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Protein expression profiling in the serum is used to identify novel biomarkers and investigate the signaling pathways in various diseases. The aim of the present study was to evaluate serum biomarkers associated with coronary artery stenosis resulting from atherosclerosis. The study included 4 groups of subjects: group A and B with and without coronary lesions, respectively, were selected from a previously reported cohort study on coronary atherosclerosis, control group C comprised of asymptomatic subjects and group D was used for independent validation of the microarray data by ELISA. Labeled serum proteins were profiled by an Explorer antibody array, which included 656 specific antibodies in two replicates (FullMoon Biosystems, USA). Cadherin-P, interleukin-5, glutathione S-transferase Mu, and neuronal nitric oxide synthase were sex-independently increased in Group A compared with those in group B. The microarray data on cadherin-P were externally validated in an independent group D using ELISA. Fibroblast growth factor-1, FGF-2, collagen II, granulocyte-macrophage colony-stimulating factor, IL-1 alpha, angiopoietin-2, granulocyte colony-stimulating factor, lymphocyte cell-specific protein tyrosine kinase, and IkappaB kinase b were increase in men in group A compared with group B. Cyclin-dependent kinase 1, DNA fragmentation factor subunit alpha DFF45/ICAD, adenovirus type 2 E1A, calponin, ADP-ribosylation factor-6, muscle-specific actin, thyroid hormone receptor alpha, and alpha-methylacyl-CoA racemase were specifically increased in women in Group A compared with group B. Alterations in the levels of specific proteins may point to the signaling pathways contributing to coronary atherosclerosis, and these proteins will be useful biomarkers for the progression of cardiovascular diseases.

Aims. The goal of the present case-control study was to assess associations of a panel of routine and experimental biomarkers with the risk for cardiovascular death and acute myocardial infarction (AMI) in a cohort recruited from the multicenter study “Cardiovascular Epidemiology in Russian Federation” (ESSE-RF).Methods and Results. A total of 222 subjects included cardiovascular death (N=48) and AMI cases (N=63) detected during 6.5-year follow up with corresponding numbers of matched healthy controls. The levels of 15 routine and experimental biochemical markers were assayed to analyze associations with the outcomes using logistic and Cox proportional hazard regressions validated by comparison between various groups to assess the estimated hazard ratio (eHR). Elevated levels of cardiac troponin I (cTnI), C-reactive protein (CRP), and metabolites of nitric oxide (NOx) were independently associated (P

Objectives Endothelial dysfunction contributes to the onset and progression of cardiovascular diseases. However, direct associations of vasoactive mediators with cardiovascular risk are poorly understood. Methods We have determined associations of circulating levels of stable metabolites of nitric oxide, nitrate and nitrite (NOx), endothelin-1, and the endothelin-1/NOx ratio with blood pressure in 177 asymptomatic subjects without signs of coronary atherosclerosis; associations with blood pressure and with presence of coronary lesions were also evaluated in 457 patients suspected to have coronary heart disease with or without coronary lesions confirmed by coronary angiography. All participants were on a low nitrate diet 24 h prior to blood sampling. Results In men, NOx levels were inversely correlated with blood pressure similar to women with low (0–4%) European Systematic Coronary Risk Estimation (SCORE). However, the correlation was not significant in women with high SCORE (5–8%). High systolic blood pressure over 140 mm Hg was negatively associated with NOx levels in asymptomatic men (p=0.05) but not in women. This association is disrupted in male and female patients with coronary atherosclerosis. In male patients, NOx (p=0.05), endothelin (p=0.01), and the endothelin/NOx ratio (p=0.04) were associated with presence of coronary lesions. Conclusions Thus, elevated cardiovascular risk according to SCORE over 4% in asymptomatic women, but not in men, is associated with a shift in markers of endothelial dysfunction. Presence of coronary lesions in patients is associated with significant changes in circulating levels of markers of endothelial dysfunction in men but not in women.

