Participants
The study included randomly selected groups of male and female participants 25–80 years of age from two cohorts 1 and 2.
Apparently healthy subjects (cohort 1)
Cohort 1 included healthy volunteers (N = 307; 24–69 years of age; 44.8 ± 8.6 years; 80.4% men) without prior record of cardiovascular diseases or other significant comorbidities. The cohort 1 subjects were volunteers over 18 years of age participating in the microcirculation study that started in 2019 in National Research Center for Preventive Medicine (NRCPM), Moscow, Russia [6]. Evaluation of the participants included assessment of baseline cardiovascular characteristics and biochemical parameters in the blood at the time of the recruitment visit. The following exclusion criteria were applied: any acute inflammation, including oral or dental inflammation, at the enrollment date; hematological diseases; left ventricular ejection fraction below 40%; diabetes mellitus; chronic kidney, liver, or heart failure; oncological diseases; mental illness; autoimmune diseases; any blood sugar lowering therapy; any cholesterol-lowering therapy; pregnancy; and lactation. The protocol of the study was approved by the local Independent Ethics Committee of NRCPM (protocol number 01/01-2019 of February 12, 2019). The study was compliant with the guidelines of the Helsinki Declaration and WHO. All participants gave their written informed consent to participate in the study and to grant access to their personal data.
Body weight of the participants was measured at a 0.1-kg precision using an electronic scale (Seca Ltd., Hamburg, Germany). Height was measured using a stadiometer to the nearest 0.1 cm (Seca Ltd.). The body mass index (BMI) was calculated according to the equation: weight/height2 (kg/m2). Cohort 1 participants were examined for preventive counseling and estimation of SCORE according to the European Guidelines on cardiovascular disease prevention [7]. SCORE assessment included gender, age, systolic blood pressure (SBP), total cholesterol, and use of tobacco.
Patients with CHD (cohort 2)
Cohort 2 included 218 patients (63 ± 10.9 years of age, 54% men) treated at NRCPM as described previously [8]. Eligible patients were over 25 years of age and signed an informed consent for inclusion in the study and the collection and biobanking of the blood. Inclusion criteria were as follows: over 18 years of age, signed an informed consent, and underwent coronary angiography. All patients of cohort 2 had clear indications for coronary angiography performed by the method of Judkins [9] according to current European Society of Cardiology guidelines [10, 11] by using Philips Integris Allura Cath Lab and the GE Innova 4100 IQimaging system (General Electric, Fairfield, CT, USA). Stenosis was quantified using the Advantage Workstation software version 4.4 (General Electric). Indications for angiography included positive exercise test, positive stress echocardiography, symptoms of advanced angina pectoris, arrhythmia, pathological changes in electrocardiogram with physical inability to perform exercise or stress tests, or high Duke score.
Exclusion criteria for cohort 2 were as follows: myocardial infarction within 6 months of admission; any acute inflammatory disease; chronic kidney failure stage III and higher with rate of glomerular filtration below 60 ml/min/1.73 m2; decompensated diabetes mellitus type I or type II with levels of glycated hemoglobin over 7.5%; left ventricular ejection fraction below 40%; any oncological disease; familial hypercholesterolemia; any hematological disease; and immune and autoimmune diseases. Additional details have been provided in our previous publications [11, 12].
Blood pressure and heart rate were measured as described previously [12]. The patients were examined, diagnosed, stratified, and treated from 2011 to 2016 according to the National Guidelines for diagnosis and treatment of stable angina [13]. The study complied with all relevant national regulations and institutional policies. Study protocols have been approved by the Independent Ethics Committee of NRCPM (approval no. 09 − 05/19) according to the guidelines of the Helsinki Declaration and WHO. All participants gave their written informed consent to participate in the study and to grant access to their personal data.
Blood sampling
Blood was withdrawn from the cubital vein after 12–14-h fasting on the date of the visit at the baseline of the study. The serum was obtained by centrifugation at 1,000 g for 15 min at 4 °С. Apparently healthy asymptomatic individuals (cohort 1) were specifically asked to limit smoking and consumption of high nitrate containing foods for at least 24 h prior to their visit to the hospital. Patients of cohort 2 suspected of having CHD were admitted to the hospital at least 24 h prior to blood sampling and were fed a controlled hospital diet that limits consumption of dietary nitrate. Serum was aliquoted and stored at − 26°C.
