The Artemisinin Derivative Artesunate Inhibits Corneal Neovascularization by Inducing ROS-Dependent Apoptosis in Vascular Endothelial Cells

PURPOSE. Without therapeutic intervention, corneal neovascularization rapidly compromises visual acuity, and is a leading cause of blindness. Artesunate was reported to inhibit angiogenesis in tumors, although, the effects of artesunate on nontumor angiogenesis have not been investigated. This study was designed to investigate the effect of artesunate on corneal neovascularization and delineate its underlying mechanism of action. METHODS. Rats with alkali-burned corneas were treated with artesunate for 11 days. Corneal neovascularization was evaluated by measuring the length and area of corneal vasculature in the rats. Apoptotic cells were stained with AnnexinV and propidine iodide (PI), and measured with flow cytometry analysis. Apoptosis-related and p38 mitogen-activated protein kinases (p38MAPK) signaling were evaluated by Western blot analysis. RESULTS. Artesunate significantly inhibited corneal neovascularization and inflammation via specifically inducing apoptosis of vascular endothelial cells. In vascular endothelial cells, artesunate increased the Bax/Bcl-2 ratio, reduced mitochondrial membrane potential, stimulated release of cytochrome C, and cleavage of caspase 9 and 3, suggesting that the mitochondrial apoptotic pathway was involved. Artesunate activated p38MAPK, and specific p38MAPK inhibitors suppressed artesunate-induced apoptosis in endothelial cells. Reactive oxygen species (ROS) levels were increased by artesunate. N-acetyl-L-cysteine blocked p38MAPK activation and protected endothelial cells from artesunate-induced apoptosis. Ferrous salt increased ROS levels and elevated the cytotoxic effect of artesunate on endothelial cells, while the iron chelating agent deferoxamine decreased ROS levels and artesunate-induced apoptosis. Artesunate had no effect on expression of Fas, Fas Ligand, or caspase 8 cleavage. CONCLUSIONS. These results suggest that artesunate induces apoptosis of endothelial cells via an iron/ROS-dependent p38MAPK-mitochondrial pathway.