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ECPB 2019, 88(4): 53–62
Research articles

Impact of Nitric Oxide Synthesis Modulators on the Mechanisms of Apoptosis Development and Production of Reactive Oxygen Species in the Blood Leukocytes in Experimental Antiphospholipid Syndrome


Antiphospholipid Syndrome (APS) is an autoimmune disorder characterized by the presence of antiphospholipid antibodies, increased risk of arterial and venous thrombosis, and pregnancy pathology. The aim of the research was to investigate the effect of L-arginine and aminoguanidine on the degree of reactive oxygen species (ROS) development and processes of apoptosis and necrosis of blood leukocytes in antiphospholipid syndrome in BALB/c mice. The experimental animals were divided into 5 groups: the 1st – the control; the 2nd – the animals with experimental APS, the 3rd – the animals with APS administered with L-arginine (25 mg/kg), the 4th – the animals with APS administered with aminoguanidine (10 mg/kg), the 5th – the animals with APS administered with L-arginine in combination with aminoguanidine. L-arginine and aminoguanidine were administered intraperitoneally once a day for 10 days after APS formation. The content of living, apoptotic and necrotic cells in blood leukocytes was evaluated using Annexin V conjugated of GFP (green fluorescent protein) and propidium iodide (PI). The fluorescence signals of the samples were registered by the channel FL1 (515-535 nm) for GFP and FL3 (620-630 nm) for PI using a flow cytometer. The redistribution between the leukocytes populations was estimated by two parameters: direct (FS, cell size) and side scatter (SS, cell granularity). The ROS in blood leukocytes was determined using 2′,7′- Dichlorofluorescin diacetate. The radiation intensity of the test samples was registered by the channel FL1 (515-535 nm) with a flow cytometer. The results were processed by means of FCS Express V3. It was established that the viability of blood leukocytes of mice decreased in APS compare to the control. L-arginine and aminoguanidine only as well as their combined administration led to normalization of the blood leukocyte viability in the BALB/c mice relative to that of the APS animals. Redistribution between granulocytes and agranulocytes in APS was proved. An increase in the number of granulocytes in blood in APS was evidenced as well. In L-arginine administration to the animals with APS, a further increase in the number of granulocytes in blood was observed. Under the combined use of L-arginine and aminoguanidine, a rebalancing of the number of granulocytes and agranulocytes to the control level took place. In addition, it was established that the basal level of ROS production in granulocytes reduced by 27% and in agranulocytes – by 19% compare to the control group. It was also proved that in case of administration of L-arginine and aminoguanidine only, the animals with APS experienced a decrease in ROS content in granulocytes and agranulocytes. The combined administration of L-arginine and aminoguanidine into the APS mice led to normal ROS content in granulocytes and agranulocytes relative to those of the APS animals. Therefore, an enhanced activation of apoptosis and deficient production of ROS is significant in the pathobiochemical mechanisms of APS. Combined administration of L-arginine and aminoguanidine leads to normalization of blood leukocytes viability of the BALB/c mice, rebalancing of granulocyte and agranulocyte counts to norm, and normalization of the ROS content in granulocytes and agranulocytes relative to that of the APS animals largely than those administered alone.

Received: 16.12.2019

Keywords: Antiphospholipid Syndrome, nitric oxide, apoptosis, leukocytes, reactive oxygen species

