaluate the effects of the eNOS T-786C polymorphism on lipid parameters and the development of acute coronary syndrome (ACS) and coronary heart disease (CHD) for the first time in a Turkish study group. We have analyzed the genotype frequencies of the T-786C polymorphism of the eNOS gene in 10 ACS patients (5 men, 5 women), 20 CHD patients (14 men, 6 women), and 31 controls (10 men, 21 women), who were angiographically proven to have normal coronaries.
Results
The demographic, biochemical and left ventricule systolic dysfunction data of the ACS, CHD patients and controls were analyzed as a function of eNOS T-786C genotypes. The eNOS gene T-786C polymorphism frequencies for T/T, C/T and C/C genotypes were respectively 10%, 40%, 50% in subjects with ACS; 75%, 20%, 5% in subjects with CHD and 67.7%, 25.8%, 6.5% in the control group. Significant difference was observed in genotype frequencies between the study groups for T-786C polymorphism (p = 0.001). The CC genotype frequency was found to be the most prevalent in ACS group in comparison to CHD and control groups (p = 0.001). TT was the most frequently observed genotype in both CHD patients and controls (p = 0.001). Left ventricule systolic dysfunction frequency was found to be highest in C/T genotype carriers (66.7%) in patients (ACS+CHD). None of the patients with LVSD were carrying the normal genotype (T/T). The eNOS T-786C polymorphism was not found to be effective over any analyzed lipid variable in patients (ACS+CHD). The HDL-cholesterol levels were found to be lower in CHD group were compared to controls (p < 0.01), whereas glucose and leucocyte levels of the ACS and CHD groups were both higher than controls (p < 0.001).
Conclusion
The significantly high frequency of eNOS -786C/C genotype in ACS patients than in those of controls, indicate the genotype association with ACS. The finding of significantly high frequency of T/T genotype in the CHD group, may support the relationship of CC genotype with ACS without CHD. The high frequency of the mutant (C/C) and heterozygous (C/T) genotypes found may be linked to left ventricule remodeling after MI.
doi:10.1186/1476-511X-7-5
PMCID: PMC2267783  PMID: 18298848
3.  A Promoter Polymorphism of the Endothelial Nitric Oxide Synthase Gene is Associated with Reduced mRNA and Protein Expression in Failing Human Myocardium 
Journal of cardiac failure  2010;16(4):314-319.
Background
Alterations of endothelial nitric oxide synthase (eNOS) enzyme activity via eNOS gene polymorphisms have been associated with significant cardiovascular morbidity and mortality. Both the thymidine to cytosine transition mutation (T−786→C) in the promoter region and the missense mutation in the exon 7 coding region of the eNOS gene (G894→T) have been associated with several cardiovascular disease states. We hypothesized that heart transplant recipients who carried at least one allele of either of the polymorphisms would have reduced myocardial tissue expression of eNOS measured in the explanted heart.
Methods/Results
Genomic DNA was isolated from myocardial tissue samples obtained from 43 explanted human hearts using standard methods. Regions of the eNOS gene were amplified from genomic DNA with a polymerase chain reaction using specific primers. Protein expression of eNOS was measured by Western blot analysis. There was a statistically significant decrease in mean eNOS expression in samples containing at least one allele for the T−786→C promoter polymorphism (p = 0.04) compared to patients homozygous for the T allele. There was no change in eNOS expression associated with the G894→T exonic polymorphisms. Conclusions: Our data show in failing human myocardium that the T−786→C promoter polymorphism is associated with reduced eNOS expression whereas the G894→T polymorphism of exon 7 is not associated with change in either eNOS mRNA or protein expression. Reduced eNOS expression associated with the promoter polymorphism may contribute to the vascular, contractile, and autonomic responses to ventricular failure.
doi:10.1016/j.cardfail.2009.12.013
PMCID: PMC2848179  PMID: 20350698
Nitric Oxide; Genetics; Cardiomyopathy; Pharmacogenetics
4.  Endothelial nitric oxide synthase gene polymorphisms -786T >C and 894G >T in coronary artery bypass graft surgery patients 
Human Genomics  2010;4(6):375-383.
Polymorphisms in the endothelial nitric oxide synthase (eNOS) gene (-786T >C and 894G >T) enhance endo-thelial dysfunction and have been studied in relation to coronary artery disease (CAD). In the present study, we examined the association of the above polymorphisms with CAD, as well as with myocardial infarction (MI), hypertension, diabetes and smoking in CAD patients. Study subjects consisted of 154 consecutive coronary artery bypass graft (CABG) patients and 155 non-CAD controls. eNOS -786T >C and 894G >T polymorphisms were genotyped by polymerase chain reaction-restriction fragment length polymorphism. The estimated frequencies of the -786C and 894T alleles did not differ between the two groups (p = 0.46 and p = 0.84, respectively). The prevalence of eNOS polymorphisms was not associated with MI, hypertension or diabetes in CABG patients; however, we found that the 894TT genotype and 894T allele were significantly more frequent in current/past smoker CABG patients (16.7 per cent and 39.6 per cent, respectively) compared with never smoker CABG patients (6.1 per cent and 24.4 per cent, respectively) (p = 0.01 and p < 0.01, respectively). We found no association of eNOS -786C and 894T variant alleles with CAD; however, within CABG patients, a gene-environment interaction was found between the eNOS 894T allele and smoking.
doi:10.1186/1479-7364-4-6-375
PMCID: PMC3525225  PMID: 20846926
coronary artery disease; coronary artery bypass graft surgery; eNOS -786T > C polymorphism, eNOS 894G > T polymorphism; smoking
5.  A novel multiplex PCR-RFLP method for simultaneous detection of the MTHFR 677 C > T, eNOS +894 G > T and - eNOS -786 T > C variants among Malaysian Malays 
BMC Medical Genetics  2012;13:34.
Background
Hyperhomocysteinemia as a consequence of the MTHFR 677 C > T variant is associated with cardiovascular disease and stroke. Another factor that can potentially contribute to these disorders is a depleted nitric oxide level, which can be due to the presence of eNOS +894 G > T and eNOS −786 T > C variants that make an individual more susceptible to endothelial dysfunction. A number of genotyping methods have been developed to investigate these variants. However, simultaneous detection methods using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis are still lacking. In this study, a novel multiplex PCR-RFLP method for the simultaneous detection of MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants was developed. A total of 114 healthy Malay subjects were recruited. The MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants were genotyped using the novel multiplex PCR-RFLP and confirmed by DNA sequencing as well as snpBLAST. Allele frequencies of MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C were calculated using the Hardy Weinberg equation.
