Effects of rapamycin on number activity and eNOS of endothelial progenitor cells from peripheral blood

Abstract

The aim of this investigation is to determine whether rapamycin treatment has any effect on endothelial progenitor cells (EPCs). Total mononuclear cells (MNCs) were isolated from peripheral blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture dishes. After 7 days in culture, attached cells were stimulated with rapamycin (in a series of final concentrations: 0.1, 1.0, 2.0 and 5.0 g/ml) for 6, 12, 24 and 48 h. EPCs were characterized as adherent cells, double positive for DiLDL uptake and lectin binding by direct fluorescence staining. EPC proliferation and migration were determined using the MTT assay and a modified version of the Boyden chamber assay, respectively. An EPC adhesion assay was performed by replating the cells on fibronectin-coated dishes; adherent cells were then counted. Tube formation activity was assayed by using a tube formation assay kit and endothelial nitric oxide synthase (eNOS) was assayed by Western blot analysis. Incubation of isolated human MNCs with rapamycin decreased the number of EPCs present; rapamycin also decreased EPCs proliferative, migratory, adhesive, tube formation capacity and eNOS production in a concentration- and time-dependent manner. Rapamycin was found to decrease the number, proliferative, migratory, adhesive and tube formation capacities of the EPCs, and also was found to decreases eNOS in the EPCs.

Figure 1

Characteristics of human EPCs. MNCs were cultured for 7 days, and adherent cells lectin binding (green, exciting wavelength 477 nm) and DiLDL uptake (red, exciting wavelength 543 nm) were assessed under a laser scanning confocal microscope. Double positive cells appearing yellow in the overlay were identified as differentiating EPCs (×400). Group rapamycin 5.0 g/ml (B1, B2, B3) significantly decreased the number of EPCs compared with control group (A1, A2, A3).

Figure 2

Effects of rapamycin on EPCs’ tube formation. After 24 h, the tubes formed in group 5.0 g/ml rapamycin (D) were seen less than the control group (C) significantly.

Figure 3

Western blot analysis of eNOS of EPCs by a variety of concentrations of rapamycin. The expression of eNOS was significantly decreased in the cells treated with rapamycin in a concentration‐dependent manner. Compared to control group, #P < 0.01.