Proteasome inhibitor MG132 suppresses number and function of endothelial progenitor cells: Involvement of nitric oxide synthase inhibition

Abstract

The aim of this study was to determine whether proteasome inhibitor MG132 treatment has any effect on endothelial progenitor cells (EPCs). Total mononuclear cells (MNCs) were isolated from peripheral blood by Ficoll density gradient centrifugation. EPCs were identified as adherent cells double positive for DiLDL-up-take and lectin binding by direct fluorescent demonstrated under a laser scanning confocal microscope. After 7 days in culture, EPCs were stimulated with proteasome inhibitor MG132 in series of final concentrations of 20, 50, 100, 200 nmol/l for 12, 24, 48 h. Cell proliferation and apoptosis were determined, respectively, by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, annexin V/propidium iodide binding assay. Colony-forming capacity was performed by colony assay. EPCs adhesion and migration were assayed with adhesion assay and transwell migration assay, respectively. The expression of endothelial nitric oxide synthase (eNOS) was assayed by Western blot analysis, while nitric oxide (NO) generation was detected using the Griess method. It was found that proteasome inhibitor MG132 decreased the number of EPCs and EPC colonies, increased EPC apoptosis, decreased EPC proliferative, adhesive, migration capacity and eNOS/NO production in a concentration- and time-dependent manner. These data indicate that proteasome inhibitor MG132 suppresses the number and function of EPCs, and these actions may involve decreased eNOS/NO production in the EPCs.