Abstract P069: Microvesicles From Enos-suppressed Endothelial Cells Induce Endothelial Cell Dysfunction

Abstract

Introduction: Endothelial nitric oxide synthase (eNOS) activity is critical to vascular health. Impaired eNOS activity and diminished NO production are common characteristics of a proatherogenic, dysfunctional endothelial phenotype that is associated with cardiovascular risk factors and disease. Extracellular microvesicles, particularly endothelial cell derived microvesicles (EMVs) represent novel mechanistic mediators of endothelial dysfunction and vascular disease. It is unknown whether eNOS suppression affects EMV number and function. We tested the following hypotheses: 1) eNOS blockade increases EMV release; and 2) EMVs derived from eNOS-suppressed cells adversely affect endothelial cell inflammation, apoptosis and NO production. Methods: Human umbilical vein endothelial cells (HUVECs) were treated with the eNOS inhibitor, L-N G -Nitroarginine methyl ester (L-NAME; 300mM) for 24 h. EMVs (CD144 + ) released into the supernatant from cells treated with L-NAME or vehicle were isolated and quantified by flow cytometry. Fresh HUVECs were then treated with either L-NAME-derived or control EMVs for 24 h. To evaluate the role of endocytosis on the endothelial effects of EMVs, HUVECs were pre-incubated (12 h) with EIPA, filipin and chlorpromazine for 2 h, and all experiments repeated. Results: EMV release was markedly higher (~100%; P<0.05) in cells treated with L-NAME compared with control (81±6 vs. 40±7 EMV/μL). L-NAME-generated EMVs induced significantly higher release of IL-6 (38.4±5.1 vs. 21.0±1.9 pg/mL) and IL-8 (38.9±3.5 vs. 27.2±3.1 pg/mL) as well as greater active NF-κB p65 (Ser-536) (9.7±0.7 vs. 6.1±0.6 AU) expression than control EMVs. The expression of activated-caspase-3 was significantly higher in the cells treated with L-NAME (9.5±1.1 vs. 6.4±0.4 AU). Total eNOS (97.1±8.2 vs. 157.5±15.6 AU), activated eNOS (4.9±1.2 vs. 9.1±1.3 AU) and NO production (5.0±0.8 vs. 7.0±0.6 μmol/L) were significantly lower in endothelial cells treated with EMVs from eNOS suppressed cells. Endocytosis blockers mitigated the deleterious endothelial effects of EMVs. Conclusion: eNOS-suppression increases EMV release. Moreover, EMVs from eNOS-suppressed cells increase endothelial cell inflammation and apoptosis and decrease NO production.