Microparticles Induce Cell Cycle Arrest Through Redox-Sensitive Processes in Endothelial Cells: Implications in Vascular Senescence

Abstract

Background - Chronic disease accelerates endothelial dysfunction in aging, a process associated with cell senescence. However, the mechanisms underlying this process are unclear. We examined whether endothelial cell (EC)-derived microparticles (MPs) facilitate EC senescence and questioned the role of reactive oxygen species in this process. // Methods and Results - Senescence was induced by sequential passaging of primary mouse ECs. Cells retained phenotypic characteristics of ECs from passage 4 through passage 21. Passage 21 ECs exhibited features of senescence, including increased staining of senescence-associated ß-galactosidase (SA-ßgal), a greater percentage of cells in G1/G0 phase of the cell cycle, and increased phosphorylation of p66Shc (P<0.05). Microparticle formation from passage 21 ECs was increased versus passage 4 ECs (~2.2-fold increase versus passage 4, P<0.05), and the Rho kinase inhibitor fasudil blocked this increase. Exposure of passage 4 ECs to MPs shifted cells from a proliferating to a nonproliferating phenotype, as indicated by cell cycle analysis and increased senescence-associated ß-galactosidase staining. MPs increased EC generation of O2•- (~2.7-fold) and H2O2 (~2.6-fold), effects blocked by apocynin (nicotinamide adenine dinucleotide phosphate oxidase inhibitor) and rotenone (mitochondrial oxidase inhibitor) but not by allopurinol (xanthine oxidase inhibitor). MPs increased expression of cell cycle proteins p 21 cip1 and p16ink4a and stimulated phosphorylation of p66Shc in ECs (P<0.05 versus untreated ECs). Pretreatment with the reactive oxygen species scavenger sodium 4,5-dihydroxybenzene-1,3-disulfonate (tiron) abrogated the prosenescent effects of MPs. // Conclusions - MPs promote EC senescence through nicotinamide adenine dinucleotide phosphate oxidase- and mitochondrial-derived reactive oxygen species. Such redox-sensitive processes may be important in vascular dysfunction in aging.