Methods
The 114 healthy volunteers were recruited for this study, and their DNA was extracted. Primer pair was designed using Primer 3 Software version 0.4.0 and validated against the BLAST database. The primer specificity, functionality and annealing temperature were tested using uniplex PCR methods that were later combined into a single multiplex PCR. Restriction Fragment Length Polymorphism (RFLP) was performed in three separate tubes followed by agarose gel electrophoresis. PCR product residual was purified and sent for DNA sequencing.
Results
The allele frequencies for MTHFR 677 C > T were 0.89 (C allele) and 0.11 (T allele); for eNOS +894 G > T, the allele frequencies were 0.58 (G allele) and 0.43 (T allele); and for eNOS −786 T > C, the allele frequencies were 0.87 (T allele) and 0.13 (C allele).
Conclusions
Our PCR-RFLP method is a simple, cost-effective and time-saving method. It can be used to successfully genotype subjects for the MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants simultaneously with 100% concordance from DNA sequencing data. This method can be routinely used for rapid investigation of the MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants.
doi:10.1186/1471-2350-13-34
PMCID: PMC3511178  PMID: 22594584
6.  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  2009;331(1-2):201-205.
The T-786C, Glu298Asp, and 27 bp variable number of tandem repeats (27 bp-VNTR-a/b) polymorphsims of the endothelial nitric oxide synthase (eNOS) gene are thought to alter nitric oxide production and contribute to the development of vascular and renal disease risk. The objective of this study is to investigate whether these three polymorphisms examined previously by others are associated with cardiovascular and renal disease risk in Mexican Americans. Study participants (N = 848; 21 families) were genotyped for T-786C, Glu298Asp, and 27 bp-VNTR-a/b polymorphisms by PCR followed by restriction digestion. Association analyses were performed by a measured genotype approach implemented in the program SOLAR. Of the phenotypes (type 2 diabetes, hypertension, body mass index, waist circumference, total cholesterol, high density lipoprotein cholesterol, triglycerides, systolic and diastolic blood pressure, albumin to creatinine ratio (ACR), and estimated glomerular filtration rate) examined for association, the 27 bp-VNTR-a/b variant exhibited statistically significant association with ACR (P = 0.047) after accounting for the trait specific covariate effects. In addition, the promoter variant (T-786C) showed a significant association with triglycerides (P = 0.034) after accounting for covariate influences. In conclusion, the present study adds evidence to the role of eNOS candidate gene polymorphisms in modulating the risk factors related to cardiovascular-renal disease in Mexican Americans although the magnitude of the genetic effect is small.
doi:10.1007/s11010-009-0159-5
PMCID: PMC3428139  PMID: 19468830
eNOS; Genetic polymorphisms; Association analyses; ACR; Triglycerides; Mexican Americans
7.  Endothelial Nitric Oxide Synthase (eNOS) Gene Polymorphism is Associated with Age Onset of Menarche in Sickle Cell Disease Females of India 
Background and Objective
Females with sickle cell disease (SCD) often show late onset of menarche. In transgenic sickle cell mouse, deficiency of gene encoding endothelial nitric oxide synthase (eNOS) has been reported to be associated with late onset of menarche. Thus to explore the possible association of eNOS gene polymorphism with age of onset of menarche in SCD females, 3 important eNOS gene polymorphisms- eNOS 4a/b, eNOS 894G>T (rs1799983) and eNOS-786 T>C (rs2070744) and plasma nitrite levels were tested among three groups of females- SCD late menarche, SCD early menarche and control females.
Methodology
About 39 SCD females comprising of 18 SCD early menarche and 21 SCD late menarche groups were studied along with 48 control females. Genotyping of eNOS gene polymorphisms were done by PCR-RFLP and quantification of plasma nitrite level was performed by ELISA based commercial kits.
Results
SCD late menarche females showed significantly higher prevalence and higher association of heterozygous genotypes, higher frequency of mutant alleles .4a., .T. and .C. as compared to that of control group and SCD early menarche group. The frequency of haplotype .4a-G-C. and haplotype .4b-G-C. (alleles in order of eNOS 4a/b, eNOS 894G>T and eNOS-786 T>C respectively) were found to be significantly high in SCD late menarche compared to combined groups of SCD early menarche and controls. SCD late menarche group had significantly low level of plasma nitrite concentration for all 3 eNOS gene polymorphisms as compared to controls and SCD early menarche females.
Conclusion
eNOS gene polymorphism may influence age of onset of menarche in SCD females.
doi:10.4084/MJHID.2013.036
PMCID: PMC3684346  PMID: 23795274
8.  Endothelial Nitric Oxide Synthase Gene T-786C Polymorphism in Renal Transplant Recipients 
Background: Nitric oxide (NO) is a major mediator in vascular biology, regulating regional blood flow. NO and the enzymes required for its production contribute to ischemia-reperfusion injury. The T-786C functional polymorphism in the promoter region substantially reduces promoter activity of the endothelial nitric oxide synthase (eNOS) gene and compromises endothelial NO synthesis.
Objective: To examine the association between T-786C (rs 2070744) single nucleotide polymorphism (SNP) in eNOS gene and the development of acute rejection in renal transplant patients.
Methods: 60 renal transplant recipients (30 with episodes of acute rejection (ARs) and 30 without rejection (non-ARs)), between June 2008 and March 2010, were included in this study. The polymorphism was determined by PCR-restriction fragment-length polymorphism analysis.
Results: The distribution of the genotypes were TT/TC/CC 60%, 33.4%, 6.6%, and 43%, 46.7%, 13.3% in ARs and non-ARs, respectively (p=0.28). The frequency of T-allele was 76.7% and 66.3%; and for C-allele was 66.6% and 33.3% in ARs and non-ARs, respectively (p=0.09). There were no significant associations between these polymorphisms and acute and chronic kidney allograft rejection.
Conclusion: We could not detect any significant association between polymorphism in T-786C of eNOS gene and the development of acute rejection.
PMCID: PMC4089251  PMID: 25013599
Nitric oxide; Endothelial nitric oxide synthase; Polymorphism; Transplant; Acute rejection
9.  Genotype-Dependent Expression of Endothelial Nitric Oxide Synthase (eNOS) and Its Regulatory Proteins in Cultured Endothelial Cells 
DNA and cell biology  2005;24(4):218-224.
