Circadian Rhythms of Endothelial Nitric Oxide Synthase and Toll-like Receptors 2 Production in Females with Rheumatoid Arthritis Depending on NOS3 Gene Polymorphism.

BACKGROUND: Rheumatoid Arthritis (RA) is an autoimmune polygenic disease characterized by rapid disability progression and high prevalence. Progression of RA is closely associated with chronobiological changes in the production of some hormones and inflammatory mediators, influencing the disease course and therapy efficacy. The main pathogenetic mechanism of RA is angiogenesis, which is controlled by biological clock-genes. Further investigation of circadian rhythms of angiogenic mediators production in RA patients may be considered as important and relevant. The aim of this study was to establish daily variability of serum endothelial Nitric Oxide Synthase (NOS3) and toll-like receptors 2 (sTLR2) levels in female RA patients depending on the NOS3 gene polymorphism. METHODS: We examined 173 RA patients (100% female) aged 43.7 ± 7.35 years and 34 age-matched healthy women without joint diseases and autoimmune diseases (control). RA was diagnosed by ACR/EULAR 2010 criteria. Blood serum NOS3 and sTLR2 levels were determined at 08:00 and 20:00 using Cloud-Clone Corp kits (USA). NOS3 T-786С (rs2070744) polymorphism was determined by Real-Time PCR (Bio-Rad iCycler IQ5) using SNP-express kits. The SPSS22 software package was used for statistical processing of the results. RESULTS: Females with RA demonstrated oppositely directed serum NOS3 and sTLR2 daily changes: NOS3 level in the morning (08:00) was lower than in the evening (+ 45.5 ± 30.7%), and sTLR2 level in the evening (at 20:00) was lower than in the morning (-21.6 ± 13.1%). RA patients had differences in NOS3 and sTLR2 production depending on NOS3 T786C genotype. CC subjects had NOS3 level at 08:00, 20:00 and day average levels lower (16-25%), and sTLR2 level higher (24-27%) than those of TT subjects. RA patients, carriers of CC genotype, had higher chances of NOS3 and sTLR2 aberrant production compared to TT and TC genotype carriers (OR = 2.99 and 4.79, respectively). CONCLUSION: RA patients demonstrated oppositely directed circadian changes of serum NOS3 and sTLR2. CC genotype carriers had lower NOS3 and higher sTLR2 production rates than TT and TC genotype carriers.

Circadian fluctuations of endothelial nitric oxide synthase activity in females with rheumatoid arthritis: a pilot study.

Rheumatoid arthritis (RA) is a disease associated with circadian disorders of steroid hormones or cytokine secretion which induce inflammatory, destructive and proliferative processes in the synovial joints. Angiogenesis plays an important role in RA, but circadian rhythms of the angiogenic mediator production, especially endothelial nitric oxide synthase (NOS3), are still unclear. NOS3 takes part in regulation of endothelial functions, inflammation, and bone remodeling process. Studying circadian rhythms of NOS3 production in RA patients will make an improvement in understanding the angiogenic-inflammatory pathways relevant to rheumatic diseases. The aim of the study was to test the hypothesis of a diurnal variation in circulating levels of NOS3 in RA patients. A cross-sectional monocentric pilot study of circadian variability of endothelial nitric oxide synthase in a Ukrainian population was conducted between March and July 2017. We examined 36 RA patients (100% women) and 34 age-matched healthy women without joint diseases and autoimmune diseases (control). Blood samples were collected four times per day (at 08:00; 14:00; 20:00 and 02:00) for two consecutive days. Serum NOS3 concentration was measured by ELISA (Cloud-Clone Corp kit). The study was conducted in compliance with bioethical standards. The SPSS22 software package was used for statistical processing of the results. A diurnal variation in circulating levels of NOS3 in healthy women was established, with peak values appearing in the evening and acrophase at 20:00, and low values in the morning, with batiphase at 08:00. In patients with RA serum, NOS3 levels were substantially decreased throughout the day compared to the control. In RA patients, a diurnal variation in circulating levels of NOS3 was also established. However, the variability of NOS3 production was higher in RA patients than in the control group. For example, in RA patients the difference between morning/evening values of NOS3 was 1.3 times higher (p < 0.05) than in the control. Negative correlations were found between the morning NOS3 levels and RA activity markers such as DAS28 and the number of tender and swollen joints. The diurnal variation in circulating levels of NOS3 in women with RA as well as in healthy women was found. However, in RA patients, a decrease in NOS3 production was observed, especially in the morning, which was associated with an increase in the disease activity. Thus, the circadian rhythm of circulating NOS3 can be opposite to the circadian rhythm of secretion of main inflammatory regulators in RA.