Platelet-derived exosomes of septic individuals possess proapoptotic NAD(P)H oxidase activity: A novel vascular redox pathway.
OBJECTIVE
Vascular dysfunction in sepsis may involve apoptosis of vascular cells through redox signaling mechanisms, which are still poorly investigated. Platelets have been shown to produce reactive oxygen species and to release microparticles, related to thrombotic and inflammatory processes. The present study was undertaken to investigate whether, in severe sepsis, platelet-derived microparticles could produce reactive oxygen species through a phagocyte-type nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and if such particles may induce vascular cell apoptosis through a reactive oxygen species-dependent mechanism.


METHODS
Experimental study.


METHODS
Molecular and cell biology laboratories related to tertiary hospitals.


METHODS
Microparticles obtained from septic patients and from healthy individuals were investigated concerning their biochemical properties and their effects on vascular endothelial and smooth muscle cells in culture.


METHODS
Microparticle surface antigens were studied by flow cytometry and the presence of NADPH oxidase subunits by Western blot analysis. Microparticle reactive oxygen species generation was investigated through superoxide dismutase-inhibitable cytochrome c reduction and 5 microM lucigenin chemiluminescence. The effects of microparticles on vascular cell apoptosis rates were analyzed by immunofluorescence microscopy based on annexin V-fluorescein 5(6)-isothiocyanate assay.


RESULTS
Flow cytometry analysis of microparticles obtained from septic patients and healthy individuals showed a surface antigenic pattern similar to exosomes and strongly suggestive of platelet origin. Those microparticles also displayed the p22 and gp91 subunits of phagocyte-simile NADPH oxidase and exhibited intrinsic reactive oxygen species production. Incubation of endothelial and vascular smooth muscle cells with microparticles enhanced apoptosis rates. Reactive oxygen species generation and apoptosis-inducing activity were markedly greater with exosomes from septic individuals than with exosomes from healthy subjects. These effects were diminished by the addition of superoxide dismutase or the NADPH oxidase inhibitors diphenylene iodonium and phenilarsine oxide.


CONCLUSIONS
Platelet-derived exosome NADPH oxidase activity seems to contribute to vascular cell apoptosis and may represent a new vascular redox-signaling pathway involved in the pathophysiology of sepsis.
Janiszewski
Mariano
M
Cellular Biology Laboratory, Instituto de Ensino e Pesquisa, Hospital Israelita Albert Einstein, Avenida Albert Einstein, São Paulo, Brazil.
Do Carmo
Alípio O
AO
Pedro
Marcelo A
MA
Silva
Eliézer
E
Knobel
Elias
E
Laurindo
Francisco R M
FR
D000704:Analysis of Variance; D017209:Apoptosis; D001792:Blood Platelets / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000293:injuries; D006801:Humans; D066298:In Vitro Techniques; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000293:injuries; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D019255:NADPH Oxidases; D010084:Oxidation-Reduction; D017382:Reactive Oxygen Species / Q000378:metabolism; D018805:Sepsis / Q000150:complications / Q000503:physiopathology*; D015398:Signal Transduction; D014652:Vascular Diseases / Q000209:etiology / Q000503:physiopathology*
D017382:Reactive Oxygen Species; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases