Extracellular neutrophil traps in cardiovascular diseases: problems and prospects of research

One of the links in the pathogenesis of cardiovascular diseases is chronic low-intensity systemic inflammation. In 2004, a previously unknown process was discovered — the formation of extracellular neutrophil traps (NETs) — NETosis (n eutrophil extracellular traps). NETs play a role in antimicrobial immunity though in certain cases they become a factor in the development of pathology. This review presents data on the effect of extracellular neutrophil traps on individual pathologies of the cardiovascular system (atherosclerosis, atrial fibrillation, thrombosis). The authors describe the mechanisms of NET formation (vital NETosis, suicidal NETosis) and their role in thrombus formation (as a framework for thrombus formation, initiation of coagulation), in the development of endothelial dysfunction, and electrical heterogeneity of the atrial myocardium. Data are presented indicating the connection between atherosclerosis, thrombosis, and atrial fibrillation with the activity of NETosis. Most studies demonstrate existing correlations on laboratory models, while the determination of NETs in patients with cardiovascular pathology in real clinical practice is almost absent. At the same time, understanding the processes associated with NETosis can help to identify specific markers and further strategies for the therapy of cardiovascular diseases.

1. Federal State Statistics Service. (In Russian). URL: https://rosstat.gov.ru (date of access: 03/26/2023)

2. Rosales C. Neutrophil: A cell with many roles in inflammation or several cell types? Front. Physiol. 2018;9:113. DOI: 10.3389/FPHYS.2018.00113

3. Brinkmann V., Reichard U., Goosmann C., Fauler B., Uhlemann Y, Weiss D.S., Weinrauch Y., Zychlinsky A. Neutrophil extracellular traps kill bacteria. Science. 2004;303:1532–1535. DOI: 10.1126/SCIENCE.1092385

4. Ravindran M., Khan M.A., Palaniyar N. Neutrophil Extracellular Trap Formation: Physiology, Pathology, and Pharmacology. Biomolecules. 2019;9(8):365. DOI: 10.3390/BIOM9080365

5. Ley K., Hoffman H.M., Kubes P., Cassatella M.A., Zychlinsky A., Hedrick C.C., Catz S.D. Neutrophils: New insights and open questions. Sci. Immunol. 2018;3(30):eaat4579. DOI: 10.1126/SCIIMMUNOL.AAT4579

6. Zhou X., Jin M., Liu L., Yu Z., Lu X., and Zhang H. Trimethylamine N-oxide and cardiovascular outcomes in patients with chronic heart failure after myocardial infarction. ESC Hear. Fail. 2020;7(1):188–193. DOI: 10.1002/EHF2.12552

7. Mortaz E., Alipoor S.D., Adcock I.M., Mumby S., Koenderman L. Update on Neutrophil Function in Severe Inflammation. Front. Immunol. 2018;9:2171. DOI: 10.3389/FIMMU.2018.02171

8. Papayannopoulos V. Neutrophil extracellular traps in immunity and disease. Nat. Rev. Immunol. 2018;18(2):134–147. DOI: 10.1038/NRI.2017.105

9. Belambri S.A., Rolas L., Raad H., Hurtado-Nedelec M., Dang P.M.C., El-Benna J. NADPH oxidase activation in neutrophils: Role of the phosphorylation of its subunits. Eur. J. Clin. Invest. 2018;48(2):e12951. DOI: 10.1111/ECI.12951

10. Rada B. Neutrophil extracellular traps. Methods Mol. Biol. 2019;1982:517–528. DOI: 10.1007/978-1-4939-9424-3_31

11. Huang S.U.S, O’Sullivan K.M. The expanding role of extracellular traps in inflammation and autoimmunity: the new players in casting dark webs. Int. J. Mol. Sci. 2022;23:7. DOI: 10.3390/IJMS23073793

12. Ingelsson B., Söderberg D., Strid T., Söderberg A., Bergh A.C., Loitto V., Lotfi K., Segelmark M., Spyrou G., Rosén A. Lymphocytes eject interferogenic mitochondrial DNA webs in response to CpG and non-CpG oligodeoxynucleotides of class C. Proc. Natl. Acad. Sci. U. S. A. 2018;115(3):E478–E487. DOI: 10.1073/PNAS.1711950115

13. Fukuchi M., Kamide Y., Ueki S., Miyabe Y., Konno Y., Oka N. et al. Eosinophil ETosis-mediated release of galectin-10 in eosinophilic granulomatosis with polyangiitis. Arthritis Rheumatol. (Hoboken, N.J.). 2021;73(9):1683–1693. DOI: 10.1002/ART.41727