DNA polymorphisms in endothelial nitric oxide synthase (eNOS) gene have been shown to be associated with constitutive eNOS expression and coronary artery disease (CAD). In the present study we explored the hypothesis whether genotype-dependent effects can be maintained in vitro during replication, or the effect is conditional on in vivo biological environments. Human umbilical vein endothelial cells (HUVEC) were collected and cultured from 89 normal deliveries of Mexican Americans. The cells were treated with or without cigarette smoking extracts (CSE) and genotypes of eNOS polymorphisms were determined by PCR. We measured the levels of eNOS by ELISA and its binding proteins including heat-shock protein 90 (Hsp-90) and caveolin-1 by Western blotting. The rare C allele for the promoter T786C polymorphism (0.2), and the rare 4 × 27-bp repeat allele in the intron 4 (0.30) were different from those reported in other populations. Yet, the rare T allele in the exon 7 (G894T polymorphism) was similar as others. After four passages in vitro, both the in-tron 4 and promoter polymorphisms maintained significant effects on eNOS mRNA levels in HUVECs (P < 0.05). However, the effects on eNOS protein and enzyme activity were less consistent. Although primary smokers had significantly lower eNOS protein levels (P < 0.05), the in vitro CSE treatment on cultured HUVECs only resulted in a significant reduction in NO levels as measured by the stable metabolites of nitrite/nitrate (P < 0.001). Neither Hsp-90 nor caveolin-1—important eNOS regulators—appears to mediate the genotype-smoking effects on eNOS expression although HUVECs did produce more Hsp-90 when exposed to CSE. Our study demonstrates that endothelial cells maintain genotype-dependent expression even after the deprivation of in vivo environment. However, the cigarette smoking–genotype interaction may require such in vivo conditions to be manifested.
doi:10.1089/dna.2005.24.218
PMCID: PMC1350115  PMID: 15812238
10.  Is there a relationship between endothelial nitric oxide synthase gene polymorphisms and ankylosing spondylitis? 
Clinics  2013;68(3):305-309.
OBJECTIVE:
Nitric oxide is produced by endothelial nitric oxide synthase, and its production can be influenced by polymorphisms of the endothelial nitric oxide synthase gene. Because candidate genes responsible for susceptibility to ankylosing spondylitis are mostly unknown and available data suggest that there may be problems related to the nitric oxide pathway, such as endothelial dysfunction and increased asymmetric dimethylarginine, this study aimed to assess the association of common endothelial nitric oxide synthase gene polymorphisms with ankylosing spondylitis.
METHODS:
One hundred ninety-four unrelated Turkish ankylosing spondylitis patients and 113 healthy without apparent cardiovascular disease, hypertension or diabetes mellitus were included. All individuals were genotyped by PCR-RFLP for two single-nucleotide polymorphisms, namely 786T>C (rs2070744, promoter region) and 786 Glu298Asp (rs1799983, exon 7). Variable numbers of tandem repeat polymorphisms in intron 4 were also studied and investigated by direct electrophoresis on agarose gel following polymerase chain reaction analysis. The Bath ankylosing spondylitis metrology index of the patients was calculated, and human leukocyte antigen B27 was studied.
RESULTS:
All studied polymorphisms satisfied Hardy-Weinberg equilibrium. Sex distributions were similar between the patient and control groups. No significant differences were found in the distributions of allele and genotype frequencies of the studied endothelial nitric oxide synthase polymorphisms between patients and controls. There were no correlations between endothelial nitric oxide synthase polymorphisms, disease duration, Bath ankylosing spondylitis metrology index or human leukocyte antigen B27.
CONCLUSION:
The results presented in this study do not support a major role of common endothelial nitric oxide synthase polymorphisms in Turkish ankylosing spondylitis patients.
doi:10.6061/clinics/2013(03)OA04
PMCID: PMC3611897  PMID: 23644848
Ankylosing Spondylitis; Endothelial Nitric Oxide Synthase; Nitric Oxide; Inflammation; Atherosclerosis
11.  Endothelial nitric oxide synthase gene polymorphisms and risk of diabetic nephropathy: a systematic review and meta-analysis 
BMC Medical Genetics  2014;15:9.
Background
Nitric oxide (NO) has numerous functions in the kidney, including control of renal and glomerular hemodynamics, by interfering at multiple pathological and physiologically critical steps of nephron function. Endothelial NOS (eNOS) gene has been considered a potential candidate gene to diabetic nephropathy (DN) susceptibility. Endothelial nitric oxide synthase gene (eNOS-3) polymorphisms have been associated with DN, however some studies do not confirm this association. The analyzed polymorphisms were 4b/4a, T-786C, and G986T.
Methods
The Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) statement was used in this report. Case–control studies that had diabetic patients with DN as cases and diabetic patients without nephropathy as controls, as well as that evaluated at least one of the three polymorphisms of interest were considered eligible. All studies published up until December 31st, 2012 were identified by searching electronic databases. Hardy-Weinberg equilibrium assessment was performed. Gene-disease association was measured using odds ratio estimation based on the following genetic contrast/models: (1) allele contrast; (2) additive model; (3) recessive model; (4) dominant model and (4) co-dominant model.
Results
Twenty-two studies were eligible for meta-analysis (4b/a: 15 studies, T-786C: 5 studies, and G984T: 12 studies). Considering 4b/a polymorphism, an association with DN was observed for all genetic models: allele contrast (OR = 1.14, CI: 1.04-1.25); additive (OR = 1.77, CI: 1.37-2.28); recessive (OR = 1.77, CI: 1.38-2,27); dominant (OR = 1.12, CI: 1.01-1.24), with the exception for co-dominance model. As well, T-786C polymorphism showed association with all models, with exception for co-dominance model: allele contrast (OR = 1.22, CI: 1.07-1.39), additive (OR = 1.52, CI: 1.18-1.97), recessive (OR = 1.50, CI: 1.16-1.93), and dominant (OR = 1.11, CI: 1.01-1.23). For the G894T polymorphism, an association with DN was observed in allelic contrast (OR = 1.12, CI: 1.03-1.25) and co-dominance models (OR = 1.13, CI: 1.04-1.37).
Conclusions
In the present study, there was association of DN with eNOS 4b/a and T-786C polymorphism, which held in all genetic models tested, except for co-dominance model. G894T polymorphism was associated with DN only in allele contrast and in co-dominance model. This data suggested that the eNOS gene could play a role in the development of DN.
doi:10.1186/1471-2350-15-9
PMCID: PMC3900462  PMID: 24433471
12.  Endothelial Nitric Oxide Synthase Single Nucleotide Polymorphism and Left Ventricular Function in Early Chronic Kidney Disease 
PLoS ONE  2015;10(1):e0116160.
