Autor: Mariano Janiszewski, Elias Knobel, Frm Laurindo, Expedito Eustáquio Ribeiro da Silva, Alipio O. Carmo, MA Pedro
Rok vydání: 2003
Předmět:
Zdroj: Critical Care. 7:P117
ISSN: 1364-8535
DOI: 10.1186/cc2313
Popis: Cells release microparticles following apoptosis (apoptotic bodies) or for signaling purposes (exosomes). In contrast to the bigger apoptotic bodies (> 400 nm), exosomes (diameter ~100 nm) do not present phosphatidylserine on their surface and expose major histocompatibility complex components, CD9 and CD63. Prognosis of some thrombotic-inflammatory diseases has been related to microparticle release. Sepsis is an abnormal immuno-inflammatory response to an infection, including dysregulation of apoptotic mechanisms in vascular cells. The major redox signaling pathway in vessels involves the enzymatic complex superoxide generating NADPH oxidase. In previous work we showed that, in sepsis, there is augmented platelet release of microparticles when compared with healthy controls. Those septic microparticles also possess greater NADPH oxidase activity, which can be responsible for vascular cell apoptosis. Our objective was to better characterize those microparticles obtained from septic patients and determine possible pathways related to their release. Through sequential filtration and centrifugation we separated microparticles from septic plasma (n = 16, 24 hours of diagnosis accordingly to ACCP/SCCM 1992 criteria) or from healthy controls (n = 6). Apoptotic bodies were obtained from the medium of cultured endothelial cells exposed to ultraviolet light for 30 min. Laser light scattering revealed particles with diameter between 82 and 112 nm. In contrast to the microparticles, apoptotic bodies do not have NADPH oxidase activity as assayed by lucigenin 5 μM luminescence. Western blot analysis revealed greater NADPH oxidase subunit expression in septic particles when compared with healthy controls, and none on apoptotic bodies. Flow cytometry disclosed positive phosphatidylserine exposure on apoptotic bodies, while microparticles were positive to CD9 and CD63. Washed, fresh platelets from single donors were stimulated with thrombin (0.1 U/ml), tumor necrosis factor alpha (10 μg/ml), lipopolysaccharide (LPS) (0.1 μg/ml) and with the nitric oxide (NO) donor sodium nitroprusside (2 mM) for 30 min. Particles obtained from the tumor necrosis factor-stimulated and thrombin-stimulated platelets were similar to the apoptotic bodies, while those obtained from NO-stimulated or LPS-stimulated platelets shared characteristics with the septic microparticles, with low phosphatidylserine exposure, high CD9 and CD63 expression, and oxidase activity. In conclusion, in sepsis there is a platelet release of exosomes, which possess a vascular pro-apoptotic NADPH oxidase activity. Furthermore, we showed that LPS and NO, agents related to the pathophysiology of severe sepsis, induce similar superoxide producing exosome release from healthy platelets, suggesting the existence of a new redox signaling pathway.
Databáze: OpenAIRE
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