Leukocyte-derived microparticles exaggerate endothelial senescence and vascular dysfunction induced by high glucose

Abstract

Microparticles (MPs) are plasma membrane vesicles and vascular effectors. High levels of pro-inflammatory cytokines and procoagulant endothelial-derived MPs circulate in diabetic patients. We have shown that (i) leukocyte-derived MPs shed in response to stress are pro-inflammatory, procoagulant and prosenescent endothelial effectors; (ii) high glucose induces premature endothelial senescence. To determine the possibility that leukocyte-derived MPs affect endothelial senescence and vascular function in response to high glucose. Leukocyte MPs were isolated from rat splenocytes with either 5 mg/ml LPS (MPLPS), 25 ng/ml PMA/1 mM A23187 ionophore (MPPMAi), or vehicle (MPCTL). Porcine coronary artery endothelial cells (ECs) at passage 1 were incubated for 48 h with 1–30 nM MPs in high or low glucose concentration (HG 25 mM, NG 5.5 mM). Senescence-associated β-galactosidase (SA-ß-GAL) activity was assessed by C12FDG, protein expression by Western blot analysis. Pig coronary artery rings were pre-incubated with HG or NG for 12 h prior to addition of 1–30 nM MPs for 12 h. Bradykinin (BK)-induced endothelium-dependent relaxations were assessed in organ chambers, and staining of target proteins by confocal microscopy. At 10 nM, MPLPS and MPPMAi enhanced SA-b-GAL activity both by about 2-fold in NG, and respectively 3 and 3.7-fold in HG. The expression of senescence markers p21, p16 doubled and that of eNOS decreased 2-fold. MPPMAi and MPLPS induced a concentration-dependent inhibition of BK-induced relaxation, inhibition by respectively 10 nM and 30 nM,being 65% in NG, amounting to about 85% in HG, whereas 30 nM MPCTL had no effect. MPPMAi and MPLPS reduced eNOS expression by 60% in NG and 80% in HG. Conversely, VCAM-1, COX-2 were up-regulated. Leukocyte-derived MPs enhance HG-induced alteration of the endothelial function by inducing premature senescence and might contribute to vascular dysfunction in diabetes patients.