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

Abstract

IntroductionSeveral 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.MethodsDuring 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.ResultsSize, 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.ConclusionWe 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.

CD81 and CD48 show different expression on blood eosinophils in systemic sclerosis: new markers for disease and pulmonary inflammation?

“…CD81 is also a known marker for exosomes [17], but CD81 may also inhibit degranulation in mast cells [18]. We hypothesise that reduced CD81 surface levels may reflect degranulation of eosinophils.…”

Oxidized Low-Density Lipoprotein-Dependent Platelet-Derived Microvesicles Trigger Procoagulant Effects and Amplify Oxidative Stress

“…Microvesicles are exosomes or small particles released by many cells on stimulation (4)(5)(6). The nomenclature of these vesicles lacks consensus.…”

Immune system and regulation of regeneration

“…2017; 16 (4): 94-105 100 в области патофизиологии сепсиса [65][66][67]. Ин-кубация ýндотелиальных клеток и SMCs с тром-боцитарными микрочастицами от септических больных приводит к выраженной индукции апоп-тоза в клетках, в результате выработки активных форм кислорода, что предполагает центральный механизм патогенеза сосудистой дисфункции при сепсисе [66,67].…”

“…2017; 16 (4): 94-105 100 в области патофизиологии сепсиса [65][66][67]. Ин-кубация ýндотелиальных клеток и SMCs с тром-боцитарными микрочастицами от септических больных приводит к выраженной индукции апоп-тоза в клетках, в результате выработки активных форм кислорода, что предполагает центральный механизм патогенеза сосудистой дисфункции при сепсисе [66,67]. Как бы то ни было, но доказа-но, что тромбоцитарные микрочастицы способ-ны фосфорилировать и активировать RAC-alpha serine/threonine-protein kinase, protein kinase B alpha (Akt), серин-треониновую киназу, кото-рая инактивирует промоутер смерти, ассоцииро-ванный с B-клеточной лимфомой 2 -RAC-alpha serine/threonine-protein kinase, protein kinase B alpha (BAD) [68], и оказывает антиапоптотиче-скую активность в ТНР-1 клетках, линии клеток моноцитарного лейкоза человека, зависящей от р-селектина [69].…”