Recent studies have demonstrated that microparticles (MPs) generated from T lymphocytes undergoing both activation and apoptosis correct endothelial injury, and regulate angiogenesis. These effects are mediated by an increase of NO release and a decrease of reactive oxygen species (ROS) production. Apoptosis is an important mechanism that controls endothelial cell number and regeneration. We studied the effects of these MPs on human umbilical vein endothelial cell (HUVEC) apoptosis induced by actinomycin D (actD). Engineered MPs were obtained by activation of human lymphocyte CEM-T cell line with phytohemagglutinin and then, by stimulation with phorbol-12-myristate-13-acetate and actD. HUVECs were grown for 24h in absence or presence of the proapoptotic agent actD (1μg/ml), and/or 10μg protein/ml of MPs. In another set of experiments, endothelial cells were pre-incubated either with nonselective caspases inhibitor, z-vad.fmk (50μm), PI3-kinase inhibitor, LY294002 (10μm), ERK inhibitor, U0126 (10μm), NOS inhibitor, L-NA (100μm), or the superoxide dismutase (SOD) mimetic, manganese(III)tetrakis-(1-methyl-4-pyridyl)-porphyrin pentachloride (MnTMPyP, 100μm). The proportion of nuclei with hypodiploid DNA (sub-G1 peak) corresponding to apoptotic cells was determined by flow cytometry after staining with propidium iodide, and by microscopy using TUNEL labeling. Our results clearly indicate that activated/apoptotic T lymphocytes MP treatment significantly reduces HUVEC apoptosis evoked by actD. Moreover, pancaspases inhibitor reduces the degree of cell death either in presence or in absence of MPs, indicating the implication of caspases in actD-induced apoptosis. Although, the PI3-kinase inhibitor induces apoptosis by itself, it does not blunt the capacity of MPs in reducing apoptosis induced by actD. In contrast, the inhibitory effect of MPs is prevented in the presence either of the ERK inhibitor or the NOS inhibitor. Interestingly, MnTMPyP reduces actD-evoked apoptosis and the protective effects of MPs. Thus, these MPs may act as an SOD mimetic under these experimental conditions. Altogether, T lymphocyte MPs evoked cell protection against apoptosis at the level of caspases inhibition, MAP kinases, NO and ROS formations. They underscore the potential therapeutic of such MPs against vascular pathologies in close association with apoptosis.
Supported by ANR-07-PHYSIO-010-01.Le texte complet de cet article est disponible en PDF.