14. Fukuchi M., Miyabe Y., Furutani C., Saga T., Moritoki Y., Yamada T. et al. How to detect eosinophil ETosis (EETosis) and extracellular traps. Allergol. Int. 2021;70(1):19–29. DOI: 10.1016/J.ALIT.2020.10.002

15. Vorobjeva N., Galkin I., Pletjushkina O., Golyshev S., Zinovkin R., Prikhodko A. et al. Mitochondrial permeability transition pore is involved in oxidative burst and NETosis of human neutrophils. Biochim. Biophys. acta. Mol. basis Dis. 2020;1866(5). DOI: 10.1016/J.BBADIS.2020.165664

16. Liew P.X., Kubes P. The neutrophil’s role during health and disease. Physiol. Rev. 2019;99(2):1223–1248. DOI: 10.1152/PHYSREV.00012.2018

17. Hidalgo A., Libby P., Soehnlein O., Aramburu I. V., Papayannopoulos V., Silvestre-Roig C. Neutrophil extracellular traps: from physiology to pathology. Cardiovasc. Res. 2022;118(13):2737. DOI: 10.1093/CVR/CVAB329

18. Tan C., Aziz M., Wang P. The vitals of NETs. J. Leukoc. Biol. 2021;110(4):797. DOI: 10.1002/JLB.3RU0620-375R

19. Cahilog Z., Zhao H., Wu L., Alam A., Eguchi S., Weng H., Ma D. The role of neutrophil NETosis in organ injury: novel inflammatory cell death mechanisms. Inflammation. 2020;43(6):2021. DOI: 10.1007/S10753-020-01294-X

20. Wienkamp A.K., Erpenbeck L., Rossaint J. Platelets in the NETworks interweaving inflammation and thrombosis. Frontiers in immunology. 2022;13:953129. DOI: 10.3389/FIMMU.2022.953129

21. Mawson T.L., Vromman A., Bernardes-Souza B., Franck Gr., Persson O., Nakamura M. et al. Neutrophil extracellular traps induce endothelial cell activation and tissue factor production through interleukin-1α and cathepsin G. Arteriosclerosis, thrombosis, and vascular biology. 2018;38(8):1901–1912. DOI: 10.1161/ATVBAHA.118.311150

22. Kim S.J., Carestia Ag., McDonald B. Platelet-mediated net release amplifies coagulopathy and drives lung pathology during severe influenza infection. Frontiers in immunology. 2021;12:772859. DOI: 10.3389/FIMMU.2021.772859

23. Khan M.A., Ali Z.S., Sweezey N., Grasemann H., Palaniyar N. Progression of cystic fibrosis lung disease from childhood to adulthood: neutrophils, neutrophil extracellular trap (NET) formation, and NET degradation. Genes (Basel). 2019;10(3). DOI: 10.3390/GENES10030183

24. Novotny J., Oberdieck P., Titova A., Pelisek J., Chandraratne S., Nicol P. et al. Thrombus NET content is associated with clinical outcome in stroke and myocardial infarction. Neurology. 2020;94(22):E2346–E2360. DOI: 10.1212/WNL.0000000000009532

25. Sharma S., Hofbauer T.M., Ondracek A.S., Chausheva S., Alimohammadi A., Artner T. et al. Neutrophil extracellular traps promote fibrous vascular occlusions in chronic thrombosis. Blood. 2021;137(8):1104–1116. DOI: 10.1182/BLOOD.2020005861

26. Moore S., Juo H.H., Nielsen C.T., Tyden H., Bengtsson A.A., Lood C. Role of neutrophil extracellular traps regarding patients at risk of increased disease activity and cardiovascular comorbidity in systemic lupus erythematosus. J. Rheumatol. 2020;47(11):1652–1660. DOI: 10.3899/JRHEUM.190875

27. Casey K.A. Smith M.A., Sinibaldi D., Seto N.L., Playford, M.P., Wan X. et al. Modulation of cardiometabolic disease markers by type I interferon inhibition in systemic lupus erythematosus. Arthritis Rheumatol. (Hoboken, N.J.). 2021;73(3):459–471. DOI: 10.1002/ART.41518

28. Novikov D.G., Zolotov A.N., Bikbavova G.R., Livzan M.A., Telyatnikova L.I. Neutrophil extracellular traps in a patient with ulcerative colitis. Russ. J. Evidence-Based Gastroenterol. 2022;11(2):1–38. DOI: 10.17116/DOKGASTRO20221102131

