Enhancing amplification of late‐outgrowth endothelial cells by bilobalide

Abstract

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 H2O2 were quantified with DAF‐FM DA, dihydroethidium, hydroxyphenyl fluorescein and a H2O2 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 H2O2 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.

Reactive Oxygen Species Imaging in U937 Cells

“…Our previous results showed that external oxidative stress can also lead to HO • formation being tightly linked with the production of singlet oxygen in U937 cells (Rác et al, 2015). Since HPF has been identified to be a probe that can also react with other reactive species including but not limited to peroxynitrite, EPR spectroscopy was needed to validate our results (Dunand et al, 2007;Carlisi et al, 2014;Liu et al, 2018). When other methods are not available, an alternative fluorochrome (for instance, BODIPY) with higher sensitivity to HO • could be a potential candidate (Lei et al, 2017).…”

Retracted: Bilobalide alleviates tumor necrosis factor‐alpha‐induced pancreatic beta‐cell MIN6 apoptosis and dysfunction through upregulation of miR‐153

“…Bilobalide (BB) is a sesquiterpenoid with 15 carbon skeletons and is initially found in Ginkgo biloba leaves (Liu et al, ). Ginkgo biloba leaves are largely applied in traditional Chinese medicine.…”

Bilobalide: A review of its pharmacology, pharmacokinetics, toxicity, and safety

“…Transfusion of autologous late‐outgrowth endothelial cells (OECs) is a promising treatment for restenosis after revascularization. BB can increase nitric oxide (NO) production and prolong the lifespan of available NO to enhance the growth and in vitro expansion of OEC, which may simplify the preparation of OECs for the autologous‐OECs‐transfusion therapy (Liu et al, 2018). Besides, BB can increase the expressions of β‐catenin and enhance the secretion of vascular endothelial growth factors (Zhang et al, 2019).…”