NOS2, NOS3, SONE gene transcripts levels in peripheral blood leukocytes and their relationship with markers of endothelial dysfunction in hypertension

Objective. The aim of the study was to evaluate the level of expression of the NOS2, NOS3, SONE genes in peripheral blood leukocytes (PBL) of patients with hypertension (HTN) and to study the relationship between the level of transcripts of these genes and the content of nitric oxide metabolites and markers of endothelial dysfunction.

Design and methods. The study included healthy people (25 people) and patients with HTN (stages I–II) before prescribing antihypertensive drugs (15 people) and taking cardioselective β-adrenergic receptor blockers for more than a year (metoprolol (25 mg per day) or bisoprolol (5–10 mg per day)) (20 people). The level of gene transcripts was assessed by real-time polymerase chain reaction (PCR). The level of nitric oxide metabolites was determined by the colorimetric method using the Griess reagent. The content of asymmetric dimethylarginine (ADMA), soluble forms of vascular cell adhesion molecule (sVCAM), and intercellular adhesion molecule (sICAM) in blood plasma was determined by ELISA. The content of malondialdehyde (MDA) in blood plasma was determined spectrophotometrically by color reaction with thiobarbituric acid. Statistical processing of the results was carried out using the Statgraphics Centurion XVI software package (version 16.1.11).

Results. The level of nitric oxide metabolites in the blood plasma of HTN patients without antihypertensive therapy was 2,1 times higher than in healthy individuals (p = 0,001) and 1,7 times higher than in patients with HTN taking metoprolol or bisoprolol (p = 0,002). The relative content of mRNA of the NOS3 gene in PBL of individuals included in the study did not differ (p > 0,05). The level of NOS2 gene transcripts in PBL of HTN patients before the prescription of antihypertensive drugs exceeded that in healthy individuals (p = 0,0009) and in HTN patients taking metoprolol or bisoprolol (p = 0,0002). The number of SONE transcripts in the PBL of HTN patients was higher than in people with normal blood pressure (p < 0,00001 when comparing patients before the prescription of antihypertensive therapy and individuals from the control group; p = 0,04 when comparing patients with HTN taking antihypertensive drugs and normotensive subjects). The content of MDA, ADMA, sVCAM was higher in the plasma of HTN patients without antihypertensive therapy compared with people from the control group (p = 0,005, 0,003, 0,039, respectively) and patients taking metoprolol or bisoprolol (p = 0,0006, 0,019, 0,016, respectively). The content of nitric oxide metabolites positively correlated with NOS2, SONE, VCAM1 mRNA level in PBL, the content of MDA and ADMA in blood plasma (p < 0,05). A positive correlation was found between the concentration of MDA and ADMA in plasma (p = 0,03).

Conclusions. An increase in the level of nitric oxide metabolites in HTN is associated with an increase in the transcriptional activity of the NOS2 gene, a disturbance of the redox balance of the body, and the development of endothelial dysfunction. The SONE gene is probably involved in the modulation of nitric oxide levels in HTN not only as an antisense transcript that destabilizes the mRNA of the NOS3 gene in vascular endothelial cells, but also indirectly, namely, through the regulation of homeostasis of immune system cells through autophagy.

1. Ambrosino P, Bachetti T, D’Anna SE, Galloway B, Bianco A, D’Agnano V et al. Mechanisms and clinical implications of endothelial dysfunction in arterial hypertension. J Cardiovasc Dev Dis. 2022;9(5):136. doi:10.3390/jcdd9050136

2. Zheng D, Liu J, Piao H, Zhu Z, Wei R, Liu K. ROStriggered endothelial cell death mechanisms: Focus on pyroptosis, parthanatos, and ferroptosis. Front Immunol. 2022;13:1039241. doi:10.3389/fimmu.2022.1039241

3. Förstermann U, Sessa WC. Nitric oxide synthases: regulation and function. Eur Heart J. 2012;33(7):829–837. doi:10.1093/eurheartj/ehr304

4. Lundberg JO, Weitzberg E. Nitric oxide signaling in health and disease. Cell. 2022;185(16): 2853–2878. doi:10.1016/j.cell.2022.06.010

5. Carnicer R, Crabtree MJ, Sivakumaran V, Casadei B, Kass DA. Nitric oxide synthases in heart failure. Antioxid Redox Signal. 2013;18(9):1078–1099. doi:10.1089/ars.2012.4824

6. Lusov VA, Metelskaya VA, Oganov RG, Evsikov EM, Teplova NV. Level of nitrous oxide in peripheral blood serum in patients with various severity of arterial hypertension. Kardiologiia. 2011;51(12):23–28. In Russian.

7. Metelskaya VA, Oganov RG, Evsikov EM, Teplova NV. Serum NO levels, cardiovascular disease, and concomitant internal pathology in patients with primary arterial hypertension. Rossijskij Kardiologicheskij Zhurnal = Russian Journal of Cardiology. 2011;(4):23–31. In Russian.

