Platelet-derived exosomes induce endothelial cell apoptosis through peroxynitrite generation: experimental evidence for a novel mechanism of septic vascular dysfunction

Abstract\ud \ud \ud \ud Introduction\ud \ud 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.\ud \ud \ud \ud Methods\ud \ud During septic shock there is increased generation of thrombin, TNF-α and nitric oxide (NO). Human platelets were exposed for 1 hour to the NO donor diethylamine-NONOate (0.5 μM), lipopolysaccharide (LPS; 100 ng/ml), TNF-α (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 μM) or coelenterazine (5 μM) 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.\ud \ud \ud \ud Results\ud \ud 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-α 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-α.\ud \ud \ud \ud Conclusion\ud \ud 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.LRL and MJ have research grants from Fundação de Amparo a Pesquisa do Estado de São Paulo – FAPESP. MJ received a research grant from Sociedade Beneficente Israelita-Brasileira Hospital Albert Einstein.LRL and MJ have research grants from Fundação de Amparo a Pesquisa do Estado de São Paulo – FAPESP. MJ received a research grant from Sociedade Beneficente IsraelitaBrasileira Hospital Albert Einstein

Generation of reactive oxygen species by platelet-derived exosomes: a possible novel mechanism of vascular dysfunction in sepsis

Sepse, a resposta do organismo a uma infecção, está associada a altas taxas de mortalidade. A razão pela qual um mecanismo protetor resulta num quadro clínico fatal permanece inexplicada. Em trabalho prévio nosso grupo demonstrou que exossomos de origem plaquetária são os mais freqüentes em plasma de pacientes com choque séptico e que estes podem induzir apoptose em células musculares lisas vasculares e células endoteliais em cultura. Demonstramos ainda que tais exossomos possuíam uma fonte enzimática de ROS, uma NADPH oxidase cuja atividade poderia estar associada à indução da apoptose (Janiszewski et al., 2004). No presente trabalho, nós buscamos criar um modelo de geração ex vivo de exossomos similares aos encontrados em pacientes sépticos e identificar possíveis vias responsáveis pela liberação destes e seus efeitos. Choque séptico é uma condição relacionada com exposição a lipopolissacarídeo (LPS) e geração de alta quantidade de trombina, TNF e espécies reativas de nitrogênio. Através de citometria de fluxo revelamos que plaquetas humanas expostas ao doador de NO dietilamina-NONOato e ao LPS geraram exossomos similares àqueles encontrados em pacientes com choque séptico, expondo alta quantidade de tetraspaninas CD9, CD63 e CD81 mas pouca fosfatidilserina. Por outro lado, plaquetas expostas à trombina ou TNF liberaram partículas com características claramente distintas, com alta exposição de fosfatidilserina e baixa de tetraspaninas. Assim como os exossomos sépticos, os exossomos obtidos pela exposição de NO e LPS geraram radical superóxido e NO, como demonstrado pela quimioluminescência da lucigenina (5M) e celenterazinina (5M) e pela fluorescência da 4,5-diaminofluoresceína (10mM) e 2,7-diclorofluoresceína (10mM). A análise por Western Blot nos permitiu identificar as subunidades Nox1, Nox2 e p22phox da NADPH oxidase e a isoforma induzível da enzima NO sintase (NOS) nesses exossomos. Como esperado, inibidores da NOS e da NADPH oxidase reduziram significamente os sinais fluorescentes e quimioluminescentes. Em adição, as células endoteliais em cultura expostas aos exossomos gerados por dietilamina-NONOato e LPS sofreram significativo aumento da taxa de apoptose quando comparadas àquelas expostas a exossomos controle. A inibição da NADPH oxidase assim como da NOS reduziu expressivamente tal efeito. Adição de urato (1mM), mostrou efeito aditivo sobre a inibição do sinal fluorescente, assim como redução adicional da taxa apoptótica, sugerindo papel importante do radical peroxinitrito. Nós propomos, assim, que exossomos derivados de plaquetas podem representar papel adicional no já complexo cenário da sinalização vascular redox. Nesse sentido, uma abordagem baseada em exossomos pode fornecer novas ferramentas para o entendimento e até tratamento da disfunção vascular na sepseSepsis, the bodys response to infection, is associated with high mortality rates. Why a protective mechanism turns into a deadly clinical picture is a matter of debate, and goes largely unexplained. In previous work we demonstrated that plateled derived exosomes are found in the plasma of septic patients with septic shock and can induce endothelial and vascular smooth muscle cell apoptosis in culture through an enzymatic superoxide source (Janiszewski et al., 2004). In this work we sought to create a model for ex vivo generation of exosomes, and to identify the pathways responsible for ROS release by exosomes and their effects. Septic shock is a condition related to exposure of lipopolysaccharide (LPS), generation of high amounts of thrombin, TNF and nitrogen reactive species. Through flow cytometry we demonstrated that human platelets exposed to the NO-donor diethylamine-NONOate, and to LPS, generated exosomes similar to those found in the blood of septic shock patients, with high exposure of the tetraspanin CD9, CD63, and CD81, but little phosphatidylserine. On the other hand, platelets exposed to thrombin or TNF released particles with clearly distinct characteristics, such as high phosphatidylserine and low tetraspanin. Like the septic exosomes, the exosomes obtained by NO and LPS exposure generated superoxide radical and NO, as disclosed by lucigenin and coelenterazine chemiluminescence and by 4,5-diaminofluorescein and 2,7-dichlorofluorescein fluorescence. Western Blot analysis revealed the presence of Nox1, Nox2 and p22phox NADPH oxidase subunits and the inducible isoform of NO synthase (NOS) in these exosomes. As expected, NOS inhibitors or NADPH oxidase inhibitors significantly reduced the fluorescence and chemiluminescente signals. In addition, endothelial cells exposed to NO or LPS generated exosomes underwent apoptotic death, while control exosomes had no effects on apoptosis. NADPH oxidase as well as NOS inhibition significantly reduced apoptosis rates. Concomitant generation of NO and superoxide suggests biological effects of the highly reactive radical peroxynitrite. In fact, the peroxynitrite scavenger urate (1 mM) showed an additive effect on fluorescent signal inhibition, as well as on endothelial apoptosis rate reduction. We thus propose that platelet-derived exosomes may be another class of actors in the complex play known as vascular redox signaling. In this sense, an exosome-based approach can provide novel tools for further understanding and even treating vascular dysfunction related to sepsi