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

Effects of oxidized low density lipoprotein (LDL) on sinaling process in human monocyts

A modificacao oxidativa da LDL humana tem um papel importante no desenvolvimento da lesao aterosclerotica. A captacao de LDL oxidada (LDLox) pelo macrofago derivado de monocito conduz ao desenvolvimento da estria gordurosa. As celulas cheias de lipideos sao denominadas celulas espuma. A proliferacao e a diferenciacao celular podem ocorrer no local da lesao aterosclerotica em consequencia das interacoes entre LDLox e celulas vasculares. As mudancas nos niveis intracelulares de proteinas tirosina fosforiladas foram associadas ao crescimento e a diferenciacao celular. Alem disso, achados recentes estabeleceram um papel para LDLox e outros oxidantes como mediadores ou moduladores de processos de sinalizacao celular dependentes tirosina. Baseado nestas observacoes sugerimos que LDLox poderia mediar a diferenciacao dos macrofagos derivados de monocitos ate a celula espumosa, atraves das mudancas na morfologia das celulas, alteracao nos niveis de fosforilacao em tirosina e na liberacao do NO. O tratamento da celula THP1 com a LDL minima modificada (LDmm), ou LDL extensivamente modificada (LDLox) em uma concentracao de 20 mg/ml foi seguido ate 5 dias. A diferenciacao das celulas THP1 por 4OnM de PMA e GM-CSF foi usada como controle positivo. As celulas THP1 foram mantidas na cultura na ausencia de lipoproteina para o mesmo periodo e as usamos como um controle negativo. Os realces progressivos em niveis do fosforilacao em tirosina foram observados ate 3 dias nas celulas incubadas com LDLmm e LDLox. Apos 5 dias de incubacao, a fosforilacao retorna aos niveis basais. Sob as mesmas circunstancias experimentais e usando anticorpos especificos que reconhecem varios tipos de proteinas tirosinas quinases, demonstramos que a expressao de ERKl/ERK2, p56, p85 e Src, alcanca seu maximo em 3 dias. Apos 5 dias de incubacao houve a producao de oxido nitrico, avaliada pelo acumulo de nitrito no sobrenadante das culturas de celulas, aumentado substancialmente nas celulas tratadas com LDLmm. A microscopia optica revelou mudancas morfologicas nas celulas tratadas com o PMA e o LDLox apos 3 dias de incubacao. Concluimos que os resultados apresentados sugerem que LDL oxidada induz mudancas morfologicas nas celulas THPL que sao acompanhadas por mudancas em niveis celulares da fosforilacao em tirosina e da expressao de proteinasBV UNIFESP: Teses e dissertaçõe