We propose an optimized single-step assay of total nitrite and nitrate concentrations based on serum deproteinization by ultrafiltration. After deproteinization, the residual protein concentration was 0.0800±0.0007 mg/ml, which secured necessary transparency of the specimens in contrast to methanol deproteinization. The optimized assay for serum nitrite and nitrate ions is characterized by high reproducibility and accuracy (variation coefficient

Adiponectin is encoded by the ADIPOQ gene and participates in the pathogenesis of cardiovascular and metabolic diseases. The goal of the study was to assess associations of rs17300539, rs266729, rs182052, rs2241766, and rs17366743 single nucleotide polymorphisms (SNPs) of the ADIPOQ gene with concentrations of serum adiponectin and with coronary atherosclerosis and type 2 diabetes mellitus in 447 patients (316 men and 131 women) subjected to coronary angiography. SNPs of the ADIPOQ gene of the study participants were genotyped using real-time PCR. Multivariate linear regression adjusted for covariates revealed significant association between rs182052 SNP and serum adiponectin concentration (&beta, = &ndash, 0.11, 95% confidence interval (95%CI): &ndash, 0.19, &ndash, 0.03, p = 0.016). Regression analysis revealed an increase in prevalence of unstable angina (OR (odds ratio) = 2.55, 95%CI 1.4&ndash, 4.82, p = 0.018) and coronary artery disease (OR = 1.55, 95%CI 1.15&ndash, 2.09, p = 0.021) per copy of the rs182052 A allele. Prevalence of type 2 diabetes mellitus was higher in subjects with the rs182052 A allele (OR = 2.29, 95%CI 1.29-4.21, p = 0.024). Regression analysis of rs266729 showed that prevalence of unstable angina was increased (OR = 3.59, 95%CI 1.17&ndash, 10.01, p = 0.045) in the subjects with the GG genotype and prevalence of coronary artery disease (CAD) was significantly increased (OR = 1.48, 95%CI 1.09&ndash, 2.03, p = 0.045) per copy of the G allele. Haplotype analysis revealed that the subjects with the GCATT haplotype have lower adiponectin levels (&beta, 0.15, p = 0.042) and higher prevalence of unstable angina (OR = 3.597, p = 0.007) compared with reference haplotype carriers. Thus, the results indicate that minor A allele of rs182052 of the ADIPOQ gene is significantly associated with a decrease in serum adiponectin levels, and two SNPs (rs182052 and rs266729) of the ADIPOQ gene are significantly associated with cardiovascular and metabolic diseases.

Adiponectin, endothelin and nitric oxide (NO) are major regulators of vascular function. An imbalance of vasoactive factors contributes to the onset and progression of atherosclerosis. Various single nucleotide polymorphisms (SNPs) are considered to be risk factors for coronary heart disease. However, the molecular mechanisms of their associations with the components of endothelial dysfunction are poorly understood. In the present study, rs17366743, rs17300539, rs266729, rs182052 and rs2241766 SNPs of the adiponectin (ADIPOQ) gene and rs2070699, rs1800542 and rs1800543 SNPs of the endothelin-1 (EDN1) gene were genotyped in 477 patients with coronary heart disease who were subjected to coronary angiography, in order to determine the presence or absence of coronary atherosclerosis. The serum levels of adiponectin, endothelin and stable metabolites of NO, (nitrate and nitrite NOx), were assayed and their associations with the SNP genotypes and coronary lesions were calculated. The results indicated that rs17366743 of the ADIPOQ gene and rs2070699 and rs1800543 of the EDN1 gene were associated with the levels of NOx in women, which in turn was associated with cardiovascular mortality. In men, rs182052 and rs266729 of the ADIPOQ gene were associated with adiponectin levels, whereas rs17366743 of the ADIPOQ gene was associated with endothelin levels. Additionally, these SNPs were indirectly associated with the prevalence of coronary lesions in men. Therefore, the tested SNPs can be considered potential risk factors that lead to imbalance of vasoactive mediators in a gender-specific manner and contribute to the development of clinical manifestations of atherosclerosis.