29. Yang L., Liu Q., Zhang X., Liu X., Zhou B., Chen J. et al. DNA of neutrophil extracellular traps promotes cancer metastasis via CCDC25. Nature. 2020;583(7814):133–138. DOI: 10.1038/S41586-020-2394-6

30. Masucci M.T., Minopoli M., Vecchio S. Del, Carriero M.V. The emerging role of neutrophil extracellular traps (NETs) in tumor progression and metastasis. Front. Immunol. 2020;11:1749. DOI: 10.3389/FIMMU.2020.01749

31. de Buhr N., von Köckritz-Blickwede M. Detection, visualization, and quantification of neutrophil extracellular traps (NETs) and NET Markers. Methods Mol. Biol. 2020;2087:425–442. DOI: 10.1007/978-1-0716-0154-9_25

32. Moonen C.G.J., Hirschfeld J., Cheng L., Chapple I..LC., Loos B.G., Nicu E.A. Oral Neutrophils Characterized: Chemotactic, Phagocytic, and Neutrophil Extracellular Trap (NET) Formation Properties. Front Immunol. 2019;10:635. DOI: 10.3389/fimmu.2019.00635

33. Yu X., Diamond S.L. Fibrin modulates shear-induced netosis in sterile occlusive thrombi formed under haemodynamic flow. Thromb. Haemost. 2019;119(4):586–593. DOI: 10.1055/s-0039-1678529

34. Kravcov A.L., Goncharova A.Yu., Bugorkova S.A., Devdariani Z.L., Kozhevnikov V.A. Formation of neutrophil extracellular traps during modeling of plague infection in mice immunized with Yersinia pestis EV NIIEG. Problemy osobo opasnyh infekcij. 2020;(4):70–74. (In Russian). DOI: 10.21055/0370-1069-2020-4-70-74

35. Tong M., Abrahams V.M. Visualization and quantification of neutrophil extracellular traps. Methods Mol. Biol. 2021;2255:87. DOI: 10.1007/978-1-0716-1162-3_9

36. Clemente-Moragón A., Martínez-Milla J., Oliver E. Metoprolol in critically ill patients with COVID-19. Journal of the American College of Cardiology. 2021;78(10):1001–1011. DOI: 10.1016/J.JACC.2021.07.003

37. Skendros P., Mitroulis I., Ritis K. Autophagy in neutrophils: From granulopoiesis to neutrophil extracellular traps. Front. Cell Dev. Biol. 2018;6:109. DOI: 10.3389/FCELL.2018.00109

38. Breda S.V. Van, Vokalova L., Neugebauer C., Rossi S.W., Hahn S.P., Hasler P. Computational methodologies for the in vitro and in situ quantification of neutrophil extracellular traps. Front. Immunol. 2019;10:1562. DOI: 10.3389/FIMMU.2019.01562

39. Patent No. 2768152 C1 Russian Federation, IPC G01N 33/569, G01N 33/533, G01N 33/577. Method for detecting neutrophil extracellular traps in a supravitally stained blood preparation: No. 2021129097: application 06.10.2021: publ. 23.03.2022 / D.G. Novikov, A.N. Zolotov, N.A. Kirichenko, A.V. Mordyk; applicant Federal State budgetary educational institution of Higher Education “Omsk State Medical University” of the Ministry of Health of the Russian Federation. EDN ARQUHO. (In Russian).

40. Bonaventura A., Montecucco F., Dallegri F., Carbone F., Lüscher T.F.,Camici G.G., Liberale L. Novel findings in neutrophil biology and their impact on cardiovascular disease. Cardiovasc. Res. 2019;115(8):1266–1285. DOI: 10.1093/CVR/CVZ084

41. Wu M.Y., Li C.J., Hou M.F., Chu P.Y. New insights into the role of inflammation in the pathogenesis of atherosclerosis. Int. J. Mol. Sci. 2017;18(10):2034. DOI: 10.3390/IJMS18102034

42. Pertiwi K.R., Van Der Wal A.C., Pabittei D.R., Mackaaij C., Van Leeuwen M.B., Li X., De Boer O.J. Neutrophil extracellular traps participate in all different types of thrombotic and haemorrhagic complications of coronary atherosclerosis. Thromb. Haemost. 2018;118(6):1078–1087. DOI: 10.1055/S-0038-1641749

