Atrial fibrillation recurrence after catheter ablation is associated with RAD51 and p63 proteins.

Catheter ablation has been demonstrated to reduce atrial fibrillation (AF) recurrence. The mechanisms of AF recurrence after catheter ablation are unknown, and the present study aimed to identify serum proteins associated with AF recurrence. The present prospective study comprised a cohort of patients with AF, which was divided into two groups after one-year follow-up: group 1 included patients with compensated AF after catheter ablation and group 2 included patients with AF recurrence after catheter ablation. Initial microarray profiling of the serum proteins was performed in small subgroups M1 and M2 recruited from groups 1 and 2, respectively, by an antibody microarray to evaluate potentially relevant proteins. The data of initial proteomic profiling identified candidate proteins in groups 1 and 2, and their levels were then measured by ELISA. The data of profiling suggested an overall increase in the levels of RAD51 and p63 proteins in the M2 subgroup versus that in the M1 subgroup, indicating potential relevance of these two proteins to AF recurrence. The results of ELISA of the levels of RAD51 and p63 in the groups 1 and 2 demonstrated an increase in the levels of RAD51 (11.11 ± 4.36 vs 8.45 ± 4.85 ng/mL; P = 0.009) and p63 (165.73 ± 113.75 vs 100.05 ± 37.56 units of normalized optical density; P = 0.0007) in the group 2 (with AF recurrence or substrate AF) compared with that in the group 1 (compensated AF). Thus, RAD51 and p63 were associated with AF recurrence after catheter ablation and may represent possible etiological factors for subsequent outcomes.

Proteomic biomarker evaluation using antibody microarrays: association between analytical methods such as microarray and ELISA.

OBJECTIVE: To evaluate the associations between analytical methods, such as microarray and enzyme-linked immunosorbent assay (ELISA); expedient cutoffs; and the lowest possible number of microarrays in analysis for target biomarker estimation in case-control studies. METHODS: This study included 321 serum specimens, gathered in different case-control studies to test for atherosclerosis and atrial fibrillation. Among them, 48 serum specimens were analyzed using microarray technology. We used ELISA and commercial kits for confirmation of the results. RESULTS: Three proteins-cadherin-P, neuronal nitric oxide synthase, and adenovirus fiber-were shown to have distinctly different values in the case group vs the control group. As a result, we used those proteins as the target for confirmation using our alternative analytical method. Also, these protein values represented the limiting range between the highest and lowest differences in case-control groups. The results of microarray assay were confirmed using ELISA and commercial kits in the same specimens, in which microarray profiling was performed, and also in separate large case-control groups. CONCLUSIONS: A 1.5-fold difference in the protein content, as measured using microarray technology, was shown to be sufficient for further investigation of the candidate proteins. As few as 3 microarrays were considered sufficient for perspective evaluation of the target proteins. Microarray serum profiling, therefore, provides semiquantitative determination of protein in serum.

Effects of COVID-19 Infection in Healthy Subjects on Cardiac Function and Biomarkers of Oxygen Transport, Blood Coagulation and Inflammation.

BACKGROUND: The manifestations, severity, and mortality of COVID-19 are considered to be associated with the changes in various hematological parameters and in immunity. Associations of immunoglobulin G antibodies against severe acute respiratory syndrome-linked coronavirus (IgG-SARS)-positive status with cardiac function and hematological and biochemical parameters in apparently health subjects are poorly understood. METHODS: The present cross-sectional study included 307 healthy volunteers (24-69 years of age; 44.8 ± 8.6 years; 80.4% men) and was initiated in 2019 before the COVID-19 pandemic. COVID-19 episodes were confirmed by detection of IgG-SARS against SARS-CoV-2 S1 RBD to reveal 70 IgG-SARS-positive and 237 negative participants. Numerous ultrasound characteristics were assessed by echocardiography, and 15 hematological and biochemical parameters were assayed in the blood. Descriptive and comparative analysis was based on the IgG-SARS status of the participants. RESULTS: The left ventricular mass index, mitral ratio of peak early to late diastolic filling velocity or flow velocity across the mitral valve, and deceleration time of early mitral inflow were decreased (p < 0.05) in IgG-SARS-positive participants versus those in IgG-SARS-negative participants according to multivariate logistic regression analysis. Erythrocyte sedimentation rate and platelet count were slightly increased, and blood hemoglobin was decreased in IgG-SARS-positive participants compared with those in IgG-SARS-negative participants. CONCLUSIONS: LV filling, inflammation, blood coagulation, and hemoglobin appear to be influenced by COVID-19 infection in healthy participants. Our observations contribute to the definition of vulnerabilities in the apparently healthy subjects with long COVID-19. These vulnerabilities may be more severe in patients with certain chronic diseases.

Associations of adenovirus-reactive immunoglobulins with atrial fibrillation and body mass index.

Adenovirus (AdV) has been suggested to be involved in pathogenesis of atrial fibrillation (AF). We aimed to evaluate an association between AdV-specific immunoglobulins G in the serum (AdV-IgG) and AF. The present case-control study comprised two cohorts, including cohort 1 of patients with AF and cohort 2 of asymptomatic subjects. Initially, two groups, MA and MB, were selected from the cohorts 1 and 2, respectively, for serum proteome profiling using an antibody microarray to identify possible relevant protein targets. The data of microarray analysis indicated a possible overall increase in the total adenovirus signals in the group MA vs. group MB, suggesting potential relevance of adenoviral infection to AF. Then, the groups A (with AF) and B (control) were selected from the cohorts 1 and 2, respectively, to assay the presence and levels of AdV-IgG- by ELSA. The prevalence of AdV-IgG-positive status demonstrated a 2-fold increase in the group A (AF) compared with that in the group B (asymptomatic subjects); odds ratio 2.06 (95%CI: 1.11-3.84; P = 0.02). The prevalence of obesity demonstrated an approximately 3-fold increase in AdV-IgG-positive patients of the group A compared with that in AdV-IgG-negative patients of the same group A (odds ratio 2.7; 95% CI: 1.02-7.1; P = 0.04). Thus, AdV-IgG-positive reactivity was independently associated with AF, and AF was independently associated with BMI, indicating that adenoviral infection may be a possible etiological factor for AF.

