Platelet-derived exosomes induce endothelial cell apoptosis through peroxynitrite generation: experimental evidence for a novel mechanism of septic vascular dysfunction.
BACKGROUND
Several studies link hematological dysfunction to severity of sepsis. Previously we showed that platelet-derived microparticles from septic patients induce vascular cell apoptosis through the NADPH oxidase-dependent release of superoxide. We sought to further characterize the microparticle-dependent vascular injury pathway.


METHODS
During septic shock there is increased generation of thrombin, TNF-alpha and nitric oxide (NO). Human platelets were exposed for 1 hour to the NO donor diethylamine-NONOate (0.5 microM), lipopolysaccharide (LPS; 100 ng/ml), TNF-alpha (40 ng/ml), or thrombin (5 IU/ml). Microparticles were recovered through filtration and ultracentrifugation and analyzed by electron microscopy, flow cytometry or Western blotting for protein identification. Redox activity was characterized by lucigenin (5 microM) or coelenterazine (5 microM) luminescence and by 4,5-diaminofluorescein (10 mM) and 2',7'-dichlorofluorescein (10 mM) fluorescence. Endothelial cell apoptosis was detected by phosphatidylserine exposure and by measurement of caspase-3 activity with an enzyme-linked immunoassay.


RESULTS
Size, morphology, high exposure of the tetraspanins CD9, CD63, and CD81, together with low phosphatidylserine, showed that platelets exposed to NONOate and LPS, but not to TNF-alpha or thrombin, generate microparticles similar to those recovered from septic patients, and characterize them as exosomes. Luminescence and fluorescence studies, and the use of specific inhibitors, revealed concomitant superoxide and NO generation. Western blots showed the presence of NO synthase II (but not isoforms I or III) and of the NADPH oxidase subunits p22phox, protein disulfide isomerase and Nox. Endothelial cells exposed to the exosomes underwent apoptosis and caspase-3 activation, which were inhibited by NO synthase inhibitors or by a superoxide dismutase mimetic and totally blocked by urate (1 mM), suggesting a role for the peroxynitrite radical. None of these redox properties and proapoptotic effects was evident in microparticles recovered from platelets exposed to thrombin or TNF-alpha.


CONCLUSIONS
We showed that, in sepsis, NO and bacterial elements are responsible for type-specific platelet-derived exosome generation. Those exosomes have an active role in vascular signaling as redox-active particles that can induce endothelial cell caspase-3 activation and apoptosis by generating superoxide, NO and peroxynitrite. Thus, exosomes must be considered for further developments in understanding and treating vascular dysfunction in sepsis.
Gambim
Marcela Helena
MH
Division of Rheumatology, University of São Paulo School of Medicine, Avenida Doutor Arnaldo, 455, 01246-903, São Paulo, SP. mgambim@gmail.com
do Carmo
Alipio de Oliveira
Ade O
Marti
Luciana
L
Veríssimo-Filho
Sidney
S
Lopes
Lucia Rossetti
LR
Janiszewski
Mariano
M
D000328:Adult; D000818:Animals; D017209:Apoptosis; D053148:Caspase 3 / Q000378:metabolism; D002478:Cells, Cultured; D022162:Cytoplasmic Vesicles / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000378:metabolism / Q000502:physiology / Q000503:physiopathology*; D006801:Humans; D008070:Lipopolysaccharides / Q000378:metabolism; D008875:Middle Aged; D009569:Nitric Oxide / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000378:metabolism*; D011817:Rabbits; D012016:Reference Values; D018805:Sepsis / Q000097:blood / Q000503:physiopathology*; D014652:Vascular Diseases / Q000097:blood / Q000503:physiopathology*
D008070:Lipopolysaccharides; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; D053148:Caspase 3