Significance of neutrophil microparticles in ischaemia-reperfusion: Pro-inflammatory effectors of endothelial senescence and vascular dysfunction.

Endothelial senescence is an emerging cause of vascular dysfunction. Because microparticles are effectors of endothelial inflammation and vascular injury after ischaemia-reperfusion, we examined leucocyte-derived microparticles of spleen origin as possible contributors. Microparticles were generated from primary rat splenocytes by either lipopolysaccharide or phorbol-myristate-acetate/calcium ionophore, under conditions mimicking innate and adaptive immune responses. Incubation of primary porcine coronary endothelial cells with either type of microparticles, but not with those from unstimulated splenocytes, leads to a similar threefold raise in senescence-associated β-galactosidase activity within 48 hours, indicating accelerated senescence, to endothelial oxidative stress, and a fivefold and threefold increase in p21 and p16 senescence markers after 24 hours. After 12-hour incubation, the endothelial-dependent relaxation of coronary artery rings was reduced by 50%, at distinct optimal microparticle concentration. In vitro, microparticles were pro-thrombotic by up-regulating the local angiotensin system, by prompting tissue factor activity and a secondary generation of pro-coagulant endothelial microparticles. They initiated an early pro-inflammatory response by inducing phosphorylation of NF-κB, MAP kinases and Akt after 1 hour, and up-regulated VCAM-1 and ICAM-1 at 24 hours. Accordingly, VCAM-1 and COX-2 were also up-regulated in the coronary artery endothelium and eNOS down-regulated. Lipopolysaccharide specifically favoured the shedding of neutrophil- and monocyte-derived microparticles. A 80% immuno-depletion of neutrophil microparticles reduced endothelial senescence by 55%, indicating a key role. Altogether, data suggest that microparticles from activated splenocytes prompt early pro-inflammatory, pro-coagulant and pro-senescent responses in endothelial cells through redox-sensitive pathways. The control of neutrophil shedding could preserve the endothelium at site of ischaemia-reperfusion-driven inflammation and delay its dysfunction.