Full text: PDF (Ukr) 0.95M

  1. 1. Arachchillage DRJ, Laffan M. Pathogenesis and management of antiphospholipid syn- drome. British Journal of Haematology 2017;178:181-95.
  2. 2. Giannakopoulos B, Krilis SA. The pathogenesis of the antiphospholipid syndrome. N Engl J Med. 2013;368:1033-44.
  3. 3. Velasquez M, Rojas M, Abrahams VM. Escudero C, Cadavid AP. Mechanisms of en- dothelial dysfunction in antiphospholipid syndrome: association with clinical manifestations. Front. Physiol. 2018;9:1840.
  4. 4. Graham A, Ford I, Morrison R, Barker RN, Greaves M, Erwig LP. Anti-endothelial antibodies interfere in apoptotic cell clearance and promote thrombosis in patients with an- tiphospholipid syndrome. J Immunol. 2009;182(3):1756-62.
  5. 5. Ames PR, Batuca JR, Ciampa A, Iannaccone L, Delgado Alves J. Clinical relevance of nitric oxide metabolites and nitrative stress in thrombotic primary antiphospholipid syndrome. J.Rheumatol. 2010;37:2523-2530.
  6. 6. Ferreira EI, Serafim RAM. Nitric Oxide Synthase Inhibitors, Nitric Oxide Synthase - Simple Enzyme-Complex Roles, Saravi SSS, IntechOpen,. 2017. doi: 10.5772/67027. Available at http-
  7. 7. Zhang N, Diao Y, Hua R, Wang J, Han S, Li J, Yin Y. Nitric oxide-mediated pathways and its role in the degenerative diseases. Front Biosci (Landmark Ed). 2017;22:824-34.
  8. 8. Leon-Bollotte L, Subramaniam S, Cauvard O, Plenchette-Colas S, Paul C, Godard C et al. S-nitrosylation of the death receptor fas promotes fas ligand-mediated apoptosis in cancer cells. Gastroenterology. 2011;140(7): 2009-18.
  9. 9. Kienhоfer D, Boeltz S, Hoffmann MH. Reactive oxygen homeostasis - the balance for preventing autoimmunity. Lupus. 2016;25(8):943-954.
  10. 10.Reshetnyak DV, Kuznetsova TV, Kobylyansky AG, Klyukvina NG. Serum nitrate levels in patients with systemic lupus erythematosus and antiphospholipid syndrome. Therapeutic Archive. 2004; 76 (5): 19-22. 
  11. 11. Mineo C, Lanier L, Jung E, Sengupta S, Ulrich V, Sacharidou A et al. Identification of a monoclonal antibody that attenuates antiphospholipid syndrome-related pregnancy complications and thrombosis. PLoS One. 2016;11(7):e0158757.
  12. 12. Sacharidou A, Chambliss KL, Ulrich V, Salmon JE, Shen YM, Herz J et al. Antiphospholipid antibodies induce thrombosis by PP2A activation via apoER2-Dab2- SHC1 complex formation in endothelium. Blood. 2018; 131(19):2097-110.
  13. 13. Kelkka T, Kienhоfer D, Hoffmann M, Linja M, Wing K, Sareila O et al. Reactive oxygen species deficiency induces autoimmunity with type 1 interferon signature. Antioxid Redox Signal. 2014;21(16):2231-45.
  14. 14. Urbonaviciute V, Luo H, Sjоwall Ch, Bengtsson A, Holmdahl R. Low Production of Reactive Oxygen Species Drives Systemic Lupus Erythematosus. Trends in Molecular Medicine. 2019;25(10):826-835.
  15. 15. Zaichenko GV, Laryanovskaya YB, Deyeva TV, Shevchenko OI, Starokozhko VY, Kudina OV et al. Morphological status of the uterus and placenta in experimental model of gestational antiphospholipid syndrome on mice. Ukrainian Medical Almanac 2011;14(4):136-41).
  16. 16. Posokhova KA, Sampara SR, Sak I.Yu. Effect of tivortin on pregnancy, fetal and newborn status in experimental antiphospholipid syndrome. Medical chemistry. 2013;15(4):26-9.
  17. 17. Oleshchuk OM. Influence of aminoguanidine on state of nitric oxide's system at experimental liver cirrhosis. World of Medicine and Biology. 2014;3(45):133-7.
  18. 18. Rieger AM, Nelson KL, Konowalchuk JD, Barreda DR. Modified annexin v/propidium iodide apoptosis assay for accurate assessment of cell death. Journal of Visualized Experiments: JoVE. 2011;50:2597.
