Circulating microparticles derived from different types of blood cells have been reported to impair endothelial function and to induce pro-inflammatory and prothrombotic endothelial phenotypes. Although the number of monocyte-derived microparticles (M-MPs) is elevated in the blood of patients with various inflammatory conditions, their interaction with endothelial cells has been poorly investigated so far. In this study, we produced microparticles in vitro from apoptotic human monocytes and examined the effects of their interaction with cultured human umbilical vascular endothelial cells (HUVECs). We found that low concentrations of M-MPs induced the production of reactive oxygen species (ROS), mainly anion superoxide, by the endothelial cells. At sub-toxic concentrations, M-MPs induced a rapid expression of von Willebrand factor at the cell surface, which mediated the transient attachment of non-activated platelets to the endothelium in flow conditions. In parallel, M-MPs up-regulated the expression of functional tissue factor by the endothelial cells. ROS controlled these two major changes and the process involved the phosphorylation of p38 mitogen activated protein kinase. We conclude that M-MPs may contribute to thrombotic events by producing redox signalling in endothelial cells.

BackgroundAccelerated atherosclerosis is the leading cause of morbidity and mortality in diabetic patients. Hyperglycemia is a recognized independent risk factor for heightened atherogenesis in diabetes mellitus (DM). However, our understanding of the mechanisms underlying glucose damage to the vasculature remains incomplete.Methodology/Principal FindingsHigh glucose and hyperglycemia reduced upregulation of the NF-κB inhibitory and atheroprotective protein A20 in human coronary endothelial (EC) and smooth muscle cell (SMC) cultures challenged with Tumor Necrosis Factor alpha (TNF), aortae of diabetic mice following Lipopolysaccharide (LPS) injection used as an inflammatory insult and in failed vein-grafts of diabetic patients. Decreased vascular expression of A20 did not relate to defective transcription, as A20 mRNA levels were similar or even higher in EC/SMC cultured in high glucose, in vessels of diabetic C57BL/6 and FBV/N mice, and in failed vein grafts of diabetic patients, when compared to controls. Rather, decreased A20 expression correlated with post-translational O-Glucosamine-N-Acetylation (O-GlcNAcylation) and ubiquitination of A20, targeting it for proteasomal degradation. Restoring A20 levels by inhibiting O-GlcNAcylation, blocking proteasome activity, or overexpressing A20, blocked upregulation of the receptor for advanced glycation end-products (RAGE) and phosphorylation of PKCβII, two prime atherogenic signals triggered by high glucose in EC/SMC. A20 gene transfer to the aortic arch of diabetic ApoE null mice that develop accelerated atherosclerosis, attenuated vascular expression of RAGE and phospho-PKCβII, significantly reducing atherosclerosis.ConclusionsHigh glucose/hyperglycemia regulate vascular A20 expression via O-GlcNAcylation-dependent ubiquitination and proteasomal degradation. This could be key to the pathogenesis of accelerated atherosclerosis in diabetes.

Fragile atherosclerotic plaques are rich in apoptotic smooth muscle cells (SMCs) and macrophages, generating microparticules (MPs) which accumulate locally and may be released in blood in case of mechanical or spontaneous plaque disruption. Besides being highly procoagulant, this material may interact with downstream endothelium. Using a model of mouse aorta vaso-reactivity, we have investigated the effects of apoptotic MPs prepared in vitro from Fas-ligand sensitive SMCs. Short-term preincubation of aorta rings with the MPs dose-dependently reduced the vasodilatory response to acetylcholine dependent on the endothelium. This effect was prevented by the addition of abxicimab or eptifibatide, indicating a role for a beta3 integrin in this process. We further investigated its mechanism using cultured endothelial cells. The MPs were found to bind to the cells and to inhibit the production and the release of nitric oxide (NO) in response to bradykinin. This phenomenom was redox sensitive, independent of the generation of activated coagulation proteases, and was abrogated when the MPs were pretreated by trypsin. The metabolic effects of MPs were prevented by the addition of eptifibatide. Taken together, these results suggest a potential, platelet-independent, mechanism for the improvement of microvascular perfusion observed with beta3-integrin antagonists.