Background
Chronic kidney disease (CKD) is associated with accelerated cardiovascular disease and heart failure. Endothelial nitric oxide synthase (eNOS) Glu298Asp single nucleotide polymorphism (SNP) genotype has been associated with a worse phenotype amongst patients with established heart failure and in patients with progression of their renal disease. The association of a cardiac functional difference in non-dialysis CKD patients with no known previous heart failure, and eNOS gene variant is investigated.
Methods
140 non-dialysis CKD patients, who had cardiac magnetic resonance (CMR) imaging and tissue doppler echocardiography as part of two clinical trials, were genotyped for eNOS Glu298Asp SNP retrospectively.
Results
The median estimated glomerular filtration rate (eGFR) was 50mls/min and left ventricular ejection fraction (LVEF) was 74% with no overt diastolic dysfunction in this cohort. There were significant differences in LVEF across eNOS genotypes with GG genotype being associated with a worse LVEF compared to other genotypes (LVEF: GG 71%, TG 76%, TT 73%, p = 0.006). After multivariate analysis, (adjusting for age, eGFR, baseline mean arterial pressure, contemporary CMR heart rate, total cholesterol, high sensitive C-reactive protein, body mass index and gender) GG genotype was associated with a worse LVEF, and increased LV end-diastolic and systolic index (p = 0.004, 0.049 and 0.009 respectively).
Conclusions
eNOS Glu298Asp rs1799983 polymorphism in CKD patients is associated with relevant sub-clinical cardiac remodelling as detected by CMR. This gene variant may therefore represent an important genetic biomarker, and possibly highlight pathways for intervention, in these patients who are at particular risk of worsening cardiac disease as their renal dysfunction progresses.
doi:10.1371/journal.pone.0116160
PMCID: PMC4303420  PMID: 25612295
13.  Allelic polymorphism in the promoter (T–786→C), but not in exon 7 (G894→T) or the variable number tandem repeat in intron 4, of the endothelial nitric oxide synthase gene is positively associated with acute coronary syndrome in the Ukrainian population 
BACKGROUND
Different allelic variants of endothelial nitric oxide synthase (eNOS) can have different effects on the development of certain forms of ischemic heart disease, depending on the specific human population. The frequency of the polymorphism T–786 → C in the promoter, G894→T in exon 7 and the variable number tandem repeat in intron 4 were assayed in patients with acute coronary syndrome compared with clinically healthy individuals in the Ukrainian population.
METHODS
Polymerase chain reaction and restriction fragment length polymorphism analysis were used to detect the above mentioned variants of the eNOS gene in 221 patients with acute coronary syndrome and in 83 control subjects.
RESULTS
It was shown that the percentage of normal homozygotes, heterozygotes and pathological homozygotes for the T–786→C promoter polymorphism was 47.5%, 36.2% and 16.3%, respectively (controls: 48.2%, 45.8% and 6.0%; P<0.05 by χ2 test); for the G894→T polymorphism in exon 7, the percentages were 34.4%, 57.9% and 7.7%, respectively (controls: 28.9%, 67.5% and 3.6%; P>0.05); and, for the 4a/4b polymorphism in intron 4, the percentages were 64.7%, 31.2% and 4.1%, respectively (controls: 62.7%, 32.5% and 4.8%; P>0.05).
CONCLUSIONS
The C/C promoter variant of eNOS can be considered a risk factor for acute coronary syndrome in the Ukrainian population.
PMCID: PMC2274837  PMID: 18651012
Acute coronary syndrome; Allelic polymorphism; Endothelial NO synthase
14.  Endothelial nitric oxide synthase polymorphism (-786T>C) and increased risk of angiographic vasospasm after aneurysmal subarachnoid hemorrhage 
Background and Purpose
Vasospasm following aneurysmal subarachnoid hemorrhage (SAH) remains a leading cause of death and disability after aneurysm rupture. Decreased availability of nitric oxide (NO) may be crucial in the pathogenesis. We hypothesize that endothelial NO synthase (eNOS) polymorphisms may determine susceptibility to vasospasm in SAH patients.
Methods
We conducted a prospective cohort study of SAH patients, and determined vasospasm by cerebral angiography. We genotyped three eNOS polymorphisms: intron 4 variable-number-tandem-repeat (VNTR), promoter single-nucleotide-polymorphism (-786T>C SNP), and coding SNP in exon 7 (894G>T encoding E298D). Using multivariable logistic regression, we quantified the association of eNOS polymorphisms in patients with vasospasm confirmed by cerebral angiogram.
Results
For the eNOS promoter -786T>C SNP, the presence of the CC genotype compared to any T genotype (CT or TT) was associated with increased odds of vasospasm (OR 2.97, 95%CI 1.32-6.67, p=0.008). No association with vasospasm was observed for the eNOS 894G>T or VNTR polymorphisms.
Conclusions
These findings suggest that genetic variation influencing NO regulation contributes to risk of angiographic vasospasm in patients with SAH. The specific role of the promoter SNP (-786T>C) may determine the effect of NO regulated by this pathway distinct from other known eNOS polymorphisms.
doi:10.1161/STROKEAHA.107.496596
PMCID: PMC2650384  PMID: 18309169
Endothelial nitric oxide; Genetics; Subarachnoid hemorrhage; Vasospasm
15.  Influence of Endothelial Nitric Oxide Synthase Gene Intron-4 27bp Repeat Polymorphism on Its Expression in Autoimmune Diseases 
Disease markers  2013;34(5):349-356.