43. Zhou Z., Zhang S., Ding S. Abudupataer M., Zhang Z., Zhu X. et al. Excessive neutrophil extracellular trap formation aggravates acute myocardial infarction injury in apolipoprotein E deficiency mice via the ROS-dependent pathway. Oxidative Medicine and Cellular Longevity. 2019;2019:1209307. DOI: 10.1155/2019/1209307

44. Hofbauer T.M., Ondracek A.S., Lang I.M. Neutrophil extracellular traps in atherosclerosis and thrombosis. Handb. Exp. Pharmacol. 2022;270:405–425. DOI: 10.1007/164_2020_409

45. An Z., Li J., Yu J., Wang X., Gao H., Zhang W. et al. Neutrophil extracellular traps induced by IL-8 aggravate atherosclerosis via activation NF-κB signaling in macrophages. Cell Cycle. 2019;18(21):2928–2938. DOI: 10.1080/15384101.2019.1662678

46. Döring Y., Libby P., Soehnlein O. Neutrophil extracellular traps participate in cardiovascular diseases — recent experimental and clinical insights. Circ. Res. 2020;126(9):1228. DOI: 10.1161/CIRCRESAHA.120.315931

47. Josefs T., Barrett T.J., Brown E.J., Quezada A., Wu Xi., Voisin M. et al. Neutrophil extracellular traps promote macrophage inflammation and impair atherosclerosis resolution in diabetic mice. JCI Insight. 2020;5(7):e134796. DOI: 10.1172/JCI.INSIGHT.134796

48. Calvo D., Filgueiras-Rama D., Jalife J. Mechanisms and drug development in atrial fibrillation. Pharmacol. Rev. 2018;70(3):505–525. DOI: 10.1124/PR.117.014183

49. Obrezan A.G., Kulikov V.D. Atrial fibrillation and diabetes mellitus: the control of thromboembolic risk. Kardiologiia, 2020;60(7):108–114. DOI: 10.18087/CARDIO.2020.7.N1146

50. Blum S., Meyre P., Aeschbacher S., Berger S., Auberson C., Briel M., Osswald S., Conen D. Incidence and predictors of atrial fibrillation progression: A systematic review and meta-analysis. Hear. Rhythm. 2019;16(4):502–510. DOI: 10.1016/J.HRTHM.2018.10.022

51. Reese-Petersen A.L., Olesen M.S., Karsdal M.A., Svendsen J.H., Genovese F. Atrial fibrillation and cardiac fibrosis: A review on the potential of extracellular matrix proteins as biomarkers. Matrix Biol. 2020;91–92:188–203. DOI: 10.1016/J.MATBIO.2020.03.005

52. Abe I., Teshima Y., Kondo H., Kaku H., Kira S., Ikebe Y. et al. Association of fibrotic remodeling and cytokines/chemokines content in epicardial adipose tissue with atrial myocardial fibrosis in patients with atrial fibrillation. Heart Rhythm. 2018;15(11):1717–1727. DOI: 10.1016/J.HRTHM.2018.06.025

53. Scott L., Li N., Dobrev D. Role of inflammatory signaling in atrial fibrillation. Int. J. Cardiol., 2019;287:195–200. DOI: 10.1016/J.IJCARD.2018.10.020

54. Zacharia E., Papageorgiou N., Ioannou A., Siasos G., Papaioannou S., Vavuranakis M. et al. Inflammatory biomarkers in atrial fibrillation. Current Medicinal Chemistry. 2019;26(5):837–854. DOI: 10.2174/0929867324666170727103357

55. Oikonomouv E., Zografos T., Papamikroulis G., Siasos G., Vogiatzi G., Theofilis P. et al. Biomarkers in atrial fibrillation and heart failure. Current Medicinal Chemistry. 2019;26(5):873–887. DOI: 10.2174/0929867324666170830100424

56. Tilly M.J., Geurts S., Donkel S.J., Ikram M.A, de Groot N.M.S., de Maat M.P.M. Kavousi M. Immunothrombosis and new-onset atrial fibrillation in the general population: the Rotterdam Study. Clin. Res. Cardiol. 2022;111(1):96. DOI: 10.1007/S00392-021-01938-4

57. Mołek P., Ząbczyk M., Malinowski K.P., Natorska J., Undas A. Enhanced neutrophil extracellular traps formation in AF patients with dilated left atrium. Eur. J. Clin. Invest. 2023. DOI: 10.1111/ECI.13952

58. Vallés Lago J.A., Santo M.T., Latorre A. M., Tembl J.I., Salom J. B., Nieves C., Moscardó A. Neutrophil extracellular traps are increased in patients with acute ischemic stroke: prognostic significance. Thromb. Haemost. 2017;117(10):1919–1929. DOI: 10.1160/TH17-02-0130

