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NETs and cardiomyocytes


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Neutrophil extracellular traps and it's possible role in cardiac pathology. The experience of cocultivation of neutrophils and cardiomyocytes. The role of proteasomal proteolysis in NETs formation.

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NETs and cardiomyocytes

  1. 1. Neutrophil extracellular traps in pathology: focus on heart diseases Bogomoletz Institute of Physiology, Kiev, Ukraine Department of General and Mollecular Pathophysiology Vasyl Nagibin, MD,PhD
  2. 2. Neutrophils Young polymorph nuclear (PMN) Microphagocytosis Enzyme exocytosis Necrosis Apoptosis Inflammation in tissues Normaly
  3. 3. • Neutrophil extracellular traps are formed as the result of specific type of programmed cell death called NETosis. • The term “ETosis” is more wide and used to determine this type of cell death in other granulocytes (eosinophils etc.)
  4. 4. The study of neutrophils: sensation after more the 100 years of investigation. Ilya Ilyich Mechnikov (1845-1916), Nobel prize in 1908. Arturo Zychlinsky Discovered NETs in 2004,
  5. 5. Components and triggers of NETs Extracellar trap components DNA Neutrophil Elastase Histones Myeloperoxidase Lactoferrin MMP9/Gelatinase B Cathepsin G Triggers of NETs formation Et cetera Рhorbol 12-myristate 13-acetate (PMA) Activator of PKC, proinflammatory agent LPS IFNg IL-8
  6. 6. NET in pathology - antibacterial defense - antiviral defense - anticancer defense - autoimmune diseases (systemic lupus erythematosus, rheumatism, psoriasis); - metastasis - secondary alteration at inflammation of different genesis myocardial infarction
  7. 7. Myocardial infarction and NET “There is an inflammation, lets go there” Proinflammatory Normal factors conditions Myocardial infarction Situation after myocardial infarction The goal of the work: Can NETs provoke secondary alteration at MI? How to regulate it?
  8. 8. Materials and Methods Isolation of neutrophils from rat blood,using Percoll gradient Cultivation of neutrophils in RPMI w/o Phenol red and serum for 3 hours with or without (control) PMA (5 nM) Measurment of DNA released (PicoGreen, fluorescence measurment), and other NETs components (myeloperoxidase, immunohistochemistry) (A.Zychlinsky et al; JCB,Vol176,N2,2007) AND Isolation of rat neonatal cardiomyocytes using Collagenase II and Tripsin Cocultivation of rat neonatal cardiomyocytes with neutrophils Use of specific proteasome inhibitor (clasto-Lactacystin β-lactone) in dose of 5 nM
  9. 9. DNA concentration in media after cultivation of 200 000 neutrophils without (control) or with PMA 35 30 25 20 15 10 5 0 control PMA % of DNA released
  10. 10. Fluorescent microscopy of PMN using Hoechst dye control PMA PMA PMA was used for activatio9n of NETs formation in dose 5 nM for 3 hours Hoechst was used for detection of DNA at fluorescent microscopy 40x
  11. 11. Fluorescent microscopy of PMN using Hoechst dye control PMA cL PMA+cL and anti-Myeloperoxidase Ab
  12. 12. The percentage of neutrophils with NET under different conditions 120 100 80 60 40 20 0 Control PMA cL PMA+cL percentage of cells, % live foul of NET NET
  13. 13. Coculture of rat neonatal cardiomyocytes and PMN Hoehst 3334, propidium iodide 40 x
  14. 14. The percentage of necrotic cardiomyocytes in coculture with PMN at different conditions of NETs formation (%) 30 25 20 15 10 5 0 контроль PMA лактацистин PMA+лактацистин
  15. 15. The percentage of necrotic cardiomyocytes in coculture with PMN at different conditions of NETs formation after anoxia (%) 60 50 40 30 20 10 0 контроль PMA лактацистин PMA+лактацистин
  16. 16. Thanks for some of my colleagues, participating in this project Prof. Moibenko O.O. Prof. Dosenko V.E. MD. PhD. Pashevin D.O. BD. Tumanovska L.V.