BackgroundChronic 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 ResultsSenescence 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 β-galactosidase (SA-βgal), 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 β-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.ConclusionsMPs 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. (J Am Heart Assoc. 2012;1:e001842 doi: 10.1161/JAHA.112.001842.)

chemokine receptors that are upregulated in the vascular wall during deoxycorticosterone acetate (DOCA)/salt-induced hypertension in mice, and to evaluate the impact of pharmacological inhibition of these receptors on blood pressure (BP) and leukocyte accumulation. Methods and ResultsMice treated with DOCA/salt for 21 d displayed markedly elevated systolic BP (158 Ϯ 2 mmHg vs 114 Ϯ 5 mmHg shamtreatment; P Ͻ 0.0001; n Ն 11). In DOCA/salt-treated mice, fl ow cytometry revealed a 2.4-fold increase in numbers of CD45 ϩ leukocytes in the aortic wall (P Ͻ 0.05; n Ն 5). PCR screening via a gene panel containing 20 different chemokine receptors suggested an increase in expression of CCR2 in aortas of DOCA/salt-treated mice (n ϭ 3). Real-time PCR confi rmed upregulation of CCR2 in aortas from DOCA/salt-treated animals, and also levels of the CCR2 ligands CCL2, CCL7, CCL8 and CCL12 ( Ͼ 2-fold for all genes; n Ն 7; P Ͻ 0.05). Administration of a CCR2 antagonist, INCB3344 (30 mg/kg/d, i.p.), to DOCA/salt-treated mice, reduced elevated aortic expression of CCR2 by 55% (n ϭ 8, P Ͻ 0.05). INCB3344 also appeared to reduce leukocyte accumulation in the aortic wall by ~50%, although this effect was not signifi cant (P Ͼ 0.05, n ϭ 5). Importantly, INCB3344 reversed DOCA/salt-induced elevations in systolic BP by ~45% (n ϭ 11, P Ͻ 0.01). ConclusionsOur fi ndings highlight CCR2 as a promising therapeutic target to reduce leukocyte accumulation and BP in hypertension.