Nitric oxide (NO) is an important functional regulator that contributes to progression of various cardiovascular diseases. We studied associations between nitric oxide metabolites, nitrite and nitrate (NOx), and cardiovascular mortality in a prospective 3-year follow-up cohort study in 1,869 elderly patients aged over 55 years. The Cox proportional hazard regression model was adjusted for multiple factors including sex, age, risk corresponding to preexisting cardiovascular conditions, and serum inflammatory markers (C-reactive protein, interleukin-6, fibrinogen, and leucocytes count). During the follow-up period, there were a total of 348 deaths including 216 deaths unrelated to cardiovascular events and 132 cardiovascular deaths. Cox regression adjusted for factors related to cardiovascular disease risks and inflammatory markers showed a significant association between high levels of serum nitric oxide metabolites, NOx, and increased cardiovascular mortality (hazard ratio 2.21; 95% confidence interval 1.13-4.31) but there was no association with non-cardiovascular mortality. Analysis of adjusted hazard ratios demonstrates that association of serum nitric oxide metabolites with cardiovascular mortality was independent of levels of inflammatory markers. Thus, elevated concentrations of serum nitric oxide metabolites are a predictor of cardiovascular mortality and may be used as an integral marker of cardiovascular death. © 2016 BioFactors, 43(1):82-89, 2017.

Introduction and purpose. Endothelial glycocalyx is a protective structure of the vascular bed and is damaged in early stages of cardiovascular diseases. Dark field microscopy can determine the depth of penetration of red blood cells in endothelial glycocalyx to estimate the perfusion boundary region (PBR). The aim of this study was to test and validate this method. Materials and methods. The study involved 38 healthy volunteers aged from 28 to 65 years (18 women and 20 men). The sidearm dark field microscopy of the mucosal microvessels in the sublingual area was used to determine the depth of penetration of red blood cells in endothelial glycocalyx to estimate PBR. Results end discussion. The results indicate good tolerability of the test by patients. Our data on the structure of microvessels were highly reproducible and did not depend on the sampling region within sublingual area and on time intervals between sampling. PBR value was not gender-dependent. Conclusion. The dark field microscopy method passed the first step of validation, showing a good feasibility and tolerability, as well as high reproducibility of measurements that are independent of the sampling region and time intervals between sampling.

Analysis of serum proteome by antibody microarray is used to identify novel biomarkers and to study signaling pathways including protein phosphorylation and protein-protein interactions. Labeling of serum proteins is important for optimal performance of the antibody microarray. Proper choice of fluorescent label and optimal concentration of protein loaded on the microarray ensure good quality of imaging that can be reliably scanned and processed by the software. We have optimized direct serum protein labeling using fluorescent dye Arrayit Green 540 (Arrayit Corporation, USA) for antibody microarray. Optimized procedure produces high quality images that can be readily scanned and used for statistical analysis of protein composition of the serum.

Aim. To study the relationship between serum endothelin levels and lipid-lowering therapy in patients with confirmed coronary atherosclerosis. Material and methods. Patients (n=447; 320 men and 127 women; mean age 62.7±8.8 years) with coronary atherosclerosis, confirmed by coronary angiography, were included into the study. Serum endothelin levels were assessed by enzyme immunoassay (ELISA). Results. Negative correlation between the statins receiving and serum endothelin level was found in men (r=-0.11; p=0.04). Serum endothelin level was 1.8 times lower in men who received statins, compared with men without statin therapy. The relationship between statins receiving and serum endothelin level was not found in women with coronary atherosclerosis. Conclusion. The statins receiving in men with coronary atherosclerosis, in contrast to women, negatively correlated with serum endothelin level and is associated with its almost twofold reduction. Relationship between serum endothelin level and receiving other drugs was not found.