The purpose of this study was to analyse the effect of the T-786C polymorphism and intron 4 27 bp variable number tandem repeat(VNTR) eNOS markers for their potential association with Systemic Lupus Erythematosus(SLE), Hashimotos thyroiditis(HT) and Rheumatoid arthritis(RA) as well as to explore their effect on eNOS mRNA expression and nitrate production(NOx). Kuwaitis (n = 383) matched by age, gender and ethnicity were genotyped by fluorescent-labelled-restriction fragment length polymorphism (RFLP) and fragment analysis. Expression of eNOS mRNA was analysed using RT-PCR and sera from subjects were screened for NOx using ELISA. Analysis of the allelic frequency revealed a significant association of the 4b allele with susceptibility to SLE (p = 0.0092, OR = 1.76). The 4bb genotype was found to be associated with SLE (p = 0.0076, OR = 1.97) and HT (p = 0.05, OR = 1.81). Allelic and genotypic distribution did not differ between RA patients and healthy control subjects. The 4bb genotype resulted in reduced expression of eNOS mRNA in SLE, RA and HT, but only the reduction in HT was significant (p = 0.05). The 4ab genotype revealed a significant association with increased eNOS expression in HT (p = 0.03) and RA (p = 0.014) patients, and elevated NOx levels were detected in the autoimmune disease cohorts (p < 0.05) when compared to healthy control subjects. The T-786C SNP failed to show a significant association (p > 0.05) with SLE, HT, and RA patients. This study is the first to reveal a significant association between the 4bb genotype of the 27 bp VNTR and susceptibility to HT. The expression of eNOS is related to the number of 27 bp repeats, with heterozygous 4bb repeats showing a decrease in eNOS expression. eNOS – endothelial nitric oxide synthase.
doi:10.3233/DMA-130983
PMCID: PMC3810104  PMID: 23481632
eNOS; SLE; RA; HT
16.  Protein Kinase-C Beta Contributes to Impaired Endothelial Insulin Signaling in Humans with Diabetes Mellitus 
Circulation  2012;127(1):86-95.
Background
Abnormal endothelial function promotes atherosclerotic vascular disease in diabetes. Experimental studies indicate that disruption of endothelial insulin signaling through the activity of protein kinase C-β (PKCβ) and nuclear factor κB (NFκB) reduces nitric oxide availability. We sought to establish whether similar mechanisms operate in the endothelium in human diabetes mellitus.
Methods and Results
We measured protein expression and insulin response in freshly isolated endothelial cells from patients with Type 2 diabetes mellitus (n=40) and non-diabetic controls (n=36). Unexpectedly, we observed 1.7-fold higher basal endothelial nitric oxide synthase (eNOS) phosphorylation at serine 1177 in patients with diabetes (P=0.007) without a difference in total eNOS expression. Insulin stimulation increased eNOS phosphorylation in non-diabetic subjects but not in diabetic patients (P=0.003) consistent with endothelial insulin resistance. Nitrotyrosine levels were higher in diabetic patients indicating endothelial oxidative stress. PKCβ expression was higher in diabetic patients and was associated with lower flow-mediated dilation (r=−0.541, P=0.02) Inhibition of PKCβ with LY379196 reduced basal eNOS phosphorylation and improved insulin-mediated eNOS activation in patients with diabetes. Endothelial NFκB activation was higher in diabetes and was reduced with PKCβ inhibition.
Conclusions
We provide evidence for the presence of altered eNOS activation, reduced insulin action and inflammatory activation in the endothelium of patients with diabetes. Our findings implicate PKCβ activity in endothelial insulin resistance.
doi:10.1161/CIRCULATIONAHA.112.127514
PMCID: PMC3572725  PMID: 23204109
endothelium; diabetes; insulin resistance; nitric oxide
17.  Endothelial Function and Insulin Resistance in Early Postmenopausal Women with Cardiovascular Risk Factors: Importance of ESR1 and NOS3 Polymorphisms 
PLoS ONE  2014;9(7):e103444.
Cardiovascular benefits from estradiol activation of nitric oxide endothelial production may depend on vascular wall and on estrogen receptor alpha (ESR1) and nitric oxide synthase (NOS3) polymorphisms. We have evaluated the microcirculation in vivo through nailfold videocapillaroscopy, before and after acute nasal estradiol administration at baseline and after increased sheer stress (postocclusive reactive hyperemia response) in 100 postmenopausal women, being 70 controls (healthy) and 30 simultaneously hypertensive and diabetic (HD), correlating their responses to PvuII and XbaI ESR1 polymorphisms and to VNTR, T-786C and G894T NOS3 variants. In HD women, C variant allele of ESR1 Pvull was associated to higher vasodilatation after estradiol (1.72 vs 1.64 mm/s, p = 0.01 compared to TT homozygotes) while G894T and T-786C NOS3 polymorphisms were connected to lower increment after shear stress (15% among wild type and 10% among variant alleles, p = 0.02 and 0.04). The G variant allele of ESR1 XbaI polymorphism was associated to higher HOMA-IR (3.54 vs. 1.64, p = 0.01) in HD and higher glucose levels in healthy women (91.8 vs. 87.1 mg/dl, p = 0.01), in which increased waist and HOMA-IR were also related to the G allele in NOS3 G894T (waist 93.5 vs 88.2 cm, p = 0.02; HOMA-IR 2.89 vs 1.48, p = 0.05). ESR1 Pvull, NOS3 G894T and T-786C polymorphism analysis may be considered in HD postmenopausal women for endothelial response prediction following estrogen therapy but were not discriminatory for endothelial response in healthy women. ESR1 XbaI and G894T NOS3 polymorphisms may be useful in accessing insulin resistance and type 2 diabetes risks in all women, even before menopause and occurrence of metabolic disease.
doi:10.1371/journal.pone.0103444
PMCID: PMC4117493  PMID: 25077953
18.  Overexpression of Endothelial Nitric Oxide Synthase Prevents Diet-Induced Obesity and Regulates Adipocyte Phenotype 
Circulation research  2012;111(9):1176-1189.
Rationale
Endothelial dysfunction is a characteristic feature of diabetes and obesity in animal models and humans. Deficits in nitric oxide production by endothelial nitric oxide synthase (eNOS) are associated with insulin resistance, which is exacerbated by high fat diet. Nevertheless, the metabolic effects of increasing eNOS levels have not been studied.
Objective
The current study was designed to test whether overexpression of eNOS would prevent diet-induced obesity and insulin resistance.
Methods and Results
In db/db mice and in high fat-fed wild-type (WT) C57BL/6J mice, the abundance of eNOS protein in adipose tissue was decreased without significant changes in eNOS levels in skeletal muscle or aorta. Mice overexpressing eNOS (eNOS-TG mice) were resistant to diet-induced obesity and hyperinsulinemia, although systemic glucose intolerance remained largely unaffected. In comparison with WT mice, high fat-fed eNOS-TG mice displayed a higher metabolic rate and attenuated hypertrophy of white adipocytes. Overexpression of eNOS did not affect food consumption or diet-induced changes in plasma cholesterol or leptin levels, yet plasma triglycerides and fatty acids were decreased. Metabolomic analysis of adipose tissue indicated that eNOS overexpression primarily affected amino acid and lipid metabolism; subpathway analysis suggested changes in fatty acid oxidation. In agreement with these findings, adipose tissue from eNOS-TG mice showed higher levels of PPAR-α and PPAR–γ gene expression, elevated abundance of mitochondrial proteins, and a higher rate of oxygen consumption.