8. Topchieva LV, Balan OV, Malysheva IE, Korneva VA, Pankrasheva KA. The nitric oxide metabolite level and NOS2 and NOS3 gene transcripts in patients with essential arterial hypertension. Biology Bulletin. 2020;47(3):247–252. doi:10.31857/S0002332920010166

9. Kazak MV, Romanenko TS, Omelyanenko MG, Lebedeva AV, Tomilova IK, Vyatkin VN et al. Endothelial function and lipid peroxidation in patients with arterial hypertension and its cerebral complications. Kardiovaskulyarnaya Terapiya i Profilaktika = Cardiovascular Therapy and Prevention. 2009;8(2):28–32. In Russian.

10. Miyata S, Noda A, Hara Y, Ueyama J, Kitaichi K, Kondo T et al. Nitric oxide plasma level as a barometer of endothelial dysfunction in factory workers. Exp Clin Endocrinol Diabetes. 2017;125(10):684–689. doi:10.1055/s-0043-110054

11. Topchieva LV, Balan OV, Korneva VA, Malysheva IE. The role of inducible nitric oxide synthase gene variants in essential arterial hypertension (I–II stage) development. Byulleten’ Eksperimental’noj Biologii i Mediciny = Bulletin of Experimental Biology and Medicine. 2019;168(7):91–95. In Russian.

12. Searles CD. Transcriptional and posttranscriptional regulation of endothelial nitric oxide synthase expression. Am J Physiol Cell Physiol. 2006;291(5):C803–C816. doi:10.1152/ajpcell.00457.2005

13. Robb GB, Carson AR, Tai SC, Fish JE, Singh S, Yamada T et al. Post-transcriptional regulation of endothelial nitric-oxide synthase by an overlapping antisense mRNA transcript. J Biol Chem. 2004;279(36):37982–37996. doi:10.1074/jbc.M400271200

14. Kalinowski L, Janaszak-Jasiecka A, Siekierzycka A, Bartoszewska S, Wozniak M, Lejnowski D et al. Posttranscriptional and transcriptional regulation of endothelial nitric-oxide synthase during hypoxia: the role of microRNAs. Cell Mol Biol Lett. 2016;21:16. doi:10.1186/s11658-016-0017-x

15. Fish JE, Matouk CC, Yeboah E, Bevan SC, Khan M, Patil K et al. Hypoxia-inducible expression of a natural cis-antisense transcript inhibits endothelial nitric-oxide synthase. J Biol Chem. 2007;282(21):15652–15666. doi:10.1074/jbc.M608318200

16. Zhang X, Yang X, Lin Y, Suo M, Gong L, Chen J et al. Antihypertensive effect of Lycium barbarum L. with down-regulated expression of renal endothelial lncRNA sONE in a rat model of saltsensitive hypertension. Int J Clin Exp Pathol. 2015;8(6):6981–6987.

17. Azizi F, Gargari SS, Shahmirzadi SA, Dodange F, Amiri V, Mirfakhraie R et al. Evaluation of placental mir‑155–5p and long non-coding RNA sONE expression in patients with severe preeclampsia. Int J Mol Cell Med. 2017;6(1):1–30.

18. Badimon L, Romero JC, Cubedo J, Borrell-Pagẽs M. Circulating biomarkers. Tromb Res. 2012;130 Suppl 1: S12–S15. doi:10.1016/j.thromres.2012.08262

19. Hua Y, Zhang J, Liu Q, Su J, Zhao Y, Zheng G et al. The induction of endothelial autophagy and its role in the development of atherosclerosis. Front Cardiovasc Med. 2022;9:831847. doi:10.3389/fcvm.2022.831847

20. Garton KJ, Gough PJ, Philalay J, Wille PT, Blobel CP, Whitehead RH et al. Stimulated shedding of vascular cell adhesion molecule 1 (VCAM‑1) is mediated by tumor necrosis factor-alphaconverting enzyme (ADAM 17). J Biol Chem. 2003;278(39):37459–37464. doi:10.1074/jbc.M305877200

21. Tsakadze NL, Sithu SD, Sen U, English WR, Murphy G, D’Souza SE. Tumor necrosis factor-alpha- converting enzyme (TACE/ADAM‑17) mediates the ectodomain cleavage of intercellular adhesion molecule‑1 (ICAM‑1). J Biol Chem. 2006;281(6): 3157–3164. doi:10.1074/ jbc.M510797200

22. Dowsett L, Higgins E, Alanazi S, Alshuwayer NA, Leiper FC, Leiper J. ADMA: a key player in the relationship between vascular dysfunction and inflammation in atherosclerosis. J Clin Med. 2020;9(9):3026. doi:10.3390/jcm9093026