Serum biomarkers, including nitric oxide metabolites (NOx), for prognosis of cardiovascular death and acute myocardial infarction in an ESSE-RF case-control cohort with 6.5-year follow up.

The present case-control study aimed to assess associations of routine and experimental biomarkers with risk for cardiovascular death and acute myocardial infarction (AMI) in a cohort recruited from the multicenter study "Cardiovascular Epidemiology in Russian Federation" (ESSE-RF) to identify experimental biomarkers potentially suitable for expanded evaluation. A total of 222 subjects included cardiovascular death (N = 48) and AMI cases (N = 63) during 6.5-year follow up and matched healthy controls. Seven routine and eight experimental biomarkers were assayed to analyze associations with outcomes using logistic and Cox proportional hazard regressions. Elevated levels of cardiac troponin I (cTnI), C-reactive protein (CRP), and nitric oxide metabolites (NOx) were independently associated (P < 0.001) with higher risk of cardiovascular death (estimated hazard ratio (eHR) = 1.83-3.74). Elevated levels of NOx and cTnI were independently (P < 0.001) associated with higher risk of nonfatal AMI (eHRs = 1.78-2.67). Elevated levels of angiopoietin-like protein 3 (ANGPTL3) were independently associated (P < 0.001) with lower risk of cardiovascular death (eHRs 0.09-0.16) and higher risk of nonfatal AMI (eHR = 2.07; P = 0.01). These results indicated that subsequent expanded validation should focus on predictive impact of cTnI, NOx, CRP, and ANGPTL3 to develop nationwide recommendations for individual stratification of patients with cardiovascular risks.

Application of an antibody microarray for serum protein profiling of coronary artery stenosis.

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.

Detection of Anti-SARS-CoV-2-S1 RBD-Specific Antibodies Prior to and during the Pandemic in 2011-2021 and COVID-19 Observational Study in 2019-2021.

BACKGROUND: Longitudinal surveys to monitor the seroprevalence are required to support efforts for assessment of the levels of endemic stability in certain countries. We investigated seroprevalence of anti-SARS-CoV-2-S1 receptor-binding domain (RBD)-specific antibodies in the serum samples in 2011-2021, including a cohort study of 2019-2021, to evaluate the vaccination and anti-IgG-SARS-CoV-2-S1 RBD-positive statuses to assess the resistance and severity of COVID-19. MATERIALS AND METHODS: Anti-SARS-CoV-2-S1 RBD-specific antibodies were assayed in the serum samples (N = 565) randomly selected from various cohorts previously recruited from 2011 to 2021 from the city of Moscow and Moscow Region. Among them there were the participants (N = 310) recruited in 2019-2021 with an endpoint of 30 October 2021 when these participants were interviewed over phone with relevant questionnaire. RESULTS: Obtained data indicated a percentage of 3-6% of SARS-CoV-2-S1 RBD-specific antibodies detected in participants recruited in 2011-2019. The percentage of SARS-CoV-2-S1 RBD-specific antibodies was increased to 16.5% in 2020 and to 46% in 2021. The vaccination rate of 238 respondents of this cohort was 58% from August 2020 to October 2021. In total, 12% of respondents were hospitalized. The morbidity rate in the subgroup of anti-SARS-CoV-2-S1 RBD-positive respondents was 5.4-fold higher than that in the subgroup of vaccinated respondents. CONCLUSIONS: A small percentage of SARS-CoV-2-S1 RBD-specific antibodies detected in 2011-2019 indicated possible spreading of coronaviruses during the pre-pandemic period. Collective immunity in Moscow and the Moscow region was able to reach 69% from August 2020 to October 2021 if this rate is added to the rate of not vaccinated SARS-CoV-2-S1 RBD-positive subjects.

Associations of endothelial biomarkers, nitric oxide metabolites and endothelin, with blood pressure and coronary lesions depend on cardiovascular risk and sex to mark endothelial dysfunction on the SCORE scale.

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.

Levels of nitric oxide metabolites, adiponectin and endothelin are associated with SNPs of the adiponectin and endothelin genes.

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.

Elevated levels of serum nitrite and nitrate, NOx, are associated with increased total and cardiovascular mortality in an 8-year follow-up study.

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.

Increase in perfused boundary region of endothelial glycocalyx is associated with higher prevalence of ischemic heart disease and lesions of microcirculation and vascular wall.

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.

Serum nitrate and nitrite are associated with the prevalence of various chronic diseases except cancer.

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.

Ratios of leptin to insulin and adiponectin to endothelin are sex-dependently associated with extent of coronary atherosclerosis.

OBJECTIVE: 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. PATIENTS AND METHODS: 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. RESULTS: 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). CONCLUSION: 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.

Serum nitrite and nitrate levels, NOx, can predict cardiovascular mortality in the elderly in a 3-year follow-up study.

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.

Serum nitrate and nitrite levels in patients with hypertension and ischemic stroke depend on diet: a multicenter study.

OBJECTIVES: 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. PATIENTS AND METHODS: 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. RESULTS: In groups with controlled low NOx diet, patients with moderate and malignant hypertension had significantly (p<0.01) 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 (p>0.1). CONCLUSION: 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.