Endothelial biomarkers
The endothelin/NOx ratio was used as a surrogate marker of balance between vasodilatory and vasoconstrictor mediators. Concentrations of nitrite and nitrate (NOx) were assayed in the serum deproteinized by filtration through Spin-X UF-5000 molecular weight cutoff concentrators (Corning, UK) as described previously [14]. Nitrate was reduced to nitrite with vanadium (III) chloride (Sigma, USA), and NOx levels were measured by the Griess reaction as described [14;15] with modifications (CV < 9%). Endothelin-1 was measured using an ELISA kit (Affymetrix Bioscience, Santa Clara, CA, USA). Linear range of the assay was from 0.5 to 10 fmol/mL; CV < 5%.
Detection of cGAMP
cGAMP was measured in serum samples (N = 168 and N = 88) selected from cohorts 1 and 2, respectively, by competitive ELISA using commercial ELISA kits (Invitrogen, ThermoFisher Scientific, USA) according to manufacturer instructions.
Detection of IgG antibodies against SARS-CoV-2 S1 RBD
Immunoassay for qualitative detection of IgG antibodies against SARS-CoV-2 S1 RBD (IgG-SARS) was performed using an anti-SARS-CoV-2 ELISA E111-IVD kit (Mediagnost, Germany) by two-step ELISA with recombinant SARS-CoV-2-S1 RBD according to the manufacturer instructions as described elsewhere [16]. Deionized water (Aquasmart, Russia) was used to dissolve the reagents. The samples were considered antibody-positive at > 5-fold cut-off value or if the optical density was higher than 0.830. The samples with < 3-fold cut-off value were considered antibody-negative. Samples from 3-fold to 5-fold cut-off or with the intermediate optical density values were considered borderline positive. The cut-off values were selected to ensure the exclusion of false positive results with a high probability. The assay was characterized by 95.55% sensitivity, 98.36% specificity, and 10.6% inter-assay variance [17].
Routine blood tests
Routine tests were performed in NRCPM using specific guidelines approved by Center for External Quality Control of Clinical Laboratory Testing of Russian Federation (www.fsvok.ru, accessed on September 10, 2021). Clinical blood tests were performed on an MEK-8222 K automatic hematology analyzer (Nihon Kohden, Saitama, Japan). Serum lipids were assayed as described previously [18]. In brief, serum levels of total cholesterol (mmol/L), high density lipoprotein (HDL)-cholesterol (mmol/L) after precipitation of apoB-containing low density lipoproteins (LDL), and triglycerides (mmol/L) were assayed using an Architect c8000 autoanalyzer and reagents from Abbot Diagnostics, Lake Forest, Illinois, USA. The level of LDL-cholesterol (mmol/L) was calculated according to the Friedwald equation in the samples with serum triglycerides below 4.5 mmol/L. C-reactive protein (CRP; mg/L) was assayed by high sensitivity quantitative immunoturbidimetric method enhanced with latex particles (universal range 0.3–350 mg/L; highly sensitive range 0.05–20 mg/L) using an Architect c8000 analyzer and reagents from Abbot Diagnostics. Plasma glucose level (mmol/L) was assayed by the hexokinase method using the same autoanalyzer and reagents from Abbot Diagnostics.
Statistical analysis
The data were analyzed using IBM SPSS Statistics 23 and Statistica software version 8.0 (StatSoft, Inc., USA). Some clusters of the data did not pass the Kolmogorov-Smirnov test for normality of distribution; therefore, we used non-parametric tests for all calculations. Continuous variables are presented as the median (25%; 75%), and categorical variables are presented in percentages. Sample size and power were estimated using the online calculator Sampsize https://sampsize.sourceforge.net/iface/s2.html#nm (accessed on September 10, 2021). Comparisons between the two groups were performed by a post-hoc test for Kruskal-Wallis, the Dunn’s test. P values < 0.05 were considered significant.