Inflammation is an important factor in the onset and development of pathological lesions of coronary arteries. Various cytokines, including interleukin-6 (IL-6), and pattern recognition receptors, such as C-reactive protein (CRP), are the key inflammatory mediators. We aimed to identify associations of single nucleotide polymorphisms (SNPs) rs1800795 of the IL-6 gene and rs3729960 of the IL6ST gene with levels of inflammatory markers and coronary lesions. The study included 316 men and 131 women (mean age 61.2 ± 9.4 years) subjected to coronary angiography. IL-6 levels were assayed by ELISA and high sensitivity CRP (hsCRP) levels were measured by turbidimetry. The rs1800795 and rs3729960 genotypes of the IL-6 and IL6ST genes, respectively, were determined by real time PCR. Data were analyzed by the SPSS software. In women, the CC genotype (N = 31) of rs1800795 of the IL-6 gene is associated with lower concentrations of hsCRP in the dominant model (GG + CG; N = 38 and N = 62, respectively) and the corresponding hsCRP levels were significantly different (P = .015) at 1.70 mg/l (25% 0.75; 75% 3.55) and 3.30 mg/l (25% 1.20; 75% 6.18). In men, these genotypes were not associated with hsCRP levels. Distribution of allele frequency of rs1800795 of the IL-6 gene in groups with and without coronary lesions indicate that the G allele has significantly higher frequency in men but not in women with coronary atherosclerosis (OR 2.45; 95%CI 1.31–4.72; P = .003). Thus, in women, rs1800795 of the IL-6 gene is associated with hsCRP levels and in men, rs1800795 is associated with presence of coronary lesions.

Background Nitric oxide and its metabolites, nitrate and nitrite, are important regulators linked to various diseases. We studied the association of fasting serum concentrations of nitrate and nitrite, combined as NOx, without special diet, with the prevalence of various chronic diseases. Methods Fasting concentrations of NOx were assayed in a cohort of 1087 patients recruited to Stress Aging and Health in Russia study that represents male and female population in Moscow, Russia, over 55 years of age. Chronic diseases were recorded based on anamnesis and additional assays were run to characterize immune status and lipid and carbohydrate metabolism. Odds ratios were calculated to associate NOx concentrations with prevalence of chronic diseases in pooled deciles below or above borderline. Results NOx over 44.7 µM were associated with increased prevalence of various chronic diseases such as diabetes type II, hyperthyroidism, coronary heart disease, gout and thrombosis/stroke. NOx 65.3 µM and above were associated with lowered prevalence of osteoporosis. NOx levels of 74.6 µM and above were associated with significantly higher number of patients who abstain from consumption of alcoholic beverages. NOx were not associated with cancer. Conclusions Thus, fasting concentrations of NOx in serum can be an important diagnostic parameter characteristic for specific chronic diseases.

Noninvasive diagnostics of early stages of coronary artery disease and discrimination between various extents of vascular lesions in patients is an important clinical problem especially considering wide spread use of cholesterol lowering drugs that affect lipid and lipoprotein profiling. The main goal of our study was to evaluate applicability of new combinations of noninvasive biomarkers such as leptin to insulin and adiponectin to endothelin ratios, for detection of early stages of coronary atherosclerosis versus later stages of the disease.A number of previously validated serum biomarkers were tested in a group of 500 patients with coronary artery disease and examined for their association with severity of coronary lesion according to Gensini score determined by coronary angiography.Lowest extent of coronary lesions was associated with significant increase in apoA-I levels and with significantly increased ratios of adiponectin to endothelin and leptin to insulin. In male but not in female patients, adiponectin to endothelin ratio below 7.0 was associated with Gensini score representing early to high coronary lesions (p = 0.02). In female but not in male patients, leptin to insulin ratio below 3.5 was associated with Gensini score representing early to high coronary lesions (p = 0.013).Leptin to insulin and adiponectin to endothelin ratios are novel derived biomarkers useful for noninvasive diagnostics of initial stages of coronary lesions in patients with coronary artery disease.

