NETs and cardiomyocytes

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

Neutrophils

Young polymorph nuclear (PMN)

MicrophagocytosisEnzyme exocytosisNecrosis

Apoptosis

Inflammation in tissues

Norm

a ly

• 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.)

Ilya Ilyich Mechnikov (1845-1916), Nobel prize in 1908.

Arturo Zychlinsky

Discovered NETs in 2004,

The study of neutrophils: sensation after more the 100 years of investigation.

Extracellar trap componentsDNA

Neutrophil Elastase

Histones

Myeloperoxidase

Lactoferrin

MMP9/Gelatinase B

Cathepsin G

Components and triggers of NETs

Et cetera

Рhorbol 12-myristate 13-acetate (PMA)

Activator of PKC, proinflammatory agent

Triggers of NETs formation

LPSIFNgIL-8

- antibacterial defense- antiviral defense- anticancer defense

- autoimmune diseases (systemic lupus erythematosus, rheumatism, psoriasis);- metastasis- secondary alteration at inflammation of different genesismyocardial infarction

NET in pathology

Myocardial infarction and NET“There is an inflammation, lets go there”

Proinflammatory factorsNormal

conditions

Myocardial infarction

Situation after myocardial infarction

The goal of the work:Can NETs provoke secondary alteration at MI?

How to regulate it?

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

DNA concentration in media after cultivation of 200 000 neutrophils without (control) or with

PMA

0

5

10

15

20

25

30

35

control PMA

% o

f D

NA

rel

ease

d

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

control PMA

cLPMA+cL

Fluorescent microscopy of PMN using Hoechst dye and anti-Myeloperoxidase Ab

The percentage of neutrophils with NET under different conditions

0

20

40

60

80

100

120

Contro

l

PMA cL

PMA+cL

per

cen

tag

e o

f ce

lls,

%

live

foul of NET

NET

Coculture of rat neonatal cardiomyocytes and PMN Hoehst 3334, propidium iodide

40 x

The percentage of necrotic cardiomyocytes in coculture with PMN at different conditions of NETs formation (%)

0

5

10

15

20

25

30

контроль PMA лактацистин PMA+лактацистин

The percentage of necrotic cardiomyocytes in coculture with PMN at different conditions of NETs formation after

anoxia (%)

0

10

20

30

40

50

60

контроль PMA лактацистин PMA+лактацистин

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.