  19. 19. Guzyk MM, Dyakun KO, Yanitska LV, Kuchmerovska ТМ. Influence of poly(ADP-ribose) polymerase inhibitors on some pa rameters of oxida tive stress in blood leukocytes of rats with experimental diabetes. The Ukrainian Biochemical Journal. 2013;85(1):62-70).
  20. 20. Sorice M, Circella A, Cristea IM. Cardiolipin and its metabolites move from mitochondria to other cellular membranes during death receptor-mediated apoptosis. Cell Death and Differentiation. 2004;11:1133-45.
  21. 21. Rauch J, Subang R, D'Agnillo P, Koh JS, Levine JS. Apoptosis and the antiphospholipid syndrome. J Autoimmun. 2000;15(2):231-5.
  22. 22. Manganelli V., Capozzi A., Recalchi S. Signore M, Mattei V, Garofalo T et al. Altered traffic of cardiolipin during apoptosis: exposure on the cell surface as a trigger for "antiphospholipid antibodies". Journal of Immunology Research. 2015;
  23. 23. Andreoli L., Fredi M., Nalli C. Franceschini F, Meroni PL, Tincani A. Antiphospholipid antibodies mediate autoimmunity against dying cells. Autoimmunity. 2013;46(5):302-6.
  24. 24. Saligrama PT, Fortner KA, Secinaro MA, Collins CC, Russell JQ, Budd RC. IL-15 maintains T-cell survival via S-nitrosylation-mediated inhibition of caspase-3. Cell Death Differ 2014;21:904-14.
  25. 25. Azad N, Vallyathan V, Wang L, Tantishaiyakul V, Stehlik C, Leonard SS et al. S-nitrosylation of Bcl-2 inhibits its ubiquitin-proteasomal degradation. A novel antiapoptotic mechanism that suppresses apoptosis. J. Biol. Chem. 2006;281:34124-34.
  26. 26. de Groot PG, Urbanus RT. Antiphospholipid syndrome - not a noninflammatory disease. Semin Thromb Hemost. 2015;41(6):607-14.
  27. 27. Marushchak MI. Mitochondrial apoptosis mechanisms in experimental acute lung injury. Bulletin of Scientific Research. 2017;1:121-4)
  28. 28. Wirestam L, Arve S, Linge P, Bengtsson AA. Neutrophils-Important Communicators in Systemic Lupus Erythematosus and Antiphospholipid Syndrome. Frontiers in immunology. 2019;10:2734.
  29. 29. Saran U, Mani KP, Balaguru UM, Swaminathan A, Nagarajan S, Dharmarajan AM et al. sFRP4 signalling of apoptosis and angiostasis uses nitric oxide-cGMP-permeability axis of endothelium. Nitric Oxide. 2017;66:30-42.
  30. 30. Pearce LL, Kanai AJ, Epperly MW, Peterson J. Nitrosative stress results in irreversible inhibition of purified mitochondrial complexes I and III without modification of cofactors. Nitric Oxide. 2005;13:254-63.
  31. 31. Folkes LK, O'Neill P. DNA damage induced by nitric oxide during ionizing radiation is enhanced at replication. Nitric Oxide. 2013;34:47-55.
  32. 32. Baek MW, Seong KJ, Jeong YJ, Kim GM, Park HJ,. Kim SH et al. Nitric oxide induces apoptosis in human gingival fibroblast through mitochondria-dependent pathway and JNK activation. Int. Endod. J. 2015;486287-97.
  33. 33. Brookes PS, Salinas EP, Darley-Usmar K, Eiserich JP, Freeman BA, Darley-Usmar VM et al. Concentration-dependent effects of nitric oxide on mitochondrial permeability transition and cytochrome c release. J. Biol. Chem. 2000;
  34. 34. Delgado Alves J, Mason LJ, Ames PRJ, Chen PP, Rauch J, Levine JS et al. Antiphospholipid antibodies are associated with enhanced oxidative stress, decreased plasma nitric oxide and paraoxonase activity in an experimental mouse model. Rheumatology (Oxford). 2005;44(10):1238-44.
  35. 35. Miranda S, Billoir P, Damian L, Thiebaut PA, Schapman D, Le BM et al. Hydroxychloroquine reverses the prothrombotic state in a mouse model of antiphospholipid syndrome: Role of reduced inflammation and endothelial dysfunction. PLoS ONE. 2019;14(3):e0212614.

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