Conclusions
These findings demonstrate that increased eNOS activity prevents the obesogenic effects of high fat diet without affecting systemic insulin resistance, in part, by stimulating metabolic activity in adipose tissue.
doi:10.1161/CIRCRESAHA.112.266395
PMCID: PMC3707504  PMID: 22896587
Diabetes; mitochondria; metabolism; adipose tissue; cardiovascular disease; obesity; endothelial nitric oxide synthase
19.  Endothelial nitric oxide synthase gene polymorphisms and cardiovascular damage in hypertensive subjects: an Italian case-control study 
Background
Nitric oxide (NO) synthesized by endothelial nitric oxide synthase (eNOS) plays an important role in regulation of endothelial function and in the control of blood pressure. However, the results from some studies on the association between three clinically relevant eNOS gene polymorphisms (G894T, T786C and intron 4b/a) and essential hypertension are unclear. We designed a case-control study to evaluate the influence of eNOS polymorphisms on target organ damage in 127 hypertensives and 67 normotensives. Clinical evaluation, biochemical parameters, Urinary Albumin Excretion (UAE) and echocardiogram were performed to characterize target organ damage. eNOS polymorphism were recognized by PCR method.
Results
The distribution of eNOS genotypes was similar in hypertensives and normotensives but 4aa was present in the 2.5% of hypertensives and completely absent in normotensives. Subjects with 4bb, G894T, and T786C genotypes showed an increased prevalence of target organ damage. Moreover prevalence of G894T and introne 4 variants was significantly higher in hypertensives than in normotensives both with cardiovascular damage. Logistic regression analysis didn't show any association between eNOS polymorphisms, Body Mass Index (BMI), hypertension, gender and cardiovascular damage. Only the age (OR 1.11; IC 95% 1.06–1.18) was predictive of cardiovascular damage in our population.
Conclusion
Our results seem to indicate a lack of association with eNOS variants and cardiovascular damage onset.
doi:10.1186/1742-4933-5-4
PMCID: PMC2430541  PMID: 18510731
20.  Haplotypes of NOS3 Gene Polymorphisms in Dilated Cardiomyopathy 
PLoS ONE  2013;8(7):e70523.
Dilated Cardiomyopathy (DCM) is characterized by systolic dysfunction, followed by heart failure necessitating cardiac transplantation. The genetic basis is well established by the identification of mutations in sarcomere and cytoskeleton gene/s. Modifier genes and environmental factors are also considered to play a significant role in the variable expression of the disease, hence various mechanisms are implicated and one such mechanism is oxidative stress. Nitric Oxide (NO), a primary physiological transmitter derived from endothelium seems to play a composite role with diverse anti-atherogenic effects as vasodilator. Three functional polymorphisms of endothelial nitric oxide synthase (NOS3) gene viz., T-786C of the 5′ flanking region, 27bp VNTR in intron4 and G894T of exon 7 were genotyped to identify their role in DCM. A total of 115 DCM samples and 454 controls were included. Genotyping was carried out by PCR -RFLP method. Allelic and genotypic frequencies were computed in both control & patient groups and appropriate statistical tests were employed. A significant association of TC genotype (T-786C) with an odds ratio of 1.74, (95% CI 1.14 - 2.67, p = 0.01) was observed in DCM. Likewise the GT genotypic frequency of G894T polymorphism was found to be statistically significant (OR 2.10, 95% CI 1.34–3.27, p = 0.0011), with the recessive allele T being significantly associated with DCM (OR 1.64, 95% CI 1.18 - 2.30, p = 0.003). The haplotype carrying the recessive alleles of G894T and T-786C, C4bT was found to exhibit 7 folds increased risk for DCM compared to the controls. Hence C4bT haplotype could be the risk haplotype for DCM. Our findings suggest the possible implication of NOS3 gene in the disease phenotype, wherein NOS3 may be synergistically functioning in DCM associated heart failure via the excessive production of NO in cardiomyocytes resulting in decreased myocardial contractility and systolic dysfunction, a common feature of DCM phenotype.
doi:10.1371/journal.pone.0070523
PMCID: PMC3726655  PMID: 23923002
21.  NOS3 polymorphisms, cigarette smoking, and cardiovascular disease risk 
Pharmacogenetics and genomics  2006;16(12):891-899.
Objective
Endothelial nitric oxide synthase (NOS3) activity and cigarette smoking significantly influence endothelial function. We sought to determine whether cigarette smoking modified the association between NOS3 polymorphisms and risk of coronary heart disease or stroke.
Methods
All 1085 incident coronary heart disease cases, all 300 incident ischemic stroke cases, and 1065 reference individuals from the Atherosclerosis Risk in Communities study were genotyped for the T-786C and E298D polymorphisms in NOS3. Using a case–cohort design, associations between genotype/haplotype and disease risk were evaluated by multivariable proportional hazards regression. Multiplicative scale interaction testing evaluated the influence of cigarette smoking history at baseline on these associations.
Results
In Caucasians, association between E298D genotype and risk of coronary heart disease was significantly modified by current smoking status (interaction P = 0.013), with the highest risk observed in smokers carrying the variant D298 allele relative to nonsmokers carrying two E298 alleles (adjusted hazard rate ratio 2.07, 95% confidence interval 1.39−3.07). In African-Americans, association between T-786C genotype and risk of ischemic stroke was significantly modified by pack-year smoking history (interaction P = 0.037), with the highest risk observed in ≥ 20 pack-year smokers carrying the variant C-786 allele relative to < 20 pack-year smokers carrying two T-786 alleles (adjusted hazard rate ratio 4.03, 95% confidence interval 1.54−10.6).
Conclusions
An interaction between the E298D and T-786C polymorphisms in NOS3, cigarette smoking, and risk of incident coronary heart disease and ischemic stroke events appears to exist, suggesting a potential complex interplay between genetic and environmental factors and cardiovascular disease risk.
doi:10.1097/01.fpc.0000236324.96056.16
PMCID: PMC1978174  PMID: 17108813
coronary artery disease; endothelial nitric oxide synthase; polymorphism; smoking; stroke
22.  Inflammation, Insulin Resistance, and Diabetes—Mendelian Randomization Using CRP Haplotypes Points Upstream 
PLoS Medicine  2008;5(8):e155.
