Novel relationships between the ICAD, CD53, GRM1, and GSTmu protein levels and atherosclerosis and cardiovascular outcomes
Description
The levels of glutathione S-transferase Mu1 (GSTmu), DNA fragmentation factor subunit (DFF45/ICAD), interleukin-6 (IL-6), leukocyte surface antigen (CD53), and metabotropic glutamate receptor 1 (GRM1) were shown to differ between the small selected groups of patients with severe and minor coronary lesions by microarray assays. In the total cohort, only ICAD was linked to type 2 diabetes and presence of coronary artery stenosis regardless of the severity of stenosis. CD53 was specifically associated with severe degree of coronary stenosis. GRM1 was associated with the presence of coronary stenosis and cardiovascular outcomes. IL6 was associated with femoral stenosis >70% and with diabetes, and GSTmu was associated with brachiocephalic stenosis >60% and with diabetes. Only GRM1 was a predictor of surrogate endpoint outcome, with inverse associations with coronary stenosis and surrogate endpoint outcome (r= -0.24 and P=0.025 and r= -0.31 and P=0.007, respectively).
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The study cohort included patients (men and women over 25 years of age) admitted to the National Research Center for Preventive Medicine (NRCPM) in 2018-2020. Eligible patients signed an informed consent for inclusion in the present 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. 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. Coronary angiography was performed according to the guidelines of European Society of Cardiology. Microarray analysis of serum proteome was performed in serum samples of the Group A patients with minor coronary stenosis according to Gensini score and Group B with severe coronary stenosis and high Gensini score using antibody microarrays with 656 antibodies per slide in two replicates (ASB 600, Full Moon BioSystems, USA) as described previously . In-house ELISA for DNA fragmentation factor subunit (ICAD) and CD100 DNA fragmentation factor subunit (ICAD) was measured by indirect ELISA. Protein concentration was adjusted, and the assay was performed as described previously [15]. In brief, microplates were coated with serum, blocked with BSA, and washed. Mouse anti-ICAD monoclonal antibody (100 µL; 1 mg/mL; 1:10,000, Invitrogen) was added and incubated for 1 h at 37°C. After washing, the plate was incubated with secondary anti-mouse antibodies conjugated to horseradish peroxidase (FineTest, Wuhan, China; 100 µL; 1:10,000 dilution). Optical density was measured at 450 nm after incubation with ELISA substrate using Tecan Infinite 200 PRO plate reader and Magellan software (Tecan, Switzerland). Statistica software version 8.0 and SPSS IBM statistics version 23 were used for statistical analyzes. Sample size and power were estimated using the online calculator Sampsize https://sampsize.sourceforge.net/iface/s2.html#nm (accessed on 10 September 2021) for estimation of sufficient numbers of outcomes for analysis. Kolmogorov-Smirnov criterion was used for test of normality of the distributions. The data are shown as the mean (SD) (standard deviation). Two-tailed non-parametric analysis of variance and Kruskal-Wallis and Mann-Whitney tests were used to compare the groups. The strength and direction of a monotonic relationship between two ranked variables was assessed by Spearman's rank correlation coefficient. Revealed relationship was proved by logistic regression. Multivariate logistic regression was performed with Wald test (Ɣ). The P values < 0.05 were considered significant.
Institutions
- National Research Medical Center