[Ryanodine downregulates the expression of p-eNOS (Thr495) and improves the functions of rapamycin treated endothelial outgrowth cells].

ABSTRACT

OBJECTIVE: To observe the effects of ryanodine on rapamycin treated endothelial outgrowth cells (EOCs). METHODS: The mononuclear cells were harvested from umbilical cord blood by Ficoll density gradient centrifugation, then induced into EOCs and expanded in vitro. The endothelial characteristics of EOCs were identified by immunostaining and fluorescent staining. The EOCs were pretreated with or without ryanodine (10 µmol/L) for 1 h, and then treated with or without rapamycin (10 nmol/L) for 24 h. Proliferation was evaluated by CCK8 and migration was measured by Transwell. The protein expression of EOCs was evaluated by immunobloting technique with total eNOS antibody and phospho-eNOS (Thr495) antibody. RESULTS: Compared with control group, the proliferation and migration capacities of EOCs were significantly reduced while the phosphorylation of eNOS (Thr495) protein was significantly upregulated in rapamycin group (P < 0.05), expression of total eNOS was not affected by rapamycin (P > 0.05). Compared with rapamycin group, the proliferation and migration capacities of EOCs were significantly increased and the phosphorylation of eNOS (Thr495) protein was significantly downregulated in ryanodine + rapamycin group (P < 0.05). The proliferation and migration capacities, the phosphorylation of eNOS (Thr495) protein and the expression of total eNOS were not affected by ryanodine alone (P > 0.05). CONCLUSIONS: Rapamycin reduced proliferation and migration capacities while upregulated the phosphorylation of eNOS (Thr495) protein of EOCs and these effects could be partly reversed by cotreatment with ryanodine.