Background
Raised C-reactive protein (CRP) is a risk factor for type 2 diabetes. According to the Mendelian randomization method, the association is likely to be causal if genetic variants that affect CRP level are associated with markers of diabetes development and diabetes. Our objective was to examine the nature of the association between CRP phenotype and diabetes development using CRP haplotypes as instrumental variables.
Methods and Findings
We genotyped three tagging SNPs (CRP + 2302G > A; CRP + 1444T > C; CRP + 4899T > G) in the CRP gene and measured serum CRP in 5,274 men and women at mean ages 49 and 61 y (Whitehall II Study). Homeostasis model assessment-insulin resistance (HOMA-IR) and hemoglobin A1c (HbA1c) were measured at age 61 y. Diabetes was ascertained by glucose tolerance test and self-report. Common major haplotypes were strongly associated with serum CRP levels, but unrelated to obesity, blood pressure, and socioeconomic position, which may confound the association between CRP and diabetes risk. Serum CRP was associated with these potential confounding factors. After adjustment for age and sex, baseline serum CRP was associated with incident diabetes (hazard ratio = 1.39 [95% confidence interval 1.29–1.51], HOMA-IR, and HbA1c, but the associations were considerably attenuated on adjustment for potential confounding factors. In contrast, CRP haplotypes were not associated with HOMA-IR or HbA1c (p = 0.52–0.92). The associations of CRP with HOMA-IR and HbA1c were all null when examined using instrumental variables analysis, with genetic variants as the instrument for serum CRP. Instrumental variables estimates differed from the directly observed associations (p = 0.007–0.11). Pooled analysis of CRP haplotypes and diabetes in Whitehall II and Northwick Park Heart Study II produced null findings (p = 0.25–0.88). Analyses based on the Wellcome Trust Case Control Consortium (1,923 diabetes cases, 2,932 controls) using three SNPs in tight linkage disequilibrium with our tagging SNPs also demonstrated null associations.
Conclusions
Observed associations between serum CRP and insulin resistance, glycemia, and diabetes are likely to be noncausal. Inflammation may play a causal role via upstream effectors rather than the downstream marker CRP.
Using a Mendelian randomization approach, Eric Brunner and colleagues show that the associations between serum C-reactive protein and insulin resistance, glycemia, and diabetes are likely to be noncausal.
Editors' Summary
Background.
Diabetes—a common, long-term (chronic) disease that causes heart, kidney, nerve, and eye problems and shortens life expectancy—is characterized by high levels of sugar (glucose) in the blood. In people without diabetes, blood sugar levels are controlled by the hormone insulin. Insulin is released by the pancreas after eating and “instructs” insulin-responsive muscle and fat cells to take up the glucose from the bloodstream that is produced by the digestion of food. In the early stages of type 2 diabetes (the commonest type of diabetes), the muscle and fat cells become nonresponsive to insulin (a condition called insulin resistance), and blood sugar levels increase. The pancreas responds by making more insulin—people with insulin resistance have high blood levels of both insulin and glucose. Eventually, however, the insulin-producing cells in the pancreas start to malfunction, insulin secretion decreases, and frank diabetes develops.
Why Was This Study Done?
Globally, about 200 million people have diabetes, but experts believe this number will double by 2030. Ways to prevent or delay the onset of diabetes are, therefore, urgently needed. One major risk factor for insulin resistance and diabetes is being overweight. According to one theory, increased body fat causes mild, chronic tissue inflammation, which leads to insulin resistance. Consistent with this idea, people with higher than normal amounts of the inflammatory protein C-reactive protein (CRP) in their blood have a high risk of developing diabetes. If inflammation does cause diabetes, then drugs that inhibit CRP might prevent diabetes. However, simply measuring CRP and determining whether the people with high levels develop diabetes cannot prove that CRP causes diabetes. Those people with high blood levels of CRP might have other unknown factors in common (confounding factors) that are the real causes of diabetes. In this study, the researchers use “Mendelian randomization” to examine whether increased blood CRP causes diabetes. Some variants of CRP (the gene that encodes CRP) increase the amount of CRP in the blood. Because these variants are inherited randomly, there is no likelihood of confounding factors, and an association between these variants and the development of insulin resistance and diabetes indicates, therefore, that increased CRP levels cause diabetes.
What Did the Researchers Do and Find?
The researchers measured blood CRP levels in more than 5,000 people enrolled in the Whitehall II study, which is investigating factors that affect disease development. They also used the “homeostasis model assessment-insulin resistance” (HOMA-IR) method to estimate insulin sensitivity from blood glucose and insulin measurements, and measured levels of hemoglobin A1c (HbA1c, hemoglobin with sugar attached—a measure of long-term blood sugar control) in these people. Finally, they looked at three “single polynucleotide polymorphisms” (SNPs, single nucleotide changes in a gene's DNA sequence; combinations of SNPs that are inherited as a block are called haplotypes) in CRP in each study participant. Common haplotypes of CRP were related to blood serum CRP levels and, as previously reported, increased blood CRP levels were associated with diabetes and with HOMA-IR and HbA1c values indicative of insulin resistance and poor blood sugar control, respectively. By contrast, CRP haplotypes were not related to HOMA-IR or HbA1c values. Similarly, pooled analysis of CRP haplotypes and diabetes in Whitehall II and another large study on health determinants (the Northwick Park Heart Study II) showed no association between CRP variants and diabetes risk. Finally, data from the Wellcome Trust Case Control Consortium also showed no association between CRP haplotypes and diabetes risk.
What Do These Findings Mean?
Together, these findings suggest that increased blood CRP levels are not responsible for the development of insulin resistance or diabetes, at least in European populations. It may be that there is a causal relationship between CRP levels and diabetes risk in other ethnic populations—further Mendelian randomization studies are needed to discover whether this is the case. For now, though, these findings suggest that drugs targeted against CRP are unlikely to prevent or delay the onset of diabetes. However, they do not discount the possibility that proteins involved earlier in the inflammatory process might cause diabetes and might thus represent good drug targets for diabetes prevention.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050155.
This study is further discussed in a PLoS Medicine Perspective by Bernard Keavney
The MedlinePlus encyclopedia provides information about diabetes and about C-reactive protein (in English and Spanish)
US National Institute of Diabetes and Digestive and Kidney Diseases provides patient information on all aspects of diabetes, including information on insulin resistance (in English and Spanish)
The International Diabetes Federation provides information about diabetes, including information on the global diabetes epidemic
The US Centers for Disease Control and Prevention provides information for the public and professionals on all aspects of diabetes (in English and Spanish)
Wikipedia has a page on Mendelian randomization (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
doi:10.1371/journal.pmed.0050155
PMCID: PMC2504484  PMID: 18700811
23.  Endothelial Nitric Oxide Synthase Glu298Asp Gene Polymorphism is Associated with Hypertensive Response to Exercise in Well-Controlled Hypertensive Patients 
Yonsei Medical Journal  2007;48(3):389-395.
Purpose
Hypertensive response to exercise (HRE) is known to be an adverse prognostic factor for future cardiovascular events and may be associated to endothelial dysfunction. Previous studies regarding endothelial nitric oxide synthase (eNOS) Glu298Asp polymorphism focused upon its relation to hypertension. In this study, we hypothesize that the polymorphism may be associated with inherent difference in endothelial response to exercise.
Patients and Methods
Two hundred sixty nine patients who underwent treadmill test were enrolled in this study; 77 patients (mean age 55.8 ± 9.4 years) had hypertensive response (peak systolic BP of ≥ 210mmHg in men and ≥ 190mmHg in women). Pulse wave velocity (PWV) was measured on 153 patients of them. The Glu298Asp exchange in exon 7 was determined by the methods of single base extension with amplifying primers and probes for TaqMan.
Results
The percentages of the GG, GT and TT genotypes were 81.0, 18.6 and 0.4%, respectively. The presence of GT or TT genotype was independently associated with prevention of HRE when controlled for age, sex, baseline systolic BP and homeostatic model assessment (HOMA) index (OR = 0.35, p = 0.016). Subgroup analysis showed that preventive effect for HRE of T allele was significant in females (p < 0.001) and patients without insulin resistance (p = 0.009).
Conclusion
In our study, eNOS Glu298Asp polymorphism was significantly associated with HRE. This result suggests that the presence of T allele of the Glu298Asp polymorphism may be a favorable factor to in preventing HRE, especially in female and patients without insulin resistance.
doi:10.3349/ymj.2007.48.3.389
PMCID: PMC2628106  PMID: 17594145
Endothelial nitric oxide synthase; gene polymorphism; hypertension; hypertensive response to exercise
24.  Is type 2 diabetes mellitus a vascular disease (atheroscleropathy) with hyperglycemia a late manifestation? The role of NOS, NO, and redox stress. 
Background
Cardiovascular disease accounts for at least 85 percent of deaths for those patients with type 2 diabetes mellitus (T2DM). Additionally, 75 percent of these deaths are due to ischemic heart disease.
Hypothesis
Is type 2 diabetes mellitus a vascular disease (atheroscleropathy) with hyperglycemia a late manifestation? The role of NOS, NO, and redox stress.
Testing of the hypothesis
The vulnerable three arms of the eNOS reaction responsible for the generation of eNO is discussed in relation to the hypothesis: (1). The L-arginine substrate. (2). The eNOS enzyme. (3). The BH4 cofactor.
Implications of the hypothesis
If we view T2DM as a vascular disease initially with a later manifestation of hyperglycemia, we may be able to better understand and modify the multiple toxicities associated with insulin resistance, metabolic syndrome, prediabetes, overt T2DM, and accelerated atherosclerosis (atheroscleropathy). The importance of endothelial nitric oxide synthase, endothelial nitric oxide, tetrahydrobiopterin (BH4), L-arginine, and redox stress are discussed in relation to endothelial cell dysfunction and the development and progression of atheroscleropathy and T2DM. In addition to the standard therapies to restore endothelial cell dysfunction and stabilization of vulnerable atherosclerotic plaques, this article will discuss the importance of folic acid (5MTHF) supplementation in this complex devastating disease process.
Atheroscleropathy and hyperglycemia could be early and late manifestations, respectively, in the natural progressive history of T2DM.
doi:10.1186/1475-2840-2-2
PMCID: PMC151667  PMID: 12628022
ADMA; Atherosclerosis; Folic Acid; Oxidative stress; Reactive Oxygen Species (ROS); endothelial Nitric Oxide (eNO); endothelial Nitric Oxide Synthase (eNOS) (NOS-3)
25.  Gender-related associations of genetic polymorphisms of α-adrenergic receptors, endothelial nitric oxide synthase and bradykinin B2 receptor with treadmill exercise test responses 
Open Heart  2014;1(1):e000132.
Background
Treadmill exercise test responses have been associated with cardiovascular prognosis in individuals without overt heart disease. Neurohumoral and nitric oxide responses may influence cardiovascular performance during exercise testing. Therefore, we evaluated associations between functional genetic polymorphisms of α-adrenergic receptors, endothelial nitric oxide synthase, bradykinin receptor B2 and treadmill exercise test responses in men and women without overt heart disease.
Methods
We enrolled 766 (417 women; 349 men) individuals without established heart disease from a check-up programme at the Heart Institute, University of São Paulo Medical School. Exercise capacity, chronotropic reserve, maximum heart-rate achieved, heart-rate recovery, exercise systolic blood pressure (SBP), exercise diastolic blood pressure (DBP) and SBP recovery were assessed during exercise testing. Genotypes for the α-adrenergic receptors ADRA1A Arg347Cys (rs1048101), ADRA2A 1780 C>T (rs553668), ADRA2B Del 301–303 (rs28365031), endothelial nitric synthase (eNOS) 786 T>C (rs2070744), eNOS Glu298Asp (rs1799983) and BK2R (rs5810761) polymorphisms were assessed by PCR and high-resolution melting analysis.
Results
Maximum SBP was associated with ADRA1A rs1048101 (p=0.008) and BK2R rs5810761 (p=0.008) polymorphisms in men and ADRA2A rs553668 (p=0.008) and ADRA2B rs28365031 (p=0.022) in women. Maximum DBP pressure was associated with ADRA2A rs553668 (p=0.002) and eNOS rs1799983 (p=0.015) polymorphisms in women. Exercise capacity was associated with eNOS rs2070744 polymorphisms in women (p=0.01) and with eNOS rs1799983 in men and women (p=0.038 and p=0.024).
Conclusions
The findings suggest that genetic variants of α-adrenergic receptors and bradykinin B2 receptor may be involved with blood pressure responses during exercise tests. Genetic variants of endothelial nitric oxide synthase may be involved with exercise capacity and blood pressure responses during exercise tests. These responses may be gender-related.
doi:10.1136/openhrt-2014-000132
PMCID: PMC4275769  PMID: 25544888
GENETIC POLYMORPHISM; ALPHA-ADRENERGIC RECEPTORS; EXERCISE TESTING; NITRIC OXIDE SYNTHASE; BRADYKININ RECEPTOR

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