59. Mołek P., Ząbczyk M., Malinowski K.P., Natorska J., Undas A. Markers of NET formation and stroke risk in patients with atrial fibrillation: association with a prothrombotic state. Thromb. Res. 2022;213:1–7. DOI: 10.1016/J.THROMRES.2022.02.025

60. Monte A. del, Arroyo A.B., Andrés-Manzano M.J., García-Barberá N., Caleprico M.S., Vicente V., Roldán V. et al. miR-146a deficiency in hematopoietic cells is not involved in the development of atherosclerosis. PLoS One. 2018;13(6):e0198932. DOI: 10.1371/JOURNAL.PONE.0198932

61. Reyes-García A.M. de los, Zapata-Martínez L., Águila S., Lozano M.L., Martínez C., González-Conejero R. microRNAs as biomarkers of risk of major adverse cardiovascular events in atrial fibrillation. Front. Cardiovasc. 2023;10:1135127. DOI: 10.3389/FCVM.2023.1135127

62. Arroyo A.B., De Los Reyes-García A.M., Rivera-Caravaca J.M., Valledor P., García-Barberá N., Roldán V., Vicente V., Martínez C., González-Conejero R. MiR-146a Regulates Neutrophil Extracellular Trap Formation That Predicts Adverse Cardiovascular Events in Patients With Atrial Fibrillation. Arterioscler. Thromb. Vasc. Biol. 2018;38(4):892–902. DOI: 10.1161/ATVBAHA.117.310597

63. Arroyo A.B., De Los Reyes-García A.M., Rivera-Caravaca J.M., Valledor P., García-Barberá N., Roldán V. et al. miR-146a is a pivotal regulator of neutrophil extracellular trap formation promoting thrombosis. Haematologica, 1021;106(6):1636. DOI: 10.3324/HAEMATOL.2019.240226

64. Ortmann E.W., Kolaczkowska E. Age is the work of art? Impact of neutrophil and organism age on neutrophil extracellular trap formation. Cell Tissue Res. 2018;371(3):473. DOI: 10.1007/S00441-017-2751-4

65. Chen C.L., Hui S.G, Wang Z.Y., Zhi L.Q. Influence factors of deep venous thromboembolism after knee arthroplasty and significance of changes of serum nets and sVCAM-1 levels. Zhongguo Gu Shang. 2022;35(11):1053–1059. DOI: 10.12200/J.ISSN.1003-0034.2022.11.009

66. Alkarithi G., Duval C., Shi Y., Macrae F.L., Ariëns R.A.S. Thrombus structural composition in cardiovascular disease. Arterioscler. Thromb. Vasc. Biol. 2021;41(9):2370–2383. DOI: 10.1161/ATVBAHA.120.315754

67. Ansari J., Gavins F.N.E. Neutrophils and platelets: immune soldiers fighting together in stroke pathophysiology. Biomedicines. 2021;9(12):1945. DOI: 10.3390/BIOMEDICINES9121945

68. Cha M.J., Ha J., Lee H., Kwon I., Kim S., Kim Y.D. et al. Neutrophil recruitment in arterial thrombus and characteristics of stroke patients with neutrophil-rich thrombus. Yonsei Med. J. 2022;63(11):1016. DOI: 10.3349/YMJ.2022.0328

69. Ma Y. Role of neutrophils in cardiac injury and repair following myocardial infarction. Cells. 202;10(7):1676. DOI: 10.3390/CELLS10071676

70. Hofbauer T.M., Mangold A., Scherz T., Seidl V., Panzenböck A., Ondracek A.S. et al. Neutrophil extracellular traps and fibrocytes in ST-segment elevation myocardial infarction. Basic Res. Cardiol. 2019;114(5). DOI: 10.1007/S00395-019-0740-3

71. Laridan, Martinod K., Meyer S.F. De. Neutrophil extracellular traps in arterial and venous thrombosis. Semin. Thromb. Hemost. 2019;45(1):86–93. DOI: 10.1055/S-0038-1677040

72. Wang Y., Luo L., Braun O., Westman J., Madhi R., Herwald H., Mörgelin M., Thorlacius H. Neutrophil extracellular trap-micro particle complexes enhance thrombin generation via the intrinsic pathway of coagulation in mice. Sci. Rep. 2018;8(1):4020. DOI: 10.1038/S41598-018-22156-5