Enhancing amplification of late-outgrowth endothelial cells by bilobalide

Transfusion of autologous late-outgrowth endothelial cells (OECs) is a promising treatment for restenosis after revascularization. Preparing cells by in vitro amplification is a key step to implement the therapy. This study aimed to demonstrate that bilobalide, a terpenoid, enhances the OEC amplification. Human-, rabbit- and rat OECs and a mouse femoral artery injury model were used. Expanding OECs used endothelial growth medium-2 as the standard culture medium while exploring the mechanisms used endothelial basal medium-2. Proliferation assay used MTT method and BrdU method. Migration assay used the modified Boyden chamber. Intracellular nitric oxide, superoxide anion, hydroxyl radical/peroxynitrite and H₂ O₂ were quantified with DAF-FM DA, dihydroethidium, hydroxyphenyl fluorescein and a H₂ O₂ assay kit, respectively. Activated ERK1/2 and eNOS were tested with the Western blot. Bilobalide concentration-dependently enhanced OEC number increase in vitro. Transfusion of bilobalide-based human OECs into femoral injured athymia nude mouse reduced the intimal hyperplasia. Bilobalide promoted OEC proliferation and migration and increased the intracellular nitric oxide level. L-NAME, a NOS inhibitor, inhibits but not abolishes OEC proliferation, migration and ERK1/2 activation. Bilobalide concentration-dependently enhanced the eNOS Ser-1177 phosphorylation and Thr-495 dephosphorylation in activated OECs. Bilobalide alleviates the increase in hydroxyl radical/peroxynitrite, superoxide anion and H₂ O₂ in proliferating OECs. In conclusion, nitric oxide plays a partial role in OEC proliferation and migration; bilobalide increases OEC nitric oxide production and decreases nitric oxide depletion, promoting the OEC number increase; Bilobalide-based OECs are active in vivo. The findings may simplify the preparation of OECs, facilitating the implementation of the autologous-OECs-transfusion therapy.