High glucose increases the formation and pro-oxidative activity of endothelial microparticles.

AIMS/HYPOTHESIS: Individuals with diabetes exhibit increases in circulating endothelial microparticles (eMPs, also referred to as endothelial microvesicles), which are associated with endothelial dysfunction and a heightened risk of cardiovascular complications. We have shown that eMPs are markers and mediators of vascular injury although their role in diabetes is unclear. We hypothesised that the composition and biological activity of eMPs are altered in response to high glucose exposure. We assessed the effects of high glucose on eMP formation, composition and signalling in cultured HUVECs. METHODS: eMPs were isolated from the media of HUVECs cultured under conditions of normal glucose (eMPNG), high glucose (eMPHG) or osmotic control of L-glucose (eMPLG). eMP size, concentration and surface charge were assessed by nanoparticle tracking analysis and flow cytometry. eMP protein composition was assessed by liquid chromatography-tandem mass spectrometry, and eMP-mediated effects on coagulation, reactive oxygen species (ROS) production and vessel function were assessed. RESULTS: Exposure of HUVECs to high glucose for 24 h caused a threefold increase in eMP formation, increased mean particle size (269 ± 18 nm vs 226 ± 11 nm) and decreased surface charge. Compared with eMPNG or eMPLG, eMPHG possessed approximately threefold greater pro-coagulant activity, stimulated HUVEC ROS production to a greater extent (~250% of eMPNG) and were more potent inhibitors of endothelial-dependent relaxation. Proteomic analysis of eMPs identified 1212 independent proteins of which 68 were exclusively found in eMPHG. Gene ontology analysis revealed that eMPHG-exclusive proteins were associated with signalling pathways related to blood coagulation, cell signalling and immune cell activation. CONCLUSIONS/INTERPRETATION: Our results indicate that elevated glucose is a potent stimulus for eMP formation that also alters their molecular composition leading to increased bioactivity. Such effects may contribute to progressive endothelial injury and subsequent cardiovascular complications in diabetes.