23. Antoniades C, Shirodaria C, Leeson P, Antonopoulos A, Warrick N, Van-Assche T et al. Association of plasma asymmetrical dimethylarginine (ADMA) with elevated vascular superoxide production and endothelial nitric oxide synthase uncoupling: Implications for endothelial function in human atherosclerosis. Eur Heart J. 2009;30(9):1142–1150. doi:10.1093/eurheartj/ehp061

24. Williams B, Mancia G, Spiering W, Agabiti Rosei T, Azizi M, Burnier M et al. 2018 Practice Guidelines for the management of arterial hypertension of the European Society of Cardiology and the European Society of Hypertension. Blood Press. 2018;27(6):314–340. doi:10.1080/08037051.2018.1527177

25. Pinto JP, Dias V, Zoller H, Porto G, Carmo H, Carvalho F et al. Hepcidin messenger RNA expression in human lymphocytes. Immunology. 2010;130(2):217–230. doi:10.1111/j.1365-2567.2009.03226.x

26. Rajan S, Ye J, Bai S, Huang F, Guo YL. NF-kappaB, but not p38 MAP kinase, is required for TNF-alpha-induced expression of cell adhesion molecules in endothelial cells. J Cell Biochem. 2008;105(2):477–486. doi:10.1002/jcb.21845

27. Senthilkumar M, Amaresan N, Sankaranarayanan A. Estimation of malondialdehyde (MDA) by thiobarbituric acid (TBA) assay. In: Plant-Microbe Interactions. Springer Protocols Handbooks. Humana, New York, NY. 2021. doi:10.1007/978-1-0716-1080-0-25

28. Metelskaya VA, Gumanova NG. Screening-method for nitric oxide metabolites determination in human serum. Klinicheskaya Laboratornaya Diagnostika = Clinical Laboratory Diagnostics. 2005;6:15–18. In Russian.

29. Qian M, Fang X, Wang X. Autophagy and infammation. Clin Transl Med. 2017;6(1):24. doi:10.1186/s40169-017-0154-5

30. English L, Chemali M, Duron J, Rondeau C, Laplante A, Gingras D et al. Autophagy enhances the presentation of endogenous viral antigens on MHC class I molecules during HSV‑1 infection. Nat Immunol. 2009;10(5):480–487. doi:10.1038/ni.1720

31. Lee H, Mattei LM, Steinberg BE, Alberts P, Lee YH, Chervonsky A et al. In vivo requirement for Atg5 in antigen presentation by dendritic cells. Immunity. 2010;32(2):227–239. doi:10.1016/j.immuni.2009.12.006

32. Rahtes A, Geng S, Lee C, Li L. Cellular and molecular mechanisms involved in the resolution of innate leukocyte inflammation. J Leukoc Biol. 2018;104(3):535–541. doi:10.1002/JLB.3MA0218-070R

33. Saitoh T, Fujita N, Jang MH, Uematsu S, Yang BG, Satoh T et al. Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL‑1betaproduction. Nature. 2008;456(7219):264–268. doi:10.1038/nature07383

34. Lucinda N, Figueiredo MM, Pessoa NL, da Silva Santos BS, Lima GK, Freitas AM et al. Dendritic cells, macrophages, NK and CD8+ T lymphocytes play pivotal roles in controlling HSV‑1 in the trigeminal ganglia by producing IL1‑beta, iNOS and granzyme B. Virol J. 2017;14(1):37. doi:10.1186/s12985-017-0692-x

35. Gatica D, Chiong M, Lavandero S, Klionsky DJ. The role of autophagy in cardiovascular pathology. Cardiovasc Res. 2022;118(4):934–950. doi:10.1093/cvr/cvab158

36. Scherz-Shouval R, Shvets E, Fass E, Shorer H, Gil L, Elazar Z. Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4. EMBO J. 2007;26(7):1749–1760.

37. Atamer A, Ilhan N, Kocyigit Y, Toprak G, Ozbay M, Celik Y. The role of asymmetric dimethylarginine (ADMA) and leptin in hypertensive patients. J Int Med Res. 2008;36(1):54–62. doi:10.1177/147323000803600108

38. Ibrahim MA, Eraqi MM, Alfaiz F. A. Therapeutic role of taurine as antioxidant in reducing hypertension risks in rats. Heliyon. 2020;6(1):e03209. doi:10.1016/j.heliyon.2020.e03209

39. Gorshunova NK, Rakhmanova OV. Oxidative stress and its variations in the pathogenesis of arterial hypertension. Sovremennye problemy nauki i obrazovaniya = Modern problems of science and education. 2018;3. URL: https://science-education.ru/ru/article/view?id=27701. In Russian.

40. Nesterov YuI, Teplyakov AT. Potentialities of correction of lipid peroxidation with combined antihypertensive therapy in patients with arterial hypertension. Arterial’naya Gipertenziya = Arterial Hypertension. 2004;10(1):36–38. In Russian.

41. Schulz E, Gori T, Münzel T. Oxidative stress and endothelial dysfunction in hypertension. Hypertens Res. 2011;34(6):665–673. doi:10.1038/hr.2011.39