BACKGROUND Nitric oxide (NO) is one of the key regulators of vascular function. Abnormal NO signalling is linked to various cardiovascular diseases. We studied associations between circulating levels of NO metabolites, nitrite and nitrate (NOx) and total and cardiovascular mortality in a prospective 8-year follow-up cohort study in 1869 patients aged over 55 years. MATERIALS AND METHODS The Cox proportional hazard ratio (HR) regression models were adjusted for multiple risk-related variables. Post hoc Kaplan-Meier survival curves were compared by the Log-rank test. RESULTS Proportional Cox regression analysis demonstrated that high serum levels of NOx over 70 µmol/L were associated with elevated total mortality (HR 1.4; 95% CI: 1.06-1.80; P = 0.02) and cardiovascular mortality (HR 1.4; 95% CI: 0.98-1.98; P = 0.03) when HR was adjusted for age, sex, smoking and urinary creatinine. Additional adjustments for various mortality-associated baseline comorbidities did not influence associations of elevated NOx with total and cardiovascular mortality. Association of elevated NOx with total mortality persisted in the multivariate regression model combining a number of other characteristics while association of NOx with cardiovascular mortality became non-significant in the multivariate model. Specific subset of patients contributing to these associations was determined by Kaplan-Meier survival analysis indicating that cardiovascular and total mortality were increased in men with high serum levels of NOx over 70 µmol/L (Log-rank test P = 0.01). These associations were not observed in women. CONCLUSION Elevated concentrations of serum NOx over 70 µmol/L can be used to predict mortality in men over 55 years of age.

Objective PBR characterizes penetration of red blood cells inside glycocalyx and its thickness can have profound impact on microcirculation and other vascular parameters. The goal of our study was to reliably quantify PBR and assess its potential use as a new marker of cardiovascular pathology. Methods The study included 208 patients (123 men and 85 women from 40 to 65 years of age) with various grades of cardiovascular SCORE risk index and IHD. PBR was quantified by sidestream dark field capillaroscopy with green light excitation. Cutaneous microcirculation was evaluated with laser Doppler fluorometry. Results Elevated PBR values over 2 mm were associated with morphological and functional lesions of arterial wall and microcirculation and lowered levels of ApoA1 lipoprotein. Moreover, elevated PBR values were associated with 2.07-fold increase in prevalence of cerebral atherosclerosis (P = .015) and 2.42-fold increase in prevalence of IHD (P = .024). Increase in PBR was associated with elevated systolic blood pressure. Conclusions Thus, PBR can be considered a new highly reproducible and promising marker candidate for non-invasive diagnostics of IHD and cerebral atherosclerosis suggesting important role of microcirculation in development and progression of cardiovascular diseases.

We studied the effects of antioxidants resveratrol and pQ510 on physiological parameters and the state of endothelial NO-synthase as a marker of the regulatory function of the endothelium in the aorta of rats with modeled arterial hypertension. The antioxidants promoted recovery of stable NO metabolites in rat serum and maintained expression of endothelial NO-synthase at a normal level. These effects were confirmed by correction of blood pressure and endothelium-dependent vascular dilation assessed by endothelial dysfunction coefficient.

Nitric oxide, NO, is the key vasorelaxing agent produced by vascular endothelium. Stable end products of NO oxidation, nitrate and nitrite (NOx), are considered as markers of endothelial dysfunction associated with hypertension. Our goal was to compare NOx levels in patients with hypertension on controlled low nitrate diet with NOx levels in general population without dietary restrictions.The study included 1005 participants split into 8 groups: healthy subjects (n=28) and patients with moderate (n=37) and malignant hypertension (n=24) on low nitrate diet; healthy subjects (n=9) and patients with stroke (n=44) on low nitrate diet; normotensive subjects (n=354) and patients with elevated blood pressure (n=430) and severe hypertension (n=79) without dietary restrictions.In groups with controlled low NOx diet, patients with moderate and malignant hypertension had significantly (p0.01) higher serum NOx versus healthy subject (66.9±18.8μM and 75.3±22.7μM versus 52.8±28.8μM, respectively). Patients with stroke had significantly (p=0.02) higher serum NOx levels versus corresponding group of healthy subjects (106.3±58.4μM versus 71.2±18.1μM, respectively). In the absence of dietary control, differences in serum NOx levels between normotensive subjects and patients with elevated blood pressure and severe hypertension were not significant (p0.1).Differences in serum NOx concentrations are associated with severity of hypertension only in patients on low nitrate diet. The highest concentrations of NOx were observed in patients with stroke. In the absence of dietary control, NOx levels were not